F Mariette
email: francois.mariette@cemagref.fr

Investigations of food colloids by NMR and MRI

Keywords: magnetic resonance imaging, NMR, relaxation, diffusion, food process, gel, emulsion

Since the publication of the ISO method for measurement of solid fat content using NMR relaxation at low field, the application of this technique to the characterisation of food composition and food structure on several length scales has expanded considerably.

Improvements in the electronic and computational specifications of NMR spectrometers and the use of more sophisticated signal processing methods have led to several new applications, and NMR, previously recognized as a powerful technique to provide information regarding composition, is now widely used for microstructural investigations. Moreover, MRI applications have in recent years been extended from the medical field to food science and this has opened up new opportunities for the understanding of food. We examine here recent developments in these techniques, including NMR relaxation, multidimensional NMR relaxation, diffusion NMR and MRI.

Introduction

The use of low field NMR spectrometers in the food industry began in the 70's with the commercial availability of the first benchtop NMR systems. Several applications were developed during the following years, but they were limited to the determination of water and fat content and measurement of the solid fat index. Surprisingly, the number of application did not increase much during the following twenty years. In 1992, the first International Conference on Applications of Magnetic Resonance in Food Science was organised, and since that time, with the improvements in benchtop NMR characteristics, new applications have been proposed. One of the most well-known is droplet size measurement. Moreover, the use of NMR relaxation time was proposed, not only for determination of water and fat content, but also for the investigation of food structure. Simultaneously, the use of magnetic resonance imaging techniques, in which the contrast in the image is governed by the NMR relaxation time, has emerged. MRI now is a powerful technique to monitor the composition and structural changes in foods during processing. This review focuses on the recent applications of low field NMR and MRI in food colloids and deals with recent advances such as multidimensional NMR relaxation and NMR diffusometry.

Solid and liquid-like relaxation

Most of the NMR relaxation studies on food colloids are performed with low magnetic field spectrometers typically operating at frequencies less than 25 Mhz. Since the introduction of the benchtop low magnetic field NMR equipment in the 70s, the electronic and computational specifications have improved dramatically. Short, powerful excitation pulses now permit sampling of the relaxation decay curve from 11µs up to several seconds. The number of data points available (several hundred for Free Induction Decay (FID) and several thousand for the Carr Purcell Meiboon Gill sequence (CPMG)) and the fast sampling rate (20 Mhz) are used for the simultaneous acquisition of the solid and liquid-like relaxation. Moreover, the use of continuous inverse Laplace transform methods has improved the understanding of relaxation behaviour in complex food products.

For T 2 relaxation, the signal is therefore directly fitted as a sum of Gaussian functions for the solid part and the sum of exponential functions for the liquid part:

( )         - +         - = ∑ ∑ 2 2 2 exp exp T t L T t S t Y i i eq. 1
However, the T 2 for the solid part is often very short (around 15µs) and the number of data available for the fitting is to small to allow multi-Gaussian fitting because of the limitation of the sampling rate of the acquisition board, so the solid part is mainly described by a single Gaussian function. For a specific system with a highly structured solid phase such as lipid crystals in β polymorphic form, eq 1 becomes:

( )

        - +       - = ∑ 2 2 2 exp sin 2 1 exp T t L bt bt t a S t Y i eq. 2
with the second moment M 2 calculated from the fitting parameters a and b with the following equation:

2 2 2 3 1 b a M + = eq. 3
According to the chemical composition of the product, the signal for the Gaussian relaxation component can be attributed to non-exchangeable protons from solid fat, ice, protein and polysaccharides, whereas, the relaxation component from the liquid phase is generally attributed to protons from liquid water and liquid fat, and from exchangeable protons from biopolymers (proteins, carbohydrates or polysaccharides). The simultaneous acquisition of solid and liquid-like relaxation has been extensively used to improve significantly the accuracy of standard NMR applications such as solid fat content, and also to carry out quantitative analysis of NMR signals from complex products such as ice cream, cake, cheese, etc., leading to new NMR applications.

Solid and liquid fat relaxation

Determination of solid fat content is one of the most popular applications of NMR in the food industry. Nevertheless, this international reference method is based on very crude acquisition and data processing requiring the acquisition of a pair of data points (one at 11µs, which corresponds to both the solid and liquid part, and one at 70µs, which corresponds to the liquid part only). Following the work of Van Duynhoven et al. [START_REF] Van Duynhoven | Determination of MG and TG phase composition by time-domain NMR[END_REF], Trezza et al. [START_REF] Trezza | Rapid phasecompositional assessment of lipid-based food products by time domain NMR[END_REF] recently, demonstrated that greater accuracy in the measurement of the solid fat content could be achieved from combined FID and CPMG acquisition and when the signal is analysed with semi-empirical mathematical functions for solid, semi-solid and liquid components. An extension of this method has also been proposed in order to adapt the method to non-anhydrous fat products such as emulsions [START_REF] Duval | Practical implications of the phase-compositional assessment of lipid-based food products by time-domain NMR[END_REF]. Indeed, in emulsions the contribution of non-fat solid-like protons such as non-echangeable protein protons to the solid phase has to be taken into account. Moreover, this method (based on the fitting of both the FID and CPMG signal) which takes into account the relaxation time and the signal intensity can be used to assess the polymorphic composition of lipids. The free induction decay of the α polymorph was described by a monotonously decaying Gaussian function such as eq. 1, while for the β and β' polymorph an 'Abragam sinc' function was added (eq. 2). The results demonstrated that the NMR method was potentially the first technique to provide simultaneously the amount of the solid and liquid phase and the polymorphic state of the lipid. Parallel to the development of this T 2 -based approach, an investigation into the behaviour of the T 1 from the crystallised fat phase was carried out [START_REF] Adam-Berret | Study of triacylglycerol polymorphs by nuclear magnetic resonance: effects of temperature and chain length on relaxation parameters[END_REF]. From an extended investigation of the effects of temperature and triacyglycerol chain lengths on the T 1 and the second moment M 2 , the authors proved the ability of NMR to determine the polymorphism of triacylglycerols regardless of temperature.

For example, a ratio of 1.5 to 2 between the M 2 of the β form and the α form was observed whatever the triacylglycerol, while the T 1 values for the α polymorph were 15-25 times lower than for the β polymorph. The sensitivity of the T 1 relaxation time of the fat crystal organisation has previously been suggested in fat mixtures with added surfactant and in food emulsions. For example, wide variations in T 1 were observed according to the cooling rate of milk fat [START_REF] Ollivon | Crystallisation of anhydrous milk fat: Influence of polymorphism and emulsifiers[END_REF]. A shorter T 1 was observed for a faster cooling rate, and the T 1 was reduced when surfactants were added, compared to the T 1 from the dairy fat without surfactant. In dairy emulsions, a change in T 1 relaxation time of the fat crystal was observed according to the maturation temperature of the emulsion [START_REF] Riaublanc | Impact of fat crystals on the foaming capacity and stability of whipped creams[END_REF]. For a maturation temperature of 4°C a narrow T 1 distribution was obtained, whereas a bi-modal distribution was obtained for a maturation temperature of 12°C. Changes in the T 1 relaxation time of the crystallised phase of fat have been also reported in ice cream mix and ice cream [START_REF] Lucas | NMR assessment of ice cream: Effect of formulation on liquid and solid fat[END_REF]. The aim of the above study was to investigate the effects of the nature of fats, proteins and emulsifiers on the behaviour of the fats in the emulsion. Despite the high complexity of the composition and structure of ice cream and mixes, NMR relaxation times specific to fat protons have been extracted from the NMR signal. The results showed a decrease in the T 1 relaxation from the solid fat after emulsification according to the nature of the fat. Moreover, an effect of the nature of the emulsifiers and the protein on the relaxation time T 1 of the fat crystal was revealed. The authors suggested that the T 1 relaxation was explained by local disorder of the crystal arrangement and by the size of the crystal and was related to the viscosity of the ice cream mixes.

Another improvement in the NMR relaxation method for the characterization of fat crystallisation has been proposed [START_REF] Ablett | Time domain NMR studies under controlled shear conditions[END_REF][START_REF] Mazzanti | In situ Rheo-NMR measurements of solid fat content[END_REF]. Based on the Rheo-NMR method proposed by Callaghan for the measurement of a flow under shear, the authors showed that the NMR technique can be used for the in situ measurement of solid fat content under shear. A specific mini-Couette cell was developed to crystallise fat samples under shear. The cell was tested with blends of canola stearine and canola oil melted at 80°C, and then crystallised at 40°C under different shear rates inside the NMR spectrometer. This system should contribute to understanding of the effects of shear flow on the solid fat content of lipid systems.

Surprisingly, the behaviour of the signal from lipids in the liquid state has had little attention.

In fact, the relaxation time of a mixture of lipids is often characterised by a wide continuous distribution of relaxation times both in T 2 and T 1 [START_REF] Chaland | H1 nuclear magnetic resonance relaxometric characterization of fat and water states in soft and hard cheese[END_REF] and influenced by both the length of the carbon chain, and the amount of unsaturation [START_REF] Botlan | A Novel Approach to the Analysis of fats by low resolution NMR spectroscopy: application to milk and vegetable fats[END_REF] which complicates the interpretation of relaxation times of the lipid phase. Nevertheless, the use of chemometric methods has recently been proposed for the processing of T 2 relaxation decay of intact oilseeds instead of fitting methods [START_REF] Prestes | A rapid and automated low resolution NMR method to analyze oil quality in intact oilseeds[END_REF]. The partial least square method was used directly on the CPMG data in order to predict the oil quality by its composition, cetane number, iodine value and kinematic viscosity. The use of NMR has therefore been proposed for the selection of high quality oils for biodiesel applications.

Lastly, the NMR solid fat method has also been extended to MRI to study lipid migration within chocolate [START_REF] Walter | Lipid migration in two-phase chocolate systems investigated by NMR and DSC[END_REF][START_REF] Choi | Oil migration in a chocolate confectionery system evaluated by magnetic resonance imaging[END_REF][START_REF] Choi | Oil migration in chocolate[END_REF]. In these experiments, only the liquid signal was recorded because of the limitations of echo times when using a spin echo technique. In fact, the T 2 from the solid phase (T 2 around 15 µs) is too short to detect the solid phase. This limitation has recently been overcome by the use of a 1D centric SPRITE MRI method [START_REF] Deka | Spatial mapping of solid and liquid lipid in confectionery products using a 1D centric SPRITE MRI technique[END_REF]. MR images from both the solid lipid and the liquid lipid can be acquired in a chocolate sample with this method. This new strategy will be very powerful to monitor the diffusion process of lipids in confectionery products.

Ice relaxation

The simultaneous acquisition of the solid and liquid-like relaxation decay signal has also been used to investigate the ice phase during freezing. For example, two components were identified in a binary sucrose solution for temperatures above -2°C [START_REF] Lucas | Water, Ice and Sucrose behaviour in frozen sucrose-protein solutions as studied by 1H NMR[END_REF]. The solid component was attributed to the non-exchangeable sucrose protons, and the liquid component was attributed to the water protons, the exchangeable sucrose protons and a fraction of the more mobile non-exchangeable sucrose protons. Below -2°C, which corresponds to the freezing temperature of water, the solid component described both the non-exchangeable sucrose protons and the ice protons. From acquisition of T 1 relaxation times, the authors demonstrated that the relaxation of ice protons can be distinguished from the sucrose protons. Variation in the ice T 1 relaxation time according to temperature exhibited the same behaviour as pure ice, whereas, the same authors demonstrated for the first time in a more complex product such as ice cream, that the ice relaxation time was affected by the protein types added to the formulation. The T 1 relaxation time of the ice was around 3600 ms for ice cream containing milk protein and between 1900 and 2200 ms for ice cream containing whey protein (Nollibel©). The difference was explained by the occurrence of defects in the ice crystal structure. These results open up new opportunities for the use of NMR in the characterisation of the ice in frozen food, which until now has been less fully investigated than the non-frozen water phase.

Biopolymer relaxation

The NMR relaxation behaviour of the solid components has also been investigated for biopolymers. For example, in milk protein mixture containing different amounts of lactose, two relaxation components were identified [START_REF] Dean | 1H Nuclear Magnetic Resonance Relaxometry Study of Water State in Milk Protein Mixtures[END_REF]. The short component was attributed to the non-exchangeable proton for the native protein casein, and the long component to the water proton and the lactose. Bi-exponential behaviour was also reported on T 2 for soy-protein isolate films. The short relaxation component with a solid-like behaviour was more sensitive to variations in water activity and glycerol content. The T 2 variations were related to the plasticizing effect on the biopolymer films [START_REF] Choi | Molecular dynamics of soyprotein isolate films plasticized by water and glycerol[END_REF]. The same solid-like behaviour was also reported in gelatine biopolymer films [START_REF] Kim | New approach for characterization of gelatin biopolymer films using proton behavior determined by low field H-1 NMR Spectrometry[END_REF]. In these matrices two relaxation times were found for the biopolymer, one for the non-echangeable protons and one for the exchangeable protons. However, the most fully studied system is still starch-based products. In the native state, the proton relaxation spectra of starch exhibit a complex multi-exponential behaviour [START_REF] Chatakanonda | The influence of time and conditions of harvest on the functional behaviour of cassava starch -a proton NMR relaxation study[END_REF][START_REF] Tang | The distribution of water in native starch granules -a multinuclear NMR study[END_REF]. In D 2 0, the first relaxation peak at 10µs has been attributed to rigid amylopectin in the semi-crystalline lamellae, the relaxation peaks at 1 ms and 20 ms have been assigned to the mobile fraction of amylopectin in amorphous regions of the granule, and the longest relaxation time peak at 80 ms has been assigned to amylose. A decrease in the relative area of the semi-crystalline and the 1 ms amorphous amylopectin relaxation peaks was observed upon gelatinisation while an increase in the other two peaks was observed. In water, the starch protons exhibit a solid-like behaviour which becomes liquid-like after gelatinisation. NMR relaxation has therefore been extensively used to study the effects on the dynamic state of starch during processing such as the effects of storage temperature, water content and gluten content on starch retrogradation [START_REF] Farhat | The retrogradation of waxy maize starch extrudates: Effects of storage temperature and water content[END_REF], and to investigate the gelatinisation process on rice starch [START_REF] Ritota | Proton NMR relaxation study of swelling and gelatinisation process in rice starch-water samples[END_REF]. The potential of NMR has recently been extended to the study of starch in complex systems containing fat and large amounts of sucrose such as cakes [START_REF] Assifaoui | Characterization of water mobility in biscuit dough using a low-field 1H NMR technique[END_REF][START_REF] Grand | NMR signal analysis to characterize solid, aqueous, and lipid phases in baked cakes[END_REF]. For example, wide variations in the amount of gelatinised starch have been reported between the crust and the centre of madeleines [START_REF] Grand | NMR signal analysis to characterize solid, aqueous, and lipid phases in baked cakes[END_REF]. The water gradient between the crust and the centre observed after cooking decreased during storage and was negligible after 15 days. Despite an increase in water content in the crust, no change in the starch relaxation time was observed for the solid-like component. However, an increase in the contribution of the solid starch component was observed in the centre and was related to the starch retrogradation. These results illustrated that careful analysis of the full NMR relaxation decay curves expands the potential of NMR, even in complex food system.

Water relaxation: a multiscale probe of food structure

The previous paragraphs have focused on improvements in NMR applications in analysis of the solid fraction of food colloids. However, the main NMR applications are based on the investigation of the water relaxation behaviour both from T 2 and T 1 . Several studies have been undertaken to understand the mechanisms involved in the water relaxation rate. The main controversy concerns the relative importance of three mechanisms 1) the nature of the proteinassociated water that contributes to the relaxation, 2) the contribution of the biopolymer protons exchanging with the water, and 3) the effects of the cross-relaxation between spinlattice relaxation associated with water protons and biopolymer protons [START_REF] Venu | Water 1H magnetic relaxation dispersion in protein solutions. A quantitative assessment of internal hydration, proton exchange, and cross relaxation[END_REF]. Using NMR CPMG T 2 dispersion [START_REF] Hills | The effects of proteins on the proton NMR transverse relaxation times of water. I Native bovine serum albumine[END_REF][START_REF] Hills | The effects of proteins on the proton NMR transverse relaxation time of water. II protein aggregation[END_REF][START_REF] Belton | Multinuclear NMR studies of water in solutions of simple carbohydrates. II. Oxygen-17 relaxation[END_REF][START_REF] Hills | Multinuclear NMR studies of water in solutions of simple carbohydrates. I proton and deuterium relaxation[END_REF] and quantitative analysis of the T 1 magnetic relaxation dispersion (MRD) [START_REF] Venu | Water 1H magnetic relaxation dispersion in protein solutions. A quantitative assessment of internal hydration, proton exchange, and cross relaxation[END_REF][START_REF] Denisov | Hydrogen exchange rates in proteins from water H-1 transverse magnetic relaxation[END_REF][START_REF] Halle | Protein hydration dynamics in solution: a critical survey[END_REF][START_REF] Chavez | Molecular basis of water proton relaxation in gels and tissue[END_REF][START_REF] Chavez | Hydrogen exchange and hydration dynamics in gelatin gels[END_REF][START_REF] Chavez | Internal water molecules and magnetic relaxation in agarose gels[END_REF], it is now clear that the water relaxation can be explained by taking into account only two relaxation mechanisms, the protein-exchanging protons relaxation and the relaxation from the internal water molecule. Moreover, analysis of 1 H, 2 H and 17 O MRD has demonstrated that the internal molecules involved in this mechanism are only marginally less mobile than the bulk water and should not be considered as "bound", as they were for many years [START_REF] Halle | Protein hydration dynamics in solution: a critical survey[END_REF], and the number of water molecules is very small. For example, Venu et al [START_REF] Venu | Water 1H magnetic relaxation dispersion in protein solutions. A quantitative assessment of internal hydration, proton exchange, and cross relaxation[END_REF] shown that a considerable difference in the MRD can be observed between a solution of pancreatic trypsin inhibitor and a mutant protein (G36S) lacking only one of the four internal water molecules. The water NMR relaxation in a biopolymer system has consequently to be interpreted as a probe of the biopolymer dynamics rather than a true measurement of water mobility.

Several applications have been proposed on the basis of the high sensitivity of the NMR water relaxation, i.e i) for measurement of water content with MRI, ii) for the investigation of changes in the food structure at a molecular level, iii) for the investigation of changes on a microscopic scale. Some aspects of these applications will be discussed and illustrated with some examples below.

Water relaxation and water content

The dependence of T 2 or T 1 on water content has been applied to determine oil and water content in fried food [START_REF] Hickey | Magnetic resonance relaxation measurements to determine oil and water content in fried foods[END_REF], to quantify the ice gradient in dough during freezing and thawing processes [START_REF] Lucas | MRI quantification of ice gradients in dough during freezing or thawing processes[END_REF], to quantify water distribution during curd draining [START_REF]NMR relaxometry and MRI for food quality control: application to dairy products and processes[END_REF], and to monitor water ingress during rehydration of pasta [START_REF] Hills | Radial NMR microimaging studies of the rehydration of extruded pasta[END_REF][START_REF] Lai | Water status of cooked white salted noodles evaluated by MRI[END_REF] and cereal-based products [START_REF] Ramos-Cabrer | Monitoring of moisture redistribution in multicomponent food systems by use of magnetic resonance imaging[END_REF][START_REF] Weglarz | Real-time mapping of moisture migration in cereal based food systems with A(w) contrast by means of MRI[END_REF], during drying of gelatine gels [START_REF] Chung | Water status of two gelatin gels during storage as determined by magnetic resonance imaging[END_REF], and after cooking of pasta [START_REF] Xing | NMR imaging of continuous and intermittent drying of pasta[END_REF][START_REF] Mccarthy | Change in moisture distribution in lasagna pasta post cooking[END_REF]. An example of moisture loss during drying of gels is given in figures 1 and 2 [START_REF] Ziegler | Moisture migration in starch molding operations as observed by magnetic resonance imaging[END_REF]. The authors generated a simple calibration between solid content (expressed in °Brix) and the T 1 relaxation time (Figure 1) and a T 1 -null MRI sequence, which selectively nullifies the voxel with a specific T 1 value, and a specific solid content was used. In figure 2, the signal from regions with a specified solid content has been removed, clearly showing the concentration gradient across the sample. (data taken from [START_REF] Ziegler | Moisture migration in starch molding operations as observed by magnetic resonance imaging[END_REF]. Reprinted with permission)

Water relaxation and molecular structure

The dependence of relaxation on molecular structure has been extensively investigated in studies of changes in macromolecular food structure resulting from processing operations.

The examples are numerous and have been extensively reviewed [START_REF] Hills | Magnetic resonance imaging in food science[END_REF][START_REF] Ruan | Water in foods and biological materials. A nuclear magnetic resonance approach[END_REF][START_REF]NMR relaxation of dairy products[END_REF]. The water relaxation sensitivity to changes in the molecular dynamics of biopolymers has also been at the origin of MRI investigations into food processing operations. Consequently, if voxel intensity is T 2 value-weighted (T 2 -weighted images) information can be provided by MRI at a molecular level. However, since several processing operations induce concomitant changes in water content and molecular structure, which both affect the MRI images to a certain extent, care should be taken in the design of MRI protocols in order to provide correct interpretations of the grey level intensity of the images. It is nevertheless possible, using the appropriate MRI protocol to obtain spatial distributions on the scale defined by the voxel size (between 100µm up to several millimetres) of structural changes on a molecular scale. This property has been widely exploited for the study of cheese ripening [START_REF]NMR imaging of dairy products[END_REF], potato frying [START_REF] Mariette | Effet du procédé de friture sur la répartition et l'état de l'eau mesurée par RMN et IRM[END_REF], freezing-thawing effects in dairy gels [START_REF]NMR relaxometry and MRI for food quality control: application to dairy products and processes[END_REF], thermal coagulation of egg white [START_REF] Lucas | Thermal coagulation of egg white: an MRI study of the process and a tentative NMR interpretation[END_REF] and egg quality [START_REF] Laghi | A proton NMR relaxation study of hen egg quality[END_REF][START_REF] Schwagele | Low resolution proton nuclear magnetic resonance spectroscopy -Application to determine the internal quality of intact eggs -physicochemical aspects[END_REF] and gelation of carbohydrate gels [START_REF] Potter | The gelation of sodium alginate with calcium ions studied by magnetic resonance imaging (MRI)[END_REF][START_REF] Duce | Use of NMR imaging to map spatial distribution of structure in polysaccharide gels[END_REF], and during the evolution of the bread matrix during storage [START_REF] Lodi | Characterization of water distribution in bread during storage using magnetic resonance imaging[END_REF].

For such MRI applications, the results should be validated with specific NMR studies which provide clear descriptions of the variations in relaxation time parameters in relation to processing parameters [START_REF] Lucas | Thermal coagulation of egg white: an MRI study of the process and a tentative NMR interpretation[END_REF]. For example, figure 3 describes the T 2 water relaxation time and the soluble protein concentrations in white egg according to temperature. Three phases can be distinguished to describe the T 2 variations. From 10 to 50°C, the increase in T 2 is related to temperature increase through an increase in the rotational mobility of both water molecules and protein molecules. From 50°C to 80°C the T 2 decreases. The decrease is explained by denaturation of the protein upon heating. Denaturation induces a decrease in rotational mobility probed by the T 2 relaxation. For this temperature range the T 2 evolution corresponds to changes in ovalbumin conformation, followed by its denaturation. Above 80°C, only temperature affects the T 2 values. A complete interpretation of the MRI T 2 cartography gradient observed during continuous heating of egg could therefore be proposed according to relaxation time behaviour upon changes in temperature (Figure 4). In this study the heating process was performed in one direction (from the top to the bottom), and the other walls of the container were insulated. At the beginning, uniform initial temperature (17°C) and T 2 values were observed. Eighteen minutes later, three areas could be distinguished, the first with T 2 from 450 to 550 ms which corresponded to an increase in T 2 because of the temperature. In this area the egg white was still liquid. The second area (from 550 to 250 ms) corresponded to the area where the egg white started to coagulate, and the third (with T 2 between 200 and 60ms) was attributed to the cooked egg white with a temperature above 80°C. It should be pointed out that for a quantitative description of thermal processes, separate temperature mapping has to be performed in order to correct the T 2 value. This can be also achieved using MRI [START_REF] Nott | Validation and cross-comparison of MRI temperature mapping against fibre optic thermometry for microwave heating of foods[END_REF]

Water relaxation and microscopic structure

The relaxation mechanism described above is only valid when the fast diffusive exchange limit is verified. In that case, because the exchange through thermal diffusion is fast, then the different water pool, the bulk and the hydration or internal water pool and the labile biopolymer are in exchange and the relaxation is the weight average of the three.

Nevertheless, the T 2 from water can also be complicated by the effect of translational mobility characterised by the diffusion coefficient. If we consider two different environments (1 and 2) with a size a, where the NMR characteristics of water T 21 and T 22 are different, if the diffusion coefficient rate (D (m 2 s -1 )) is fast compared to the difference in relaxation rate ∆R 2 (with R 2 = 1/T 2 ), water relaxation will be average and a single mono-exponential signal will be measured. The NMR signal is given by : ( )

        - = obs T t A t Y 2 exp eq. 4

P T obs + =

On the other hand, if the diffusion coefficient is slow compared to the relaxation rate, then the water molecule will not be exchanged and the NMR signals will be characterised by several components, each with its own relaxation time. The NMR signal then becomes:

( )         - +         - = 22 2 21 1 exp exp T t A T t A t Y eq. 5
In this case each signal will be an NMR signature of the two local environments. The limit between the fast and slow diffusion regimes is given by 1

2 2 << D R a ∆ .
Multi-exponential behaviour has been reported in gels and emulsions when non-uniform water distribution was observed, and therefore water relaxation behaviour has been used as a probe of the microstructure or used to quantify the water holding capacity [START_REF] Haiduc | Multivariate modelling of the microstructural quality of food emulsions based on NMR[END_REF][START_REF] Hinrichs | Characterisation of the water-holding capacity of fresh cheese samples by means of low resolution nuclear magnetic resonance[END_REF][START_REF] Hinrichs | Water-holding capacity and structure of hydrocolloidgels, WPC-gels and yogurts characterised by means of NMR[END_REF]. For example, in dairy products, changes in water distribution were observed during syneresis of a dairy gel [START_REF]NMR relaxometry and MRI for food quality control: application to dairy products and processes[END_REF]. A bi-exponential decay relaxation curve was observed and each component was attributed to the water expelled from the gel and to the water confined in the shrunk gel, respectively. Three water relaxation components have been identified in Mozzarella cheese, one for serum water, the water accumulated in the large open channels of the protein network, one for the water inside meshes of the casein gel-like network, and one for water trapped within the casein matrix [START_REF] Gianferri | Proton NMR transverse relaxation measurements to study water dynamic states and age-related changes in Mozzarella di Bufala Campana cheese[END_REF]. A decrease in the T 2 from the serum was observed during aging, attributed to a change in the microstructure of the cheese, induced by the rearrangement of the protein network. In an imitation cheese matrix, the water relaxation was monitored at each stage of manufacture and the changes in water relaxation were described in a four-stage model of protein hydration/matrix development [START_REF] Noronha | Investigation of imitation cheese matrix development using light microscopy and NMR relaxometry[END_REF]. In rennet-derived retentates, the effects of the composition of the fat globule surface on water holding capacity have been studied [START_REF] Metais | Influence of fat globule membrane composition on water holding capacity and water mobility in casein rennet gel: A nuclear magnetic resonance self-diffusion and relaxation study[END_REF]. Reconstituted fatty retentates were prepared from a fat-free retentate mixed with different fat-in-water emulsions stabilized with native phosphocaseinates (NPCs) or sodium caseinates. Coagulation of retentate reconstituted with native fat globules (fresh cream) and industrial fatty retentate was also investigated. The results showed that fatty products reconstituted from fresh cream and the industrial retentate presented lower water holding capacity than that obtained with native phosphocaseinates (NPCs) or sodium caseinate emulsions.

A multiexponential relaxation behaviour for water has consistently been reported in native starch-water systems because of slow diffusive exchange [START_REF] Chatakanonda | The influence of time and conditions of harvest on the functional behaviour of cassava starch -a proton NMR relaxation study[END_REF][START_REF] Tang | The distribution of water in native starch granules -a multinuclear NMR study[END_REF][START_REF] Ritota | Proton NMR relaxation study of swelling and gelatinisation process in rice starch-water samples[END_REF][START_REF] Tang | A proton NMR relaxation study of the gelatinisation and acid hydrolysis of native potato starch[END_REF][START_REF] Chatakanonda | Mobility and distribution of water in cassava and potato starches by H-1 and H-2 NMR[END_REF][START_REF] Choi | Water mobility and textural properties of native and hydroxypropylated wheat starch gels[END_REF][START_REF] Thygesen | The effects of amylose and starch phosphate on starch gel retrogradation studied by low-field H-1 NMR relaxometry[END_REF][START_REF] Doona | Molecular mobility in model dough systems studied by timedomain nuclear magnetic resonance spectroscopy[END_REF]. Four water populations have been identified in native starch, assigned to "channel water", only observed in the B-type amylopectin crystal with a T 2 around 200µs, to water in the amorphous growth rings with a T 2 around 1 ms, to water in the semi-crystalline lamellae with a T 2 around 8ms, and to the extra-granular water with a T 2 longer than 30 ms [START_REF] Tang | The distribution of water in native starch granules -a multinuclear NMR study[END_REF]. Raising the temperature to 27°C causes merging of the two middle water relaxation components because of the fast diffusion of water between the amorphous growth rings and semi-crystalline lamellae. At higher temperatures, the relative intensity of this water population increases and the relaxation time decreases. This effect is explained by the swelling of the granules. Above the melting transition, the relative intensity of this component decreases considerably because of diffusion of the amylose out of the granule. Consequently, the effects of diffusive exchange between microscopic water compartments on NMR water relaxation provide a way to investigate changes during the processing of starch-based products such as non-enzymatic browning in dehrydrated potatoes [START_REF] Acevedo | Non-enzymatic browning kinetics analysed through water-solids interactions and water mobility in dehydrated potato[END_REF], the effects of gluten on recrystallisation kinetics [START_REF] Wang | Effect of gluten content on recrystallisation kinetics and water mobility in wheat starch gels[END_REF], the effects of gluten on gelatinisation in white bread [START_REF] Wang | Water dynamics in white bread and starch gels as affected by water and gluten content[END_REF], the effects of storage on cooked rice grain [START_REF] Yano | Pulse NMR study of refrigerated cooked rice and comparison with physicochemical properties[END_REF] and the effects of water content and amylopectin crystallinity pattern on polymorphic gelatinisation [START_REF] Tananuwong | DSC and NMR relaxation studies of starch-water interactions during gelatinization[END_REF], and during rehydration of breakfast cereals [START_REF] Lucas | Kinetics of water absorption and solute leaching during soaking of breakfast cereals[END_REF].

The multiexponential analysis of MRI liquid relaxation has not to our knowledge been carried out on food colloids. However, examples have been proposed for in vivo medical applications showing that bi-exponential fitting of the T 2 decay map improved the ability of MRI for quantitative measurements. This approach would be valuable for the characterisation of food products since most of them exhibit multiexponential relaxation behaviour.

Multidimensional NMR relaxation

The NMR applications described above are all based on the analysis of 1-dimensional conventional relaxation spectra. Although T 1 -filtred T 2 or T 2 -filtred acquisition has been performed for some specific applications, the data have always been processed with a 1-dimensional Laplace inversion [START_REF] Mariette | NMR signal analysis for the attribution of the components to the solid/liquid phases in presence in mixes and ice creams[END_REF][START_REF] Witek | Investigation of starch hydration by 2D time domain NMR[END_REF]. The advent of a fast algorithm for 2-dimensional Laplace inversion [START_REF] Song | T-1-T-2 correlation spectra obtained using a fast two-dimensional Laplace inversion[END_REF] has recently led to the development of "multidimensional" NMR relaxometry. These types of measurement are valuable to improve the assignment of multiexponential relaxation behaviour, and to study dynamic processes such as chemical exchange or diffusive exchange. For example, T 2 -T 1 correlation and D-T 2 correlation experiments were performed on dairy products such as milk, yoghurt, cream and cheese [START_REF] Hurlimann | Quantitative characterization of food products by two-dimensional D-T-2 and T-1-T-2 distribution functions in a static gradient[END_REF]. For milk samples, a single peak was observed for both experiments and in that case the 2-D relaxation approach did not provide more information than a 1-D relaxation experiment. However, for the other samples a bi-modal distribution was observed for both experiments. The first peak was assigned to the water fraction and the second peak to the fat fraction. In T 1 -T 2 correlation experiments the separation between the two relaxation components was still difficult and affected by the water content of the sample. Indeed, the results showed that the fat relaxation did not change with the chemical composition of the dairy sample, while the water relaxation associated with the water phase varied from sample to sample. Consequently, for a few samples an equal relaxation time value was observed for the two fractions thus preventing any separation of the two compounds. In contrast, the D-T 2 distribution functions improved separation of the water and fat features and, because of the large difference between the water diffusion and fat diffusion coefficients, this separation was independent of the chemical composition. This approach has been proposed for robust water and fat determination in dairy products. T 1 -T 2 correlation spectroscopy has also been used to monitor the effects of high pressure and microwave processing on the microscopic water distribution and chain dynamics in starch-water systems and potato tissue [START_REF] Hills | T-1-T-2 NMR correlation studies of highpressure-processed starch and potato tissue[END_REF]. This approach showed that high pressureinduced gels were radically different from the corresponding microwave heating-induced gels.

For example, the T 2 -T 1 correlation spectra for waxy maize starch showed only a shift for the two water peaks and for the mobile starch non-exchangeable proton after high pressure treatment. Whereas after microwave heating, the three peaks were similar to those of the untreated starch-water system, but in addition three new peaks emerged. Such methods present many opportunities for sample characterisation, but it is necessary to step back from the complexities of real food systems in order to assign the peaks to particular proton pools.

As demonstrated by Marigheto et al [START_REF] Marigheto | Methods for peak assignment in low-resolution multidimensional NMR cross-correlation relaxometry[END_REF], even in simple sucrose water solutions where complex T 1 -T 2 spectra were observed, the assignment of the four peaks remained uncertain.

The authors suggested that T 1 -T 2 spectra can be used empirically as a "finger-print" for monitoring the complex changes associated with phase transformations.

We explained above how translational mobility can modify the relaxation decay curve from mono-to multi-exponential behaviour. Nevertheless, quantification of the diffusion coefficient from relaxation time experiments is still a challenge since the physical models used require several assumptions which often cannot be verified [START_REF] Price | Pulsed field gradient nuclear magnetic resonance as a tool for studying translational diffusion: Part 1. Basic Theory[END_REF]. Consequently, other NMR and MRI techniques based on the use of an external magnetic field gradient are preferred because they do not require knowledge of the mechanism involved in the relaxation behaviour. These techniques are all based on the use of well-defined linear gradient pulses which change the strength of the magnetic field probed by the molecule's protons locally.

Consequently, if a molecule diffuses spatially in this magnetic field gradient, the NMR signal is reduced: the faster the diffusion rate the higher the NMR signal reduction. A detailed description of this method, called pulsed field gradient NMR (PFG-NMR), q-NMR for NMR, and diffusion weighted MRI or diffusion tensor imaging (DTI) when using an MRI scanner, is provided by Price [START_REF] Price | Pulsed field gradient nuclear magnetic resonance as a tool for studying translational diffusion: Part 1. Basic Theory[END_REF][START_REF] Price | Pulsed-field gradient nuclear magnetic resonance as a tool for studying translational diffusion : Part II. Experimental aspects[END_REF]. The great advantage of MR diffusion spectroscopy and imaging is that quantitative measurement of the diffusion of water and metabolites can be performed non-invasively on a microscopic scale and in any direction of displacement. PFG-NMR is widely recognized as a powerful method to obtain microstructural information in emulsions [START_REF] Johns | Characterisation of emulsion systems using NMR and MRI[END_REF][START_REF] Colafemmina | Restricted diffusion: An effective tool to investigate food emulsions[END_REF]} and porous materials [START_REF] Topgaard | Probing biological tissue microstructure with magnetic resonance diffusion techniques[END_REF]. A routine PFG-NMR method has been proposed for the measurement of droplet size distribution in water/oil or oil/water emulsions [START_REF] Balinov | Determination of water droplet size in margarines and low-calorie spreads by nuclear magnetic resonance self-diffusion[END_REF][START_REF] Mooren | Determination of droplet size distributions in emulsions by pulsed field gradient NMR[END_REF][START_REF] Goudappel | Measurement of oil droplet size distributions in food Oil/Water emulsions by time domain Pulsed Field Gradient NMR[END_REF]. The accuracy of the method has been widely debated for water droplet and oil droplet size determination in food emulsions such as butter, margarine and dressings [START_REF] Van Duynhoven | Scope of droplet size measurements in food emulsions by pulsed field gradient NMR at low field[END_REF][START_REF] Van Dalen | Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy[END_REF][START_REF] Van Lent | Determination of water droplet size distribution in butter: Pulsed field gradient NMR in comparison with confocal scanning laser microscopy[END_REF][START_REF] Denkova | Evaluation of the precision of drop-size determination in oil/water emulsions by lowresolution NMR spectroscopy[END_REF]. Some extension of the method has recently been proposed for spatial determination of droplet size distribution from MRI data in emulsions [START_REF] Avila | Magnetic resonance imaging (MRI): A technique to study flow an microstructure of concentrated emulsions[END_REF] and to provide both real-time dispersion and spatially-resolved velocity measurements in a frozen drop of sucrose solution and emulsion [START_REF] Hindmarsh | Rapid measurement of dispersion and velocity in freezing drops using magnetic resonance methods[END_REF]. However, the latter method requires the use of high field NMR spectrometers.

The PFG-NMR technique has also been used to study water mobility in different protein and polysaccharide systems. The water dependence on the protein concentration has been reported in casein and whey protein solution and gels [START_REF] Colsenet | NMR relaxation and water self-diffusion studies in whey protein solutions and gels[END_REF][START_REF] Mariette | H-1 NMR diffusometry study of water in casein dispersions and gels[END_REF]. In both systems the water diffusion was reduced with increased protein concentrations in solutions and gels. The obstruction effect of the protein was explained by taking into account two self-diffusion flows: a water flow close to the protein backbone and a water flow around the casein micelle or globular whey proteins.

The effect of the change in network structure on the water diffusion coefficient was also investigated for casein gels. Independently of the method of preparing the gel, i.e acid or renneted gel, no difference in water diffusion was observed. However, heat treatment of the whey protein solution induced a slight reduction in the water diffusion. This was explained by a change in the accessibility of the whey protein aggregates to water. For casein, it was recognised that the overall accessibility to water was not significantly affected by the formation of a gel. The author concluded that water diffusion in protein systems follows a general trend whatever the protein system studied. The specific effects of casein and fat content on water diffusion have been outlined in a cheese model, and a general model describing the effects of water content on water self-diffusion has been proposed [START_REF] Metais | Influence of fat globule membrane composition on water holding capacity and water mobility in casein rennet gel: A nuclear magnetic resonance self-diffusion and relaxation study[END_REF][START_REF] Métais | Effects of casein and fat content on water self-diffusion coefficients in casein systems: a pulsed field gradient nuclear magnetic resonance study[END_REF]. This model includes the effects of aqueous phase composition, and the obstruction effects of casein and fat droplets on water diffusion. This model requires no structural information on the gel network since no effect of the structure has been observed, except when the water distribution inside the gel becomes heterogeneous and when the serum phase starts to release.

A reduction in the water diffusion was observed in soya protein suspensions, according to the protein concentration. Moreover, the range of reductions observed was the same as that previously reported for whey protein suspension, which confirmed a general tendency to a obstruction effect induced by protein. After addition of calcium, the reduction in water selfdiffusion measured in the precipitated of soy protein isolate was greater. The author suggested that in a suspension only the hydration and hydrodynamic interactions explained the change in the self-diffusion, while in the precipitate the obstruction effect of the protein particle has to be considered and included. Nevertheless, the author did not mention whether the water content was still the same in the precipitate and in the suspension. A slight difference in water content could explain difference in the water self-diffusion between the two systems.

In contrast to protein systems where slight effects of the protein structure on the water selfdiffusion have been recorded, wide variations in water self-diffusion have been reported in starch suspensions according to the granule structure [START_REF] Hills | Microstructural characterization of starch systems by NMR relaxation and Q-SPACE microscopy[END_REF]. Anomalous water diffusion behaviour was found in the case of native starch. When the internal granule structure was destroyed by gelatinisation, the water diffusion conformed to simple unrestricted diffusion in 3D space. Moreover, a similar linear variation in the water self-diffusion related to the water content was observed for a fully heated wheat starch/water system, a fully heated wheat flour/water system and a fully heated rice starch/water system [START_REF] Fukuoka | MRI observation and mathematical model simulation of water migration in wheat flour dough during boiling[END_REF]. This suggests that the difference in microstructure after gelatinisation has little effect on the water diffusion. The same tendency was recently reported during retrogradation of wheat starch gel [START_REF] Doona | Water dynamics and retrogradation of ultrahigh pressurized wheat starch[END_REF]. The water self-diffusion of heat-gelatinised starch and ultrahigh pressured gelatinised starch stayed constant during storage despite retrogradation. Moreover, the change in water selfdiffusion during storage of white bread crumb is mainly explained by moisture loss [START_REF] Baik | Water self-diffusion coefficient and staling of white bread as affected by glycerol[END_REF].

The future

Several studies are still in progress to improve the applications of NMR and MRI techniques to food colloids. Some of these, such as the use of multi-dimensional NMR relaxation are directly related to ways of manipulating the spin magnetisation , some involve the design of new magnet geometry such as the NMR-Mouse [START_REF] Haiduc | Noninvasive 'through-package' assessment of the microstructural quality of a model food emulsion by the NMR MOUSE[END_REF][START_REF] Guthausen | Measurement of fat content of food with single-sided NMR[END_REF], or open access systems such as the Halbach magnet [START_REF] Hills | A low-field, low-cost Halbach magnet array for open-access NMR[END_REF], others involve the development of techniques to measure non-NMR parameters such as flow and rheological properties, referred to as velocimetry MRI or Rheo-NMR [START_REF] Hollingsworth | Rheo-nuclear magnetic resonance of emulsion systems[END_REF][START_REF] Callaghan | Rheo-NMR and velocity imaging[END_REF], and others have proposed multi-sensor technology when NMR is associated simultaneously with other spectroscopy techniques such as NIR, impedance and ultrasound or scattering techniques [START_REF] Hills | A low-field, low-cost Halbach magnet array for open-access NMR[END_REF]. Despite the development of MRI applications for food science, extrapolations to medical applications have been few, mainly because of the specificity of the different domains. However, some studies have demonstrated that in the future we will see greater levels of connection between food science and medical science and pharmaceutical applications. For example, a connection between the rehydration/dehydration processes for a food gel can be made with the monitoring of the controlled release and hydrogel volume erosion of an implant in vivo [START_REF] Weissleder | Quantitation of slow drugrelease from an implantable and degradable gentamicin conjugate by in-vivo magneticresonance[END_REF]. Other applications involve characterisation of food in the gastro-intestinal tract in humans and animals. Marciani et al. have used MRI to investigate the behaviour of lipid emulsions in the human stomach [START_REF] Marciani | Intragastric oil-in-water emulsion fat fraction measured using inversion recovery echo-planar magnetic resonance imaging[END_REF][START_REF] Marciani | Magnetic resonance imaging of the behaviour of oil-in-water emulsions in the gastric lumen of man[END_REF]. Moreover, in order to improve the understanding of the relationship between meal viscosity and satiety, the T2 relaxation rate has been used to observe in vivo gelling of biopolymers in the stomach over time [START_REF] Hoad | In vivo imaging of intragastric gelation and its effect on satiety in humans[END_REF]. These recent studies demonstrate that promising new results can be expected from food MRI applications in the near future.

Figure 1

 1 Figure 1 Calibration curve for °Brix and 1/T 1 (data taken from [47]. Reprinted with permission).

Figure 2 MRI

 2 Figure 2 MRI transverse slice acquired with a T 1 -weighted sequence of a gel during drying. The parameters of the MRI sequence were chosen in such a way that a selective nulling of the signal within the gel was observed. The dark regions therefore correspond to systems having the solid content indicated.

Figure 3 NMR T 2

 32 Figure 3 NMR T 2 relaxation and soluble protein concentration in egg white as according to temperature (data taken from [53]. Reprinted with permission)

Figure 4

 4 Figure 4 Transverse T 2 map of egg white during heating. All the surfaces of the beaker were insulated, and the heat exchanger was place on top of the beaker in order to limit convection flow. (data taken from [53]. Reprinted with permission)


Colin Burton

José Martinez
email: jose.martinez@cemagref.fr-colin.burton@cemagref.fr

Contrasting the management of livestock manures in Europe with that practised in Asia: what lessons can be learnt?

Keywords: European agriculture, Asian agriculture, pigs, poultry, beef, fish, environmental impact, water and air pollution, abatement measures, food safety, hygiene, legislation. 1

Similarities exist in livestock farming in Europe and

Asia. There is close coupling of intensive agriculture with industrialisation and environmental impacts caused by the production of very large amounts of organic wastes in limited areas. There are also differences deriving from, geographical, climatic and cultural factors that have led to some special problems in each region. In Europe, there is still a lack of appreciation of the full agronomic value of the manures produced although most are spread in an organised way. In Asia, the solid dung is more likely to be valued and sold as a product for fish farms and vegetable or fruit production. However, land for spreading is limited and less accessible and river pollution is commonplace. In both regions, effective treatment options are available but in the absence of financial support, they are rarely popular unless a financial return is implied such as in biogas schemes.

Introduction

European livestock agriculture moved to intensive systems in the mid-twentieth century with most animal production coming from modern units by the 1960's. Industrialisation has occurred more recently in Asia and in a more patchy way with the development of major centres focussed on the principle cities in the region such as Ho Chi Minh (Vietnam) and Guangzhou (China). With the relative increase in the urban standard of living, the preferred diet moved from one of principally rice and vegetables to include more meat product. Asian countries now account for one third of cattle production and more than half of pig production [START_REF] Windhoarst | Regional patterns of livestock and poultry production in Europe[END_REF]. The period of growth in the livestock industry in Asia is far from levelling off in the way observed in Europe in recent years. One might note that, in the early nineties, Europeans consumed an average of 78 kg of meat and 178 kg of milk products per person per year. By contrast, developing Asian countries (including China), were then consuming only 18 kg and 12 kg of such products respectively. [START_REF] Ehui | The role of livestock in food security and environmental protection[END_REF]. Animal production in Asia is set to continue its expansion for the foreseeable future [START_REF] Gerber | Nitrogen losses from intensive livestock farming systems in Southeast Asia: A review of current trends and mitigation options[END_REF] with meat production from Asian countries greatly exceeding that in Europe over the coming decades. The negative impact on the environment from the growing livestock industry in Asia is evident from the visual contamination of land and surface waters. The wider impact is apparent from studies on the inevitable development of nutrient surpluses in areas of high production with the expected longer term pollution consequents [START_REF] Gerber | Geographical determinants and environmental implications of livestock production intensification in Asia[END_REF]. When comparing European agriculture to that emerging in Asia, the impression might be that Europe has largely passed through the negative phase and that it can now be used as a model for livestock agriculture in Asia and other developing areas of the world. There is certainly some scope to transfer some of the successful techniques where there is common ground. However, there are also geographical differences that require a very different approach in Asia. And in some instances, noting that balancing agriculture and the environment in Europe certainly remains to be achieved, there may yet be lessons to be learnt.

2.

Current manure handling practices in Europe Most livestock manure generated within Europe is applied direct to cropland near the farm. In principle, this can represent the simplest solution with the fewest problems so long as:

• There is enough local land to accept the applied nutrient load,

• Timely applications can be made as required by the crop; • The land is not susceptible to run-off (sloping ground) • The fields are not near residential housing • There is little risk of crop contamination or animal health problems.

In reality, this is often the situation with cattle farms where local pasture is plentiful but for pig and poultry units, land is often lacking and treatment methods are implied to deal with the surplus nutrient. However, even if there is enough land and the system appears to be in balance, problems can still arise if manure is then applied in a hasty or ill-considered way. Thus in many parts of Europe, manure management plans are encouraged to ensure a systematic spreading regime. This includes the identification of suitable fields and matching applications to the crop requirement (and reducing the use of chemical fertiliser accordingly). For liquid manure, field access problems may be overcome by pipeline transfer and irrigation systems (Figure 1). This may require prior separation of the collected slurry; efficient mixing systems are also implied. Minimum store capacity is often specified to ensure that spreading can be avoided during adverse weather conditions (eg: frozen or water-logged ground). Improvements are also possible with application equipment design including injection to reduce emissions of ammonia and odour. There has also been some development of intelligent application systems using GPS positioning to control applications automatically depending on field and season but this remains at a research stage [START_REF] Carton | Sustainable Waste Application Management Project (SWAMP). Optimal use of animal slurries for input reduction and protection of the environment in sustainable agricultural systems[END_REF]1998) . In situations where the local field capacity is exceeded, surplus nutrients must be removed if they are not to ultimately contribute to an undesirable environmental impact. Road haulage (or for shorter distances, pipeline transfer) as illustrated in Figure 1, has been used in parts of Europe, especially the Netherlands as a direct method to re-distribute manure surpluses. However, the environmental impact of the extra transport must be included in any assessment. Pipeline transport is easier once the initial investment has been made but pre-treatment to remove some suspended matter is necessary and the engineering problems increase with distance.

The destruction of components within the manure is specifically the nitrogen load (by aerobic/anoxic treatment) or organic load (by aerobic or anaerobic treatment). Nitrogen removal is via a process of nitrification (ammonia converted to nitrites and/or nitrates) followed by denitrification (nitrites and nitrates broken down to di-nitrogen gas). The technique is used in the Brittany region of France to deal with nitrogen surpluses (Beline et al, 2004). Any biological process can be expected to breakdown organic matter; for aeration, this will be to carbon dioxide and water; for anaerobic processes, this will be to acetic acid then methane. Components of manure that can not be eliminated (such as phosphorus or heavy metals) can only be removed by separation, concentration and exportation [START_REF] Daumer | The effect of phytase in pig diet and solid/liquid separation of pig slurry on phosphorous, calcium and magnesium fractionation[END_REF]. Indeed, this may be desired for all excess nutrients including nitrogen and organic matter when there is a recognised value. Separation can be by screening machines but these have a limited effect on finer suspended particles. Alternatively, there is sedimentation which is often by gravity using large shallow vessels; this typically results in sludges with concentrations between 5 and 10% dry matter [START_REF] Martinez | A study of the potential contribution of sedimentation to aerobic treatment processes for pig slurry[END_REF]. The process can be accelerated and extended by the use of a decanter centrifuge which can take the concentration of the sludge to over 30%. Solid products whether sludges, screened materials or solid litter, can be blended with wheat straw and composted to produce a potentially useful organic product sometimes saleable.

3.

Livestock manure management in Asia In Asia, the largest numbers of animals are reared close to the major population centres. This has resulted in the development of an expanding livestock industry that has become quickly swallowed up in the growing suburbs of the metropolis. However, such farms are now declining in number as government policy endeavours to relocate them away from the city to more rural regions. In time the new and larger units will benefit from the economy of scale and probably account for most of the production. Manure management differs from that in Europe in:

• The available farmland in the peri-urban environment is very limited • Manure is rarely land applied because of fears of crop contamination.

• The frequent coupling of fish and pig production • A greater value is given to biogas -often used for cooking • A greater value is given to solid dung • The importance of keeping the animals cool in a very hot and humid climate • The relative poverty of the rural region • Access and transport difficulties The main manure handling schemes are set out in Figure 2. The solid dung is often collected manually collected, dried, bagged up and sold as fertiliser for both crop and fish farms. As such the related nutrients are exported but the proportion collected varies -never a pleasant task, and even more difficult in the wet season of the monsoon. In some farms, the provision in the pig pen of shallow baths to keep the pigs cool (wallows) ensure that little solid dung can actually be collected. Thus most nutrients are still likely to remain in the liquid effluents washed from the buildings. Biogas is a popular option both with smaller farms (where it is used in local houses) and in the larger farms schemes for electricity production are encouraged. In some regions, there is the close coupling of pig production with fish ponds, the manure from the former nourishing the environment of the latter. Although solid manure products are valued in Asia, the liquid effluent is generally seen only as a nuisance and land spreading is very rare. Rice represents the main crop but both the production cycle (involving periodic land flooding) and the vulnerability of the crop to poor yields if over dosed with nitrogen make liquid manure applications very unpopular.

The situation in Thailand is typical of an economy advanced in development. Livestock farms fall into two main groups: small family units in the peri-urban areas and no land and very large modern farms (5000+ pigs) with the resources to deal more effectively with manures produced. Pig production is moving towards the latter but the pollution impact from the smaller farm is possibly more serious as inadvertent discharge to streams (direct and indirect) is commonplace. The option for biogas is limited as increasingly available electricity is preferred the odorous biogas and releases of methane may result. The exception is where the gas can be used to power fans for cooling. The reduction in pollution by lagooning systems is an option for the larger farms with available local land: the evaporation from the high temperatures leads to relatively little liquid discharge in this apparently closed system. This will enable the reduction in organic load and P (mostly accumulating in the forming sludge layer) but N losses are likely to be mostly as ammonia emissions. Vietnam represents an example of an Asian economy at an earlier stage of industrialisation: the situation is different between the north and the south. In the north, where food production is focussed on the principle metropolis of Hanoi, pig production comes from numerous farms clustered in villages and small towns; many have fewer than a dozen or so animals. Once again the solid dung is valued with quantities being collected and used to sustain adjoining fish ponds or for local vegetable production. Much of this activity takes place in the delta area of the Red River which dominates the geography of the region. Any available arable land around the edge of the village is thus given over to rice production: there is little or no structured land spreading of manures. In the relatively poor rural area the value of biogas is valued; small digesters are commonplace.

4.

Environmental impacts from intensive livestock production 4.1

Water pollution Water pollution from animal production is caused by the release of effluent through or running over soil. Nitrogen and phosphorus can be taken up by the growing crop but excesses will be released to the aquatic system; limited amounts can be immobilised in soil organic matter but only up to the system capacity. The impact of free ammonia (rather than the ammonium ion) presents the greater impact on surface water as it is toxic to many fish even at very low concentrations. In pig and cattle slurries, the proportion of N as ammonia can exceed 60 % of the total. This can be quickly nitrified after land spreading, leading to an excess of nitrates in the soil. Organic-N is not immediately available to plants but it is estimated that around half will be converted to ammonium during the first 12 months after spreading. [START_REF] Choudhary | Review of the use of swine manure in crop production : effects on yield and compositiona and on soil and water quality[END_REF]. The phosphorus content of manure is of concern because there is a direct relationship between the amount of extractable-P in the soil and the concentration of dissolved P in the surface run-off that can produce eutrophication of surface waters that are contaminated [START_REF] Withers | Phosphorus fertilizers[END_REF]. The P in manure is present in both organic and inorganic forms, but the organic fraction can be rapidly solubilised, so the mobility of P in animal manures can approach 100 %. In manures and slurries potassium is present as soluble salts, and it comes almost totally from the urine of the animals. About 90 % of K in feed is excreted by the animals. Usually, the concentration is greater in cattle slurry than for pig slurry; K availability to plants often close to 100 % [START_REF] Bernal | Nutrient balances in calcareous soils after application of different rates of pig slurry[END_REF].

Air pollution

Animal production is a major contributor to certain sources of atmospheric pollution [START_REF] Pain | Gaseous pollutants from organic waste use in agriculture[END_REF]. The ventilation air from livestock housing will contain odorous substances, methane, nitrous oxide (N 2 O) and especially ammonia all of which can detrimentally effect the environment. In Europe, CH 4 emissions originate from manure management but mostly (two thirds) from enteric fermentation of ruminants. Ammonia present in slurries largely arise from the degradation of urea. Emissions also follow field spreading and include, in addition, quantities of nitrous oxide produced from the breakdown of nitrates themselves resulting from the nitrification of ammonia in the soil.

The threat to the environment from global warming is mostly attributed to the huge amounts of carbon dioxide released into the atmosphere from anthropogenic activity. However the relatively small amounts of methane and nitrous oxide, have a disproportionate effect owing to their higher warming potentials of x25 and x300 respectively (IPCC, 1995): they now contribute around 25 and 5% of the total effect. Concentration of all three greenhouse gases have increased by 30, 145 and 15 % for carbon dioxide, methane and nitrous oxide respectively over the last 250 years [START_REF] Moisier | Soil processes and global change[END_REF] although it should also be noted that the growth rate is increasing. For its part, agriculture contributes 25, 65 and 90 % respectively of the net emission of these gases [START_REF] Duxbury | The significance of agricultural sources of greenhouse gases[END_REF]. The figure for carbon dioxide is greatly increased as a result of forest clearing but emissions of methane and nitrous oxide relate closely to agricultural production. Animal wastes contribute 10-20 % of the methane produced from agriculture. Nitrous oxide is almost entirely the result of di-nitrification of nitrates in the soil, the result of surplus applications of nitrogen whether as inorganic or organic forms. Nitrous oxide is also involved in the photochemical reactions in the stratosphere that are related to the fall in concentrations of ozone.

Disease risks and health issues

In Europe, public concern related to food hygiene have been raised in recent years due to cases of contamination of certain agricultural food products (including salmonella, e -coli, campylobacter and listeria). In addition there have been outbreaks of animal disease including foot and mouth, classical swine fever and more recently, avian influenza. Incidences of contamination of water by zoonoses are relatively few, but they often represent a very serious event with human fatalities in some cases [START_REF] Guan | Hog manure management, the environment and human health[END_REF]. In response to such incidents, there have been some additional restrictions on where and how manures may be used but so far, not mandatory treatment. Suitable treatment options do exist including aeration, anaerobic treatment, use of disinfectant chemicals and prolonged isolated storage. Thermal treatments present a more rigorous and reliable approach [START_REF] Turner | The inactivation of viruses in pig slurries: a review[END_REF] but the use of such technology is limited to specific areas where high risks occur. In response to the concerns on the quality of the farm produce, increasing limitations are applied on the use of manure on crops; in some of the most vulnerable crops (eg: leaf crops eaten raw), applications of manure, even treated, is discouraged. Such a trend if extended may ultimately make land application of manures as part of a farm cycle increasingly difficult.

The role of legislation in manure management

5.1

European environmental policy on livestock farming Most European countries have similar regulations regarding the operation of livestock farms including (i), licensing required for large housing units, (ii), mandatory storage of manures and slurries to enable a better agronomic utilization and (iii), prohibited periods for land spreading (usually the winter months from November to February) [START_REF] Mallard | Regulations on the environment[END_REF]. The European Directive represents the main regulatory tool determining national measures with each member state implementing them with its own legislation. The Nitrate Directive (91/676/EC) fixes a limit of 170 kg nitrogen for the amount of livestock manure applied to the land each year. Going further, the Water Framework Directive (2000/60/EC) lays the foundations for a new policy on water quality, integrating all previous regulations regarding specific water pollutions or specific quality standards. The WFD defines a framework for the elaboration and the implementation of water policies, but it does not contain any "operational" measures beyond the existing regulations. However a prominent role is devoted to economic instruments such as taxes and incentives. European policy derives from the Protocol to Abate Acidification, Eutrophication and Groundlevel Ozone (also known as Gothenburg Protocol) which was signed in 1999 and implemented in May 2005. This protocol fixes national annual emissions targets for different gases including ammonia and NOx to be reached by 2010. The related 2001 National Emission Ceiling (NEC) Directive (2001/81/EC) fixes national emissions ceilings for these gases. The global warming issue has been addressed, from the regulatory point of view, in a similar way. The Kyoto Protocol was adopted in Europe in December 1997, during the third session of the Conference of the Parties (COP) to the United Nations Framework Convention on Climate Change.

The 1996 IPPC directive (1996/61/EC -Integrated Pollution Prevention and Control) represents a comprehensive approach. It covers all the main categories of industrial activities which are divided into a series of sectors -one of these is livestock agriculture. Any installation coming under the scheme shall not be operated without a permit covering building and operating requirements, based on best available techniques (BAT), in order to prevent or to reduce polluting emissions. A series of detailed reference documents (BREF's) has been developed to specify what constitute BAT (EIPPCB, 2006). For livestock production, the IPPC directive currently only includes the intensive rearing of poultry or pigs with more than 40,000 places (poultry) or 2,000 places (pigs).

Environmental regulation in Asian countries

The development of environmental regulation across many parts of Asia is established with respect of limits on effluents intended for river discharge. Controls on emissions from agriculture are less well defined although there many regulations that allude to a general protection of the environment (but without specifying measures nor targets). On paper, there are set standards in existence for most countries specifying the maximum concentrations of key polluting indicators permitted in effluents for controlled discharged to rivers and surface waters. In some countries such as Thailand, concessions are offered to small livestock farms with a higher tolerable limit such as a maximum BOD (biochemical oxygen demand) of 100 ppm whereas 60 is specified more generally. However, if there is no direct discharge to rivers, the implementation of such regulation at any level becomes vague; unintentional discharges such as might arise from an overflowing lagoon in the wet season may also be overlooked. The main control on livestock farming is one via planning regulations which both restricts new development to approved areas and which actively encourages the relocation of existing farms away from over-congested peri-urban areas. In Thailand there are schemes to encourage good farm practice to gain an approval score to assist in marketing but they are usually voluntary and have only a minor influence on manure management at present (although this could be added in under a green label concept). More generally, there are schemes to encourage farmers to invest in biogas systems -these are primarily to increase energy production but an environmental benefit may also be drawn from the approach.

6.

Comparison of the two approaches in Europe and Asia 6.1

The role of manure management techniques in Europe Many European farmers will readily consider introducing equipment or techniques that simplifies the manure spreading operation such as mixers or separators to reduce blockage problems and make handling and transport easier. In some cases, these measures may coincide with a reduced environmental impact such as might be achieved by a more balanced land spreading operation. In a few cases, financial rewards such as a premium price for electricity generated from the anaerobic digestion of organic wastes has encouraged the adoption of treatment technology. Otherwise, where the benefit to the farm operation is less pronounced, the uptake of the related technology is patchy across Europe and reflects more the various pressures brought about by environmental legislation. The treatment of manure (as opposed to its handling) implies a definite physical or chemical change as the result of a processing option. This may be brought about by physical, chemical, mechanical or biological processes or a combination of these. A wide range of equipment and systems are potentially available in Europe to modify or improve manure (Burton, C.H.; Turner, C., 2003) but few of these have seen widespread uptake. This is often because they are too costly to install or run, or they are impractical for the farming system or they do not meet the actual requirement. Thus the successful options for manure treatment in Europe fall in one of three categories:

• Composting systems (or related technologies producing a useful solid product) • Biological systems for liquids that effectively breakdown some of the organic load • Separation systems that remove solids (clarification) and/or concentrate Storage, mixing and application systems by themselves do not constitute a treatment although they may be a crucial step in the management of the manure to minimize environmental impact. There is also continuing interest in the use of chemicals or biological additives for various purposes including separation, precipitation, enhancement of biological activity and odour reduction.

6.2

The future prospects of manure management in Asia

It seems unlikely that there will be widespread uptake of any manure management technology that implies other than a small cost to a farmer -and even that may only apply if there is sufficient pressure from the implementation of environmental legislation. Otherwise, a farmer will expect a financial return for any investment he makes, even if modest. Schemes that both imply farm expenditure for installation and again for operating will not be well received even if subsidized. Thus it is important to consider the financially viable options that can enable the wider implementation of appropriate technology in the current situation:

• Anaerobic digestion of manure to produce biogas for local use • Anaerobic digestion with the generation of electricity for farm use and for sale • Production of composts and other organic products for sale • Use of manure products to safely fertilise fish ponds (enhanced fish production)

• Use of manure to fertilise arable crops (in place of bought chemical fertiliser)

In the case of compost production, the transport and sale of a standardised and valued product can clearly bring a return to the farmer for an investment in manure management schemes.

There is a trade off in the extent of the operation and the value of the product produced: basic schemes for small farms can be expected to produce small amounts of a product of low value for local use. For the larger farmer, the bigger volumes of manure are likely to require transport over longer distances to find adequate markets to take all that produced. The extra transport costs will need to be covered and a higher quality product sold at a higher price would be justified. This in turn would require the inclusion of higher quality adjuncts such as rice straw itself implying a cost both for purchase and transport and the overall increase in the volume of compost produced. In any case, the export of manure products from a farm implies the removal of excess nutrients to be incorporated gainfully in arable agricultural operations.

Using manure in fish production is a widely accepted practice in Asia that provides uptake of some (but not all) of the surplus nutrients. The manure treatment issues are principally those to improve hygiene and possibly to treat run off water to reduce its nitrogen content. Aerobic treatment has some application in supporting fish production (by increasing oxygen availability) and which thus may be directed to simultaneously allowing a degree of water treatment in a modified system configuration. The sludge periodically removed from the pond will be rich in phosphorous and thus potentially a soil improver but with minimal value unless transport is relatively easy.

Organised land spreading of manure and related farm effluents is not well developed in Asia although it offers the most extensive and sustainable long term solution to the problems of animal wastes. Potentially, it can offer some savings to the arable farmer in terms of a reduced purchase of chemical fertiliser if a structured regime is followed. This means a degree of assurance that crop requirements will continue to be met. As a means of disposal, manure effluents can be transported to local farms; fears of disease can be alleviated by AD treatment. However, in any case, little monetary value can be ascribed to such a scheme unless there is some saving in the existing purchase of chemical fertiliser products and even this will not benefit the livestock farmer.

6.3

Mutual lessons for livestock farming in Europe and Asia A common factor in both Europe and Asia is that governments want sustainable agriculture with a minimal detrimental effect to the environment; however, it is the motivation of farmers themselves that ultimately determines the selection and use of such manure management processes. This will be the result of a balance of (a) what they perceive as benefit, (b) the applied pressures and inducements and (c) the available funds and resources. The perceived benefit is greater in Asia where manure (especially solid materials) are generally valued higher than in Europe. Likewise, compost schemes have a greater chance of success in a region where the cost of chemical fertilisers represents a higher proportion of the operation cost to the arable farmer. It may be thus expected then that the European practice of land spreading ought to then be more readily accepted for the same reason. This might yet be so if two issues are resolved: the first is the fear of compromising arable crop performance both from the point of view of yield and of food safety. European farmers increasingly share this concern and a more concerted attempt is needed to demonstrate targeted use of manures to provide reliably the required nutrients and thus to enable a real saving in bought in fertiliser. Health issues require both attention to an adequate storage and to specify the safer crops for manuring. The use of anaerobic treatment prior to land spreading can impart some reduction of pathogens but it is not sufficient by itself when there are local outbreaks of notifiable diseases. The second barrier to land spreading in Asia is one of transport in a region where roads can be very poor.

In most cases, there is a clear argument to apply some pressure on farmers to encourage a better environmental compliance but a stick and carrot approach would be better. In some cases, there is a strong argument for financial assistance especially to accelerate the introduction of new technologies. This would also be the case for community and joint schemes (eg: centralised facilities for biogas production) where the compliance of many partners would be necessary. Codes of good practice by themselves are unlikely to progress far but if compliance was rewarded by a green certification, some motivation would be provided. Such ideas have been around in Europe for some time but the emphasis has been weighed more on the side of food quality; the addition of an environmental credential may be an adequate reward.

Livestock farmers in Europe and Asia differ mostly in the available resources in term of both money and land area. In this respect, some compromise ought to be extended to the poorer farmers in Asia. Schemes must include some financial return that significantly defray the investment and running cost. On the positive side, the greater value given to solid products will continue to ensure that some of the nutrient load is removed from the farm. Where land is lacking, then transport and associations with local arable farmers need to be developed for mutual benefit. In this respect, there is common experience with European farmers.

7.

Conclusions • In both Europe and Asia livestock farming can cause major water and air pollution if the manure is poorly managed. There is a crucial need to bring livestock farming back into balance in terms of nutrient use in order to reduce the environmental impacts otherwise caused. Surplus nitrogen, phosphorous and organic matter must be used, removed or digested if not to contribute to pollution. • The European model is to spread most liquid and solid manures onto arable land as a source of organic fertiliser for crops. Any practice that might lead to the contamination of surface water is discouraged and penalties are being applied where spillages do occur. Some specific regulations apply on the storage and times and doses for land spreading.

• In industrialised Asia, land is rarely available for manure spreading and transport is frequently a problem. Removal of solid dung is commonplace, the material having a value for fertilising orchards, vegetable production and fish ponds. Schemes that bring a financial reward including composting draw interest but treatment of the dilute effluents produced is rare; disposal is uncontrolled.

• Treatment is an effective option but in the absence of subsidy, it is rarely used because of the implied costs which are already too high for many European farmers. On the other hand, biogas is universally popular and schemes to generate electricity are generally supported. In Asia, biogas itself is still valued for use in the home and in some farm operations.

• There is scope to use land spreading in certain areas of Asia as a means of recycling the nutrients in a joint schemes with arable farmers. In Europe, there is the real need to see the agronomic value of animal manures which amounts to millions of tonnes per year but which, for many farmers, is still viewed only as a waste product.

Figure 1 :

 1 Figure1: the main transport scenarios for the land application of liquid manures and other farm effluents. Stores can be located near to animal buildings (1, 2) or close to the point of land application (3-7). For the former, conveyance to the field can be by tanker or pipeline to irrigation equipment. Field stores located away from the farm can be supplied by tanker or pipeline. Road haulage would need to be used where long distances were involved.

Figure 2 :

 2 Figure 2: common manure handling schemes in Asia. Solid dung is often collected and dried for export (A). Large farms are considering investment in separators to enable feed for composting (B) but anaerobic digestion is more popular (C). Collection systems (D) with mixing are still rare as is the related land spreading. Lagoon storage including fish rearing (E) is common as is the addition to manures to commercial fish ponds (F). In many cases, contamination of local rivers (G) is still a common occurrence.

Acknowledgements

In the preparation of this paper, the authors wish to acknowledge with thanks assistance from the FAO and especially via their programme, LEAD coordinated by Dr Pierre Gerber. Of particular relevance is the current project "Livestock Waste Management in East Asia" managed by the FAO on behalf of the World Bank.