The purpose of this chapter is to describe the equipment (biomaterials, enzymes, reagents) food processing methods (e.g. UHT) and analytical instrumentation used to characterise the evolution of micro-constituents in oat based beverages. The analytical methods used included High Performance Liquid Chromatography (HPLC) with Diode Array or Fluorescence Detection (DAD and FD respectively), Gas Chromatography Mass Spectrometry (GC-MS) and Two Dimensional Gas Chromatography (GCxGC). This chapter also provides details of the theoretical and practical approaches for the calorimetric and rheological measurements of both liquid and solid food models undertaken as part of this work that were used to analyse the physical and chemical characteristics of biopolymers and biopolymer-dietary fibre mixtures.
2.2.1. UHT formulation: Ingredients used
The Sanitarium Health and Wellbeing Company (Cooranbong, NSW, Australia) provided micro-milled oat fibre, fine-ground orange fibre, fine-ground Kibbled wheat fibre and micro-milled oat flour for the research described in this thesis. This chapter primarily focuses on the oat fibre, which Sanitarium data showed consisted of 1% total sugar, 29.3% total dietary fibre (DF), 60.2% carbohydrate, 9.9% fat, 6.9% moisture, 16.8% protein, and 5.2% ash (w/w). The 15 kg of oat fibre provided by Sanitarium was subdivided into three-5 kg batches of d (v,0.9)182.2, 82.9, and 28.2 ?m particle sizes. The three batches were each stored at -25 ±1 °C until required.
White sugar (99.94% sucrose w/w and <0.05% ash w/w) refined from sugar cane was also obtained from Sanitarium. This form of sugar is known to be appropriate for applications in which crystal size does not damage the quality of the ultimate product (e.g. drink formulations). The material was again stored at -25 ±1 °C when it was received in lab until required. 126.96.36.199. Skim Milk Powder (SMP) The skim milk powder low/medium heat was again supplied by Sanitarium. According to their certificate of analysis the powder consisted of 54.0% carbohydrate, 1.30% fat and 34.0% protein (w/w). The material was also kept in freezer at (-25 ±1°C) until required. 2.2.2. Gelatin Gelatin with a gel strength of ~310 bloom was purchased from Sigma Aldrich (MO, USA) or (Castle Hill, NSW, Australia) batch number is (058k0109). According to the supplier determined the isoelectric point of gelatin was 8.0 and this protein also was in a powder form with a light yellow appearance. 2.2.3. Chemicals Details of the chemicals including enzymes supplied and used in this study are presented in Table 2.1. Milli-Q water obtained in house was used for the preparation of chemical solutions as well as for all of the other procedures applied in this project, such as the making of standard solutions, and mobile phases for liquid chromatography. 2.3 Methods 2.3.1. Sample preparation 188.8.131.52. UHT Samples In this project all beverages were heated using UHT technology. An indirect system via a tubular heat exchanger was used to treat liquid samples at a temperature of 145 °C ±2 °C with a holding time of 6 seconds. Tap water obtained in house was used for all UHT sample preparations. HIPEX (High Performance Heat Exchangers, Melbourne, VIC, Australia) provided the UHT system used for this part of the study and this unit is shown in Figure 2.1. The procedure for preparing samples for UHT treatment comprised of several steps. Firstly, a high shear mixer was used to thoroughly mix all ingredients at room temperature. The mixture was then heated to 60 °C and then subjected to homogenization to decrease the mean size of any fat globules it contained to < 1?m, since homogenization is known to spreads the fat globules uniformly (Chandan, 2008). Within a homogenizer, products pass through constricted orifices under intense pressure. This process breaks up and reduces the dimensions of fat globules and these are then dispersed. Within this particular study, homogenization occurred at a pressure of 3000 psi. The product then underwent sieving to enable smooth passage of the mixture through the tubular UHT system. The process led to a reduction in the original particle-size distribution of the mixture. The samples were then heated at a temperature 145 °C ± 2 °C for 6 seconds. Finally, all materials were moved into sterile containers under a composite flow cabinet. The preparation of the UHT samples involved working with a 5% (w/w) suspension of oat fibre in water. All samples were stored at between 22 °C and 30 °C for 12 weeks (see Chapter 3). For the study of polyvinyl-guaiacol (PVG) in oat-based UHT beverages, sample preparation involved aIDing 5% and 10% (w/w) concentrations of oat fibre in solution with sugar (6.7% w/w) and skim milk powder (2% w/w). This was followed by thorough mixing of the ingredients at ambient temperature, and subsequent storage at between 22 °C and 40 °C for 12 weeks (as outlined in Chapter 4). The best combination of the hydrocolloid formulations established in Chapter 6, were chosen for the rheological study after UHT treatment. The following materials were used: R1 (standard with 0% fibre), R2 (0.5% w/w of orange fibre), R3 (1% w/w of orange fibre), R4 (2% w/w of Kibbled wheat fibre), R5 (3% w/w of Kibbled wheat fibre), R6 (1.5% w/w of oat flour), R7 (3% w/w of oat fibre) and R8 (5% w/w of oat fibre). Identical procedures were carried out with respect to the packing and storage of the samples. In aIDition, duplicate samples were used for each analysis. Figure 2.1 The Ultra High Temperature (UHT) unit at the RMIT University Bundoora campus. 184.108.40.206. Preparation of gelatin and oat particle mixtures The 2% (w/w) gelatin powder was dissolved distilled water to obtain a gelatin solution. To bring about hydration, the solution was gently stirred at ambient temperature then left to stand overnight. The following day, dispersions were heated to 58 °C with gentle stirring until a clear solution was obtained. During the sample preparation process and succeeding analysis the gelatin hydration temperature remained below 60 °C. To obtain a solution of oat fibre particles, these fibres were dispersed within distilled water. Constant stirring and room temperature conditions were maintained. Binary preparations involved mixing appropriate amounts of samples prepared as described earlier at 40 °C to produce final compositions of 2% gelatin and 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0% (w/w) oat particles. 2.4 Rheological Analysis According to Barsby et al., (2002), Rheology is defined as the study of flow and distortion of materials when applied forces are applied to them. Such forces are measured using a rheometer. The determination of rheological properties can be implemented in a range of materials including fluids, dilute solutions of polymers and surfactants, all the way to saturated protein preparations and semi-solids such as pastes and creams. Most of the types of materials used in this study are known to display intricate rheological properties. The viscosity and viscoelasticity of such formulations depend on the external conditions such as temperature, stress, and timescale of the test. Internal factors such as saturation and stability of proteins and other large biomolecules also contribute to the determination of rheological properties (Mader, 2015). Rheology is essential in many scientific fields, and is very important in food science since. mouth-feel and textural properties of foods ( both very important for consumer acceptance) are mostly rheological in nature. Rheological characteristics determine also the appearance and stability of foods such as emulsions, spreads, pastes and creams (Hasenhuettl & Hartel, 2008). The rheology of food also determines many important manufacturing properties such as the flow rate through pipes, the packaging method to be used and the ease of packaging of final product. The rheological properties of food therefore have great importance to the consumer of the food and to the manufacturing processes applied to create it. Due to its importance, food scientists have developed many modern analytical techniques used in the measurement of rhetorical properties. Food rheologists aim at the development of instrumentation and compound concepts that can be applied to the analysis and description of the various forms of rheological behavior (Hasenhuettl & Hartel, 2008). In the modern food manufacturing industry measurements of food rheology are carried out to meet objectives such as the determinations of: the acceptability of food products by the consumers (as consumer acceptability of food is often affected by the texture). the design of food processing required for the individual product(s). a food products shelf life. a food products stability over time. Rheometers are used for the determination of viscosity and rheology data. High-performance rheometers have the ability to measure a wider range of shear conditions that are far greater than those that can be achieved by viscometers. Advanced rheometers can perform techniques such as viscosity profiling, zero shear measurements, and yield stress testing (Myers & John, 2006). Rheological measurements can also used in the determination of the linear viscoelastic region (LVR), and this can be performed even in the areas where the viscoelasticity properties obtained do not depend on stress or strain levels. The LVR of any material should be established before any dynamic tests are conducted and this can be done through the increase of the oscillations amplitude and by observing the magnitude of lag phase. For one to ensure that the results obtained in the dynamic oscillation tests are accurate, measurements of the amplitude have to be conducted to the same point of the sample but within the linear viscoelasticity region. In such a setup, the principle holds it that with little deformation; the molecular arrangements are closer to the equilibrium (Hasenhuettl & Hartel, 2008). Therefore, the dynamic processes at equilibrium are reflected by the instinctive reaction at the molecular level, and this constantly proceeds even when the system is at equilibrium. The domain of linear viscoelasticity helps in affirming the linear relationship of stress and strain, and also insists that the performances of fluids are explained by a single function of time. Different methods can be employed in the analysis of viscoelastic properties of materials. For rheological characterization, the storage and loss module can be measured as a function of: Temperature sweep This test gives vital information of factors such as gelation in materials made from gelatin. Also, in the test, both the loss modulus and the storage modulus are analyzed as the function of temperature provided the strain and frequency are kept constant. Time Sweep In this test, the value of temperature, strain and frequency are kept constant. The test then measures the viscoelastic properties of materials through the determination of the change of a system over a fixed period. This test is also known as the gel cure experiment. In such a case the curing time is essential as it enables the gels to attain the equilibrium state (Mader, 2015). For example, like most other biopolymer systems, the coil to helix transformation in agarose gels occur very fast which resembles a true first order phase transition. A short curing time is therefore sufficient in the case of agarose gels. However, in the case of gelatin gels, the initial phase lasts several hours thus requires a much longer curing time before a pseudo equilibrium state is achieved. Frequency Sweep Frequency is the time required to complete one oscillation. The frequency sweep is dependent on the time sweep and is important in the provision of information on viscoelastic characteristics of substances provided the temperature and strain values are kept constant. The information derived from the tests on frequency sweep is used in the classification of the tested material, for example, a firm gel or a weak gel. In this test, several parameters are derived, and theses provide essential information on the condition of the system being measured. Some of the parameters that are tested using this technique include the complex viscosity and the values of tan ?. The same data derived from the analysis can be applied in the superposition of temperature and time so as to quantify the long-term effects or changes in frequencies that are beyond the scope of the instrument or the expected test time. In this test, the central idea is the measure of equivalency between temperature and time. Strain Sweep The strain sweep test is responsible for the determination of the extent in which deformation occurs in a sample. This test is mostly essential for the identification of the LVR of the system. The test indicates that if the temperature and frequencies of the materials are kept constant, the material will always respond with an increase in amplitude. Time sweep is employed for the determination of stability before being subjected to the strain sweep. 2.4.1. Characterization of rheology measurements 220.127.116.11. Large shear deformation Large deformation in shear is most suitable for studying fluid flow process or the disruption of food products during storage and is measured using a viscometer or rheometer. Uni-directional shear is applied to the sample(s) in a large deformation rheological test. In such a test the results might be destructive to the sample (Malkin, 2015). Large deformation in shear is therefore applied in the analysis of fluids. When a shear stress is applied in a fluid, there is a resultant production of a laminar flow between two parallel surfaces that leading to the generation of a velocity complex. The velocity gradient is the change in the applied rate of shear or strain. Newtonian fluids exhibit a direct, proportional relationship between the shear stress and the shear rate with constant viscosity, whereas in the non-Newtonian fluids, the apparent viscosity values change depending on the ratio of shear stress to shear rate. Newtonian fluids comprise of liquid materials such as water, filtered juices, milk, and syrups. Rheological behavior, such as shear thinning, shear thickening, and time-dependent behaviour in Newtonian fluids are mostly exhibited in fluid and semi-solid foods (figure 2.2). In such system shear stress and shear rates exhibit a linear relationship (Singh, 2006). Non-Newtonian fluids, on the other hand, consist of foods such as Ketchup, custard and exhibit a time-dependent rheological behaviour that is a result of structural changes in the fluid composition. In this case, the shear stress and shear strain are not depicted by a linear relationship. And also the line fails to start at the origin. Shear-thinning fluids contribute in the generation of lesser that proportional shear stress. Shear-thinning fluids exhibit pseudo-plasticity and this is experienced in some fluids such as those used in salad dressings, porridge, and some concentrated fruit juices. On the other hand, Bingham plastic ( a viscoelastic material that behaves as a rigid body at low stresses but flows as a viscous fluid at high stress levels) demonstrate a linear relationship between the shear rate and shear stress with yield stress (Shaw, 2012). Such shear thickening is exhibited on gelatinized starch dispersion which exhibits a lesser than proportional increase in shear rate due to a partial increase in shear stress. In rheology, two fluid behaviours exist and are determined by time. These are the time-dependent thixotropic behaviour and the anti-thixotropic behaviour. Figure 2.2 The shear diagram of shear rate (s-1) versus shear stress of Newtonian, shear thinning, and shear-thickening. The diagram also includes Bingham and Herschel-Bulkley (H-B) with a yield stress that must be exceeded for flow to occur (Rao, 2007). 18.104.22.168. Small deformation in shear Dynamic oscillation tests carried out using a rheometer are mostly used in small deformation studies. This is the most preferred technique for the identification of gelation properties and viscoelasticity behavior of most fluids. The total opposition of materials to oscillatory shear are represented by the complex modulus (G*) which is expressed in Pascal units (Armstrong, 2015). It consists of two components storage modulus (G?), viscous modulus (G?) and these are related by the following equation: G*= (G?2+ G?2)1/2 Equation-1 The characterization of viscoelastic components such as gels depends heavily on the storage modulus (G?), which is also known as the elastic modulus and represents the strength of the network. The viscous moduli (G?), is a measure of the flow properties in the sample. This is observable in the structured state known as the loss modulus. There is a direct proportionality between the values of storage modulus and the number of interactions and the strengths involved. (Perkins, 2011) Unlike with the storage modulus, the value of the viscous modulus is independent of the force of the material and is only related to the number of interactions. Furthermore, phase angle or tan ? is a parameter associated with the degree of viscoelasticity of a sample referred to as tan ? = (G?/G?). A high value of tan ? indicates that the sample is more viscous or liquidlike. On the other hand, a low value of tan ? means that the sample is more elastic or solidlike. The following table 2.3 shows the standard rheological parameter. The ratio of viscous modulus and that of storage modulus results in the generation of the phase angle (tan ?) which gives the extent of viscoelasticity of the material. An Advanced Generation 2 (ARG-2) Rheometer system (TA instruments, New Castle, Delaware, USA) in figure 2.3 was used to generate data on both large-scale deformation and small-scale deformation oscillation experiments for the gelatin/fibre mixtures. This rheometer is able to qualitatively and quantitatively measure the viscoelasticity of fluids, semi-solids and solids. All parameters not being measured were held constant during each test; these factors included temperature, time, frequency and strain The rheological measurements were performed on UHT treated beverages to determine the level of consistency of the products in terms of a steady shear viscosity. This results highlight differences in prodect as a result of the underlying particle size distribution (372-35 µm) and level of fibre aIDition (0.5-5 % w/w ) in each formulation. The temperature used in these tests was 22 ºC and 40 mm parallel-plate geometry and 1 mm gap was also applied in all cases. The changes in viscosity of the beverages were reported for both freshly and made mixtures and thoes that had been in extended storage over 12 weeks at two temperatures (22 and 30 ºC). The ARG-2 rheometer was also used to carry out ssmall deformation oscillatory measurements in gelatin solutions and the oat/gelatin mixtures presented in chapter 5. In order to observe the gelation of gelatin mechanism of the gelatin matrix, a variable amount of gelatin (2-25%, w/w) was cooled to 10 °C at a scan rate of 1°C/min. In this experiment, the exact amount of gelatin concentration 2% (w/w) was pegged at a continuous phase to serve as a baseline for possible changes in network morphology. This is done with variable amounts of oat (0-4%, w/w) and three different particle size distributions (28.2, 82.9, and 182.2 ?m). The process also used a series of rheological tests of time sweep (3 hrs), frequency sweep (0.1-100 rad/s) and strain sweep (0.1-100%) using 40 mm parallel-plate geometry and 2 mm gap. 2.5. Micro-differential scanning calorimetry (MDSC) Calorimetry is a fundamental technique used in measuring the thermal features of the sample so as to find out the relationship between the physical properties of said sample(s) and temperature (Taylor, 2005). IDifferential scanning calorimetry (DSC) is a micro-molecular thermal analysis technique designed to measure a samples heat flow in response to time and change in temperature compared to that of a know standard. The heat capacity of the sample is displayed as a thermograph that can be viewed on connected computer. The absorption of heat and release of energy are seen as peaks and troughs in the thermograph with heat flow (mW) on the y axis against temperature on the x axis. In aIDition to identifying endothermic and exothermic reactions, the area of the peaks and troughs can be measured and is directly proportional to the change in heat enthalpy (Giri & Pal, 2014). Examples of endothermic reactions include the denaturation of protein and the gelatinization of starch. Examples of exothermic reactions include aggregation of proteins and retrogradation of starch (Fakis and Mohácsi-Farkas, 1996; Gill, Moghadam, and Ranjbar, 2010). Micro DSCs are smaller versions of traditional DSC instruments and are powerful analytical tools for characterizing the stability of proteins and other biomolecules (Schramm, 2005). This technique was applied in current the research project to determine the thermodynamic properties of the biomolecules contained in the mixtures of gelatin and oat particles. According to Li., et al (2007), DSCs have the ability to analyze the thermal properties of different components of food. It is most commonly used in the analysis of the protein, lipid and carbohydrate stability of different foods. It is also essential for the identification of the right quantity of the materials to be mixed to attain a required substance (Nishinari, 2009).Such as applications, coupled with the high sensitivity of the technology makes it useful for the determination of various factors and properties for the research described in this thesis. During the processing stages of food, processes such as mixing of different formulations occur. Some of these mixing processes can only occur if the material is in a particular state. Controlled thermal changes are therefore conducted by the application of the differential scanning calorimeters so as to achieve the state necessary for desired mixing of substances (Badea, Della Gatta & Usacheva 2012). Some of the mixtures are complex and may involve the mixing of materials in different states for example where one needs to mix a powder form with a substance in a gaseous state. During food processing, heat is involved at different stages of the preparation. During processes that involve heating, cooling or freezing food undergo different types of transformations; these include, but are not limited to, melting, crystallization, gelation, gelatinization, denaturation and oxidation. All these transformations occur at a certain range of temperature and are associated with heat variations. Thermal analysis techniques, particularly DSC, are used as a primary approach for investigating these properties of foods. However, food processing involves mixtures of food constituents and not just simple substances. The food items may be mixed or diluted with a liquid (water, milk, oil) or with a powdery substance such as sugar or fibre. For simulation of such transformations and interactions, the limited volume and the lack of in situ mixing constitute the major drawbacks of techniques involving DSC. Micro-calorimetry however, provides an ideal alternative for such investigations because it is efficient when it comes to bulk material components in diluted solutions and it has a very high sensitivity. Table 2.4 provides an overview of some endothermic or exothermic effects occurring in various types of foods. Table 2.4 (Endothermic/ Exothermic) effects for different food component (Parlouër & Benoist, 2009) Food component Endothermic effect Exothermic effect Fat, oil Melting, lipid transition Crystallisation, oxidation Protein Denaturation Aggregation, crystallization Enzyme Denaturation Aggregation, enzymatic reaction Starch Gelatinisation, glass transition Retrogradation, oxidation Milk Melting Crystallization, oxidation Hydrocolloid, gelatin Melting Gelation Carbohydrates Melting, glass transition Crystallisation, decomposition Yeast Fermentation Bacteria Growth, metabolism, fermentation The analysis of thermal profiles of liquid and solid systems such as gelatin and oat mixtures undertaken in this thesis involved a micro DSC (Figure 2.4) which was utilized since it was found to be more sensitive when it comes to revealing thermal events of the food. During the analysis of the structural characteristics of the gelatin/oat mixtures, the thermal analysis provides a firm footing on the structural behaviour of the systems in conjunction with rheological measurements. Thermal events seen in the gelatin solution at a concentration of 2% (w/w) were revealed in a cooling profile from 40 °C to 0 °C and a heating profile up to 90 °C, both carried out with oat particle inclusion (1-4% w/w) at 0.5 °C/min. Characterization on thermal transition of lipid, starch and protein present in the oat particles was examined using the micro DSC system. 2.6 Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) is useful because of its capability to evaluate samples ranging in size from nanometre up to the centimetre scale. The spacious chamber and goniometer of an SEM can accommodate relatively large samples as compared to the more traditional transmission electron microscopes. It also provides nearly unlimited points of viewing with the assistance of translational, tilting and rotary movements (Rouèche, et al 2006; Shu, et al., 2006; Zhang, et al., 2010). SEM produces an image of a sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals containing information about the characteristics of the sample. Information may include particularly about surface topography, composition as well as electrical conductivity. Secondary electrons, back scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen current and transmitted electrons are the different types of signals produced by an SEM (Kimseng and Meissel, 2001). In the most common or standard detection mode of secondary electron imaging, the SEM can produce very high resolution images of a sample surface that reveal details having a size of approximately 1 to 5 nm. SEM micrographs yield a characteristic three-dimensional appearance making them very useful for understanding the surface structure of a sample. A wide range of magnifications is also possible from about ×25 (equivalent to that of a powerful hand-lens) to ×250,000 which corresponds to 250 times the magnification limit of the best light microscopes. The beam from the electron gun is passed through electron lenses in order to decrease spot size. This then produces a clear image for analysis. Subsequently, the signals from the beam-specimen from different locations are collected by the electron detector. This then converts the collected signals to point-by-point intensity differences on the monitor providing an image of the sample (Kimseng and Meissel, 2001; FEI, 2008). The specific instrument used in the study was a Philips XL30 microscope (Figure 2.5) located at the RMIT Microscopy and Microanalysis Facility (RMMF). This instrument includes a high-vacuum feature that is the most suitable choice of operation to examine the protein and polysaccharide samples in materials such as gelatin/oat fiber mixtures used in this study. The XL30 can collect secondary electron (topography) images using a standard chamber mounted Everhart-Thornley detector. However, a backscatter detector can be mounted to the bottom of the column to collect backscatter electron images. Backscatter electron images contain atomic number contrast (Z-contrast), which is useful for elemental identification. For this thesis, the SEM was utilised to produce tangible evidence of the phase morphology of gelatin and oat fiber mixtures. Gelatin/fiber mixture solutions, which had a single gelatin content 2% (w/w) protein in the presence of varying levels of oat fiber 1-4% (w/w) were tested. The particle size distribution (d(v,0.9)) used in this work was 182.2 ?m. The sample preparation for the SEM study was the same as that used to prepare samples for the rheology study (see section 22.214.171.124). The samples for imaging were freeze-dried at -55 °C overnight and then gold plated under high-vacuum. An accelerating voltage of 30 kV was used to produce microscopic images to assisting in the characterization of the network morphology of the samples. 2.7 Particle size analysis by laser light scattering Malvern particle sizer is the most commonly used equipment for such measurements in numerous industries for characterisation and quality control purposes. This equipment can measure particle size distributions of liquid samples with insoluble dietary fibres in a few minutes. Laser light scattering is helpful because it facilitates easy and quick measurements of particle size (Kwak, Lee, Ahn, & Jeon, 2009). This technique is dependent on the fact that the diffraction angle is inversely proportional to particle size. The laser light scattering instrument comprises a laser that gives off strong light with unchanging wavelength. The instrument also comprises an appropriate detector as well as a means of conveying a laser beam through the arranged sample. Some instruments are based on the Fraunhofer approximation that supposes that particles are greatly bigger than the wavelength of the light used. The Fraunhofer approximation also supposes that, being opaque, particles do not transmit. Another assumption is that all particles scatter with identical efficiency. These assumptions are not however always accurate for numerous materials, mainly those with minute particles. This imprecision brings about errors as high as 30%, particularly if the refractive index is near to unity. The scattering transforms into some complex function having minima and maxima values when size of a particle is close to the wavelength of light (Rawle, 2012).Laser diffraction is further advantageous in that, being founded on scientific principles, it is an absolute technique. AIDitionally, this method permits significant flexibility. Moreover, laser diffraction permits measurements within the 0.1-2000 ?m range. To allow surfactants and dispersing agents to measure the primary size of a particle and to achieve high reproducibility, liquid emulsions and suspensions could be measured within re-circulating cells. Even though sub-sample amounts are usually small, meaning that the representivity of sampling could be a significant consideration, whole samples could be measured. Given that this technique is non-intrusive and non-destructive, samples may be recovered provided they are valuable. Outcomes are directly generated as volume distribution to reflect weight distribution provided that the particle density remains unchanged. This technique offers extremely replicable and rapid analyses. A high resolution of up to 100-size groups in the range of the system may be computed on the apparatus (Rawle, 2012). The particle size distribution of UHT samples was measured by the laser light scattering principle. UHT samples were analysed immediately after preparation and each week during the 12-weeks storage period. For each test 5 microliters of sample was dispersed in the sample chamber with continuous mixing. Measurements were then performed in triplicates at room temperature. The instrument used for laser light scattering was a Malvern Mastersizer X (Figure 2.6) 2.8 Sensory evaluation Sensory evaluation was undertaken to evaluate the micro-constituents of the soluble or insoluble dietary fibre systems, e.g. phenolic acids that could potentially promote the development of unpleasant flavours and therefore off-aromas. It is hypothesized that decarboxylation (removal of a carboxyl group with the of release carbon dioxide) of ferulic acid, a major phenolic acid found in oats, could produce polyvinyl guaiacol (PVG). PVG is a compound known to be responsible for off-flavours in food and can be produced during high temperature processing and subsequent prolonged storage of many food products containing insoluble dietary fibre (Refrnces). Therefore, the acceptability of UHT beverages containing either 5 or 10 % oat fibre stored at 22 and 40 °C, during a shelf life of up to 3 mouths was carried out by 8 semi-training sensory panellists using the smiling sca