Impact of a thermal treatment on the emulsifying properties of egg yolk. Part 2: Effect of the environmental conditions

The protein solubility and emulsifying properties of native and heat-treated egg yolk (EY) suspensions were investigated in various environmental conditions. Four distinct conditions were tested by combining two levels of pH, namely pH 4.0 and 6.5, and two levels of ionic strength, namely 0.15 and 0.52 M NaCl, in a model oil-in-water (O/W) emulsion containing 30% oil (v/v). Although the protein solubility was greatly reduced by the thermal denaturation in all tested environmental conditions, the average size of oil droplets obtained in emulsions made with heated EY was observed to be either similar or slightly smaller than that obtained with native EY, depending on the environmental conditions. Using heat-treated EY rather than native EY led to a significant increase of the interfacial protein concentration in all environmental conditions. This increased interfacial protein concentration was shown to have a major impact on the flocculation behaviour of the emulsions, as well as on their rheological properties and stability to creaming. Hypotheses regarding the mechanisms by which insoluble protein aggregates stabilise O/W emulsions at various pH and ionic strengths are discussed.     

Emulsifying properties of sweet potato protein: Effect of protein concentration and oil volume fraction

The effect of protein concentrations (0.1, 0.25, 0.5, 1.0, 1.5 and 2.0% w/v) and oil volume fractions (5, 15, 25, 35 and 45% v/v) on properties of stabilized emulsions of sweet potato proteins (SPPs) were investigated by use of the emulsifying activity index (EAI), emulsifying stability index (ESI), droplet size, rheological properties, interfacial properties and optical microscopy measurements at neutral pH. The protein concentration or oil volume fraction significantly affected droplet size, interfacial protein concentration, emulsion apparent viscosity, EAI and ESI. Increasing of protein concentration greatly decreased droplet size, EAI and apparent viscosity of SPP emulsions; however, there was a pronounced increase in ESI and interfacial protein concentration (P < 0.05). In contrast, increasing of oil volume fraction greatly increased droplet size, EAI and emulsion apparent viscosity of SPP emulsions, but decreased ESI and interfacial protein concentration significantly (P < 0.05). The rheological curve suggested that SPP emulsions were shear-thinning non-Newtonian fluids. Optical microscopy clearly demonstrated that droplet aggregates were formed at a lower protein concentration of <0.5% (w/v) due to low interfacial protein concentration, while at higher oil volume fractions of >25% (v/v) there was obvious coalescence. In addition, the main components of adsorbed SPP at the oil–water interface were Sporamin A, Sporamin B and some high-molecular-weight aggregates formed by disulfide linkage.     

Stabilization of multilayered emulsions by sodium caseinate and κ-carrageenan

The influence of the κ-carrageenan concentration and pH on the properties of oil-in-water multilayered emulsions was studied. Multilayered emulsions were prepared by the mixture of a primary emulsion stabilized by 0.5% (w/v) sodium caseinate (Na-CN) with κ-carrageenan solutions with different concentrations (0.05–1% w/v). The emulsions were evaluated at pH 7 and 3.5. At pH 7, there was little adsorption of κ-carrageenan onto the droplet surface and a depletion flocculation was observed when the polysaccharide concentration exceeded 0.5% (w/v). At pH 3.5, a mixed κ-carrageenan–Na-CN second layer was formed around the protein-covered droplets and the emulsions showed bridging flocculation at lower polysaccharide concentrations (0.05–0.25% w/v). Stable emulsions could be formed with the highest κ-carrageenan concentration (1% w/v) at both pH values (7.0 and 3.5). Thus, stable emulsions were successfully produced using protein–polysaccharide interfacial complexes, and the oil droplet diameter, zeta potential and rheological properties of these emulsions were not affected by changes in the pH.     

Effect of Lepidium perfoliatum seed gum addition on whey protein concentrate stabilized emulsions stored at cold and ambient temperature

In this study the effect of Lepidium perfoliatum seed gum on the properties of whey protein concentrate (WPC) stabilized corn oil-in-water emulsions at pH 7 was investigated. Various concentrations (0–0.6% w/v) of L. perfoliatum seed gum were used together with 2% (w/v) WPC to emulsify corn oil in water at a ratio of 1:5. Quality attributed such as particle size distribution, creaming profile and coalescence rate during storage at 4 and 25 °C; surface and interfacial tension; zeta potential and viscosity of the emulsions were determined. The results indicated that the addition of L. perfoliatum seed gum had no significant effect on zeta potential but the surface and interfacial tension increased with the rise of gum concentration. It was also found that the addition of L. perfoliatum seed gum to WPC emulsions at a critical concentration of 0.2% (w/v) caused flocculation of oil droplets, which resulted in marked increase in particle size and the creaming rate. However at higher gum concentrations beyond this value, the particle size remained constant, apparently because of the high viscosity of the aqueous phase. At all concentrations tested, emulsions stored at 4 °C were more stable except for those containing 0.2% L. perfoliatum seed gum.     

Assessing the potential of flaxseed protein as an emulsifier combined with whey protein isolate

The potential use of flaxseed protein isolate (FPI) as an emulsifying agent was studied in combination with whey protein isolate (WPI) or alone. All the FPI and WPI–FPI emulsions were kinetically unstable. The increase of FPI concentration (0.7% w/v) led to a higher creaming stability of the FPI emulsions due partly to a reduction in interfacial tension between aqueous and oil phases, but mainly to the gel network formation. However at this same high FPI concentration, WPI–FPI emulsions showed a decrease in droplet size and creaming stability, which could be due to the presence of flaxseed gum in the protein isolate enhancing depletion effects. A protein excess was verified in the mixed systems (0.14 or 0.7% (w/v) FPI) and the increase of FPI concentration led to an even greater surface protein content. Increasing homogenization conditions (pressure and number of passes), the creaming stability of the FPI systems increased, mainly at higher concentration (0.7% w/v). Meanwhile, in the mixed systems, the creaming stability of the emulsions containing 0.7% (w/v) FPI decreased even more, but was improved for the emulsions with 0.14% (w/v) FPI. Thus, it was observed that systems containing only FPI at higher concentration were stabilized by gel formation, while in WPI–FPI systems there was a competition by interface between biopolymers with a consequent depletion process. As a result, more stable systems were obtained with WPI addition at lower FPI concentration (0.14% w/v) and using higher homogenization pressure and number of passes (60 MPa, two passes).     

Physicochemical basis for cosolvent modulation of β-lactoglobulin functionality: Interfacial tension study

The impact of neutral cosolvents on the thermal stability of globular proteins in aqueous solutions depends on the nature of the cosolvent, e.g., sorbitol causes a pronounced increase in the thermal denaturation temperature (T_m) of β-lactoglobulin (β-lg), whereas glycerol does not. When a protein unfolds there is a change in the exposed surface area and in the type of molecular interactions that occur at the protein–solvent–cosolvent interface. These changes contribute to the free energy change associated with protein denaturation and depend on cosolvent type. In this study we measured the equilibrium interfacial tensions of aqueous glycerol (0–70% w/w) and sorbitol (0–55% w/w) solutions as a function of temperature to provide insights into the role of the interfacial energy on the thermal stability of β-lg. There was a slight increase in interfacial tension with increasing sorbitol concentration, indicating its preferential exclusion from the oil–water interface. On the other hand, there was an appreciable decrease in interfacial tension with increasing glycerol concentration, indicating its preferential accumulation at the oil–water interface. These changes were largely independent of the measurement temperature (30–80 °C). Our results suggest that sorbitol increases the T_m of β-lg mainly through a steric exclusion effect, whereas glycerol has little effect on T_m because the steric exclusion effect is counter-balanced by a differential interaction effect.     

Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model

The behaviour of cationic lactoferrin-stabilized and anionic β-lactoglobulin (β-lg)-stabilized oil-in-water emulsions (20.0% (w/w) soy oil, 1.0% (w/w) protein) in the presence of simulated intestinal fluid (SIF) containing physiological concentrations of pancreatin (0.0–10.0 mg mL^?1) and/or bile salts (0.0–25.0 mg mL^?1) at 37 °C, pH 7.5 and inorganic salts (39 mm K₂HPO₄, 150 mm NaCl and 30 mm CaCl₂) was investigated. Both emulsions showed a significant degree of coalescence and fatty acid release on mixing with SIF. Appreciably negative ζ-potential values (≥?50 mV) for both types of emulsion droplet at the highest pancreatin/bile salts concentration could be attributed to displacement of and/or binding to the interfacial proteins by bile salts, together with interfacial proteolysis by pancreatin, which enhanced the potential for lipase to act on the hydrophobic lipid core, thus generating free fatty acids and possibly mono- and/or diglycerides at the droplet surface.     

Lipid and water crystallization in protein-stabilised oil-in-water emulsions

Phase and state transitions occurring during freezing and thawing of oil-in-water emulsions with different water phase formulations, interfacial compositions and two lipid types were studied as crucial factors affecting emulsion stability. Emulsions containing 0–40% (w/w) sucrose in the water phase at pH 7, and 10, 20, 30, 40% (w/w) dispersed lipid phase (sunflower oil, SO or hydrogenated palm kernel oil, HPKO) with whey protein isolate, WPI, or sodium caseinate, NaCAS, (protein:lipid = 1:10 and 2:10) as emulsifier were prepared. Phase/state behaviour of the continuous and dispersed phases was determined by differential scanning calorimetry (DSC). Emulsion stability and morphology were derived from DSC data, gravitational separation and particle size analysis during 4 freeze-thaw cycles. Systems were stable when only lipid crystallization occurred. DSC data showed that lipid crystallization prior to water crystallization (i.e. emulsions containing HPKO) caused destabilisation at low sucrose concentrations (0, 2.5 and 5% w/w). Emulsions were stable if the dispersed oil phase crystallized after the dispersing water phase (i.e. emulsions containing SO). A concentration of sucrose ≥10% (w/w) in the aqueous phase gave stable emulsions. At 10:1 lipid to protein ratio, WPI showed better stabilising properties than NaCAS at 2.5 and 5% (w/w) sucrose. Double concentration of WPI (lipid:protein = 10:2) at 0% (w/w) sucrose significantly improved systems stability, whereas no positive effect was observed when the concentration of NaCAS was increased. From morphology study, in addition to lipid destabilisation, thickening and flocculation caused instability of the systems. These were extensive in systems containing WPI and were ascribed to interactions between whey proteins during thermal cycling.     

Characterisation of fish oil emulsions stabilised by sodium caseinate

Fish oil emulsions varying in sodium caseinate concentration (25% w/w oil and 0.1–1.0% w/w protein, giving oil-to-protein ratios of 250–25) were investigated in terms of their creaming stability, rheological properties, the mobility of oil droplets and the oil/protein interaction at the interface. The presence of excessive protein in an emulsion (i.e., at 1% w/w) caused the aggregation of oil droplets through depletion flocculation, resulting in low creaming stability and high low-shear viscosity. At a lower protein concentration (0.1% w/w), when protein was limited, the emulsion droplets were stabilised by bridging flocculation and showed good stability to creaming. Shear-thinning behaviour was observed for both flocculated emulsions. A reduction in the low-shear viscosity and a Newtonian flow was obtained for the emulsion containing an intermediate concentration of protein (0.25% w/w). At this concentration, there was relatively little excess unadsorbed protein in the continuous phase; thus the emulsion was most stable to creaming. NMR was used to characterise these emulsion systems without dilution. Shorter T₂ values (by low-field ¹H NMR), for the emulsions containing both high (1% w/w) and low (0.1% w/w) amounts of protein, indicated increased restricted mobility of oils, caused by depletion or bridging flocculation. The line broadening in oil signals in the high-field NMR spectra (¹H, ¹³C) indicated increased interaction between oil molecules and proteins at the interface with increasing protein concentration in emulsions. In addition, ³¹P NMR spectra, which reflect the mobility of the casein component only, showed increased line broadening, with reduction in protein content due to the relatively higher proportion of the protein being adsorbed to the interface of the oil droplets, compared to that in the continuous phase (i.e., as the oil-to-protein ratio was increased). The T₂ values of resonances of the individual groups on oil molecules, obtained using high-field ¹H NMR, reflected their different environments within the oil droplet.     

Flaxseed gums and their adsorption on whey protein-stabilized oil-in-water emulsions

The effect of the addition of flaxseed gum on the physicochemical properties of whey protein-stabilized (WPI) oil-in-water emulsions at pH 3.5 was investigated. Two different varieties (Emerson and McDuff) were tested at concentrations ranging from 0% to 0.33% (w/v), by measuring droplet size, ζ-potential, phase separation behavior, microstructure and apparent viscosity. With addition of flaxseed gum the ζ-potential of the droplets decreased from around +30 mV to a negative value (−10 mV) at concentrations >0.2%. These results indicated that the negatively charged polysaccharide fraction from flaxseed interacted with the protein adsorbed at the interface. An increase in apparent particle size was also noted with increasing flaxseed concentration, with destabilization becoming visually evident at concentrations higher than 0.1% (w/v). Microscopy, rheological data and size distribution analysis demonstrated for the first time that flaxseed gum interacts with protein-stabilized oil droplets at low pH, causing bridging flocculation. No significant differences were noted between flaxseed gums extracted from the Emerson and McDuff varieties. This research demonstrated that the electrostatic interactions between flaxseed gums and protein-stabilized emulsions need to be controlled when designing novel acidic beverages containing these polysaccharides.     

Characterization and acid-induced gelation of butter oil emulsions produced from heated whey protein dispersions

Whey protein isolate was dispersed at 4% or 8% (w/v) and heated at neutral pH to produce protein polymers. Butter oil, up to 20%, was homogenized in heated whey protein dispersions at pressure ranging from 10 to 120 MPa. Emulsion gelation was induced by acidification with glucono-δ-lactone. Whey protein polymers produced finely dispersed emulsions with fat droplet diameter ranging from 340 to 900 nm. Homogenization pressure was the main factor influencing droplet size. At low fat volume fraction, the emulsions exhibited Newtonian behaviour. As fat content increased, shear thinning behaviour developed as a result of depletion flocculation. Emulsion consistency index increased with protein and fat concentrations. Increasing homogenization pressure had no effect on Newtonian emulsions but promoted flocculation and significantly increased the consistency of high fat emulsions. Protein concentration was the main factor explaining emulsion gel hardness and syneresis. Syneresis decreased with increasing fat content in the gel.     

Utilization of layer-by-layer interfacial deposition technique to improve freeze–thaw stability of oil-in-water emulsions

The freeze–thaw stability of 5 wt% hydrogenated palm oil-in-water emulsions (pH 3) containing droplets stabilized by sodium dodecyl sulfate (SDS)–chitosan–pectin membranes was studied. The multilayered interfacial membranes were created using an electrostatic layer-by-layer deposition method. The ζ-potential, mean particle diameter, fat destabilization, apparent viscosity and microstructure of the emulsions were used to examine the influence of freezing on their stability. Emulsions containing oil droplets stabilized only by SDS were highly unstable to droplet coalescence when either the oil phase became partially crystallized or the water phase crystallized. Emulsions containing oil droplets stabilized by SDS–chitosan membranes were stable to droplet coalescence, but unstable to droplet flocculation. Emulsions containing droplets stabilized by SDS–chitosan–pectin membranes were stable to both droplet coalescence and flocculation. The interfacial engineering technology utilized in this study could lead to the creation of food emulsions with improved stability to freeze–thaw cycling.     

Response surface methodology and multivariate analysis of equilibrium headspace concentration of orange beverage emulsion as function of emulsion composition and structure

The influence of emulsion composition (i.e. Arabic gum, xanthan gum and, orange oil) and structural emulsion properties (i.e. average droplet size and apparent viscosity) on equilibrium headspace concentration of beverage emulsions was investigated. Increase in average droplet size led to increase the equilibrium headspace concentration of more hydrophilic volatile compounds (i.e. lower log P) such as ethyl acetate and octanal, but decrease in more hydrophobic volatile compounds such as 3-carene, myrcene and limonene. In most cases, apparent viscosity had significant positive effect on equilibrium headspace concentration. Principle component analysis (PCA) score discriminated the beverage emulsions containing the same orange oil content but different contents of emulsifiers in different classes, thus indicating the significant (p < 0.05) effect of emulsifier fraction on equilibrium headspace concentration. Beverage emulsion containing 22.2% (w/w) Arabic gum, 0.52% (w/w) xanthan gum and 14.21% (w/w) orange oil was estimated to provide the highest equilibrium headspace concentration.     

Influence of chitosan and NaCl on physicochemical properties of low-acid tuna oil-in-water emulsions stabilized by non-ionic surfactant

An influence of low molecular weight (LMW) chitosan on physicochemical properties and stability of low-acid (pH 6) tuna oil-in-water emulsion stabilized by non-ionic surfactant (Tween 80) was studied. The mean droplet diameter, droplet charge (ζ-potential), creaming stability and microstructure of emulsions (5 wt% oil) were evaluated. The added chitosan was adsorbed on the surface of oil droplets stabilized by Tween 80 through electrostatic interactions. Such addition of chitosan at different concentrations (0–10 wt%) to emulsions showed slight effect on the mean droplet diameter. However, the degree of flocculation was a function of chitosan concentration assessed by emulsions' microstructure and creaming index. The impact of chitosan on the strength of the colloidal interaction between the emulsion droplets increased with increasing chitosan concentration. The mean diameter of droplet in emulsions increased with increasing NaCl because of the electrostatic screening effect. The addition of LMW chitosan could be performed to create tuna oil emulsions with low-acid to neutral character, as well as various physicochemical and stability properties suitable for health food products.     

Influence of xanthan gum on the formation and stability of sodium caseinate oil-in-water emulsions

Studies have been made of the changes in droplet sizes, surface coverage and creaming stability of emulsions formed with 30% (w/w) soya oil, and aqueous solution containing 1 or 3% (w/w) sodium caseinate and varying concentrations of xanthan gum. Addition of xanthan prior to homogenization had no significant effect on average emulsion droplet size and surface protein concentration in all emulsions studied. However, addition of low levels of xanthan (≤0.2 wt%) caused flocculation of droplets that resulted in a large decrease in creaming stability and visual phase separation. At higher xanthan concentrations, the creaming stability improved, apparently due to the formation of network of flocculated droplets. It was found that emulsions formed with 3% sodium caseinate in the absence of xanthan showed extensive flocculation that resulted in very low creaming stability. The presence of xanthan in these emulsions increased the creaming stability, although the emulsion droplets were still flocculated. It appears that creaming stability of emulsions made with mixtures of sodium caseinate and xanthan was more closely related to the structure and rheology of the emulsion itself rather than to the rheology of the aqueous phase.     

Manipulating interfacial behaviour and emulsifying properties of myofibrillar proteins by L-Arginine at low and high salt concentration

The present work examined the impact of L-Arginine (Arg) on the emulsifying properties, interfacial behaviour and conformational characteristics of myofibrillar proteins (MPs) at high (0.6 m ) and low (0.15 m ) salt concentration to maintain good emulsifying properties of MPs at low salt concentration. The data indicated that Arg increased the emulsifying activity index/emulsion stability index (EAI/ESI) and decreased the CI and droplet size of emulsions regardless of salt concentration. Raman spectra revealed that the α-helix content decreased from 60.30% to 51.26% at high salt concentration, and from 60.20% to 54.82% at low salt concentration in the presence of Arg. In addition, MPs treated with Arg exhibited a higher interfacial pressure and more rapidly diffusion to the oil surface. Meanwhile, Arg increased the interfacial protein loading. The results demonstrated that Arg caused the unfolding of MPs, promoting the adsorption of proteins and decreasing the interfacial tension, ultimately improving the stability of emulsions at low salt concentration.     

Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides

The heat stability of emulsions (4 wt% corn oil) formed with whey protein isolate (WPI) or extensively hydrolysed whey protein (WPH) products and containing xanthan gum or guar gum was examined after a retort treatment at 121 °C for 16 min. At neutral pH and low ionic strength, emulsions stabilized with both 0.5 and 4 wt% WPI (intact whey protein) were stable against retorting. The amount of β-lactoglobulin (β-lg) at the droplet surface increased during retorting, especially in the emulsion containing 4 wt% protein, whereas the amount of adsorbed α-lactalbumin (α-la) decreased markedly. Addition of xanthan gum or guar gum caused depletion flocculation of the emulsion droplets, but this flocculation did not lead to their aggregation during heating. In contrast, the droplet size of emulsions formed with WPH increased during heat treatment, indicating that coalescence had occurred. The coalescence during heating was enhanced considerably with increasing concentration of polysaccharide in the emulsions, up to 0.12% and 0.2% for xanthan gum and guar gum, respectively; whey peptides in the WPH emulsions formed weaker and looser, mobile interfacial structures than those formed with intact whey proteins. Consequently, the lack of electrostatic and steric repulsion resulted in the coalescence of flocculated droplets during retort treatment. At higher levels of xanthan gum or guar gum addition, the extent of coalescence decreased gradually, apparently because of the high viscosity of the aqueous phase.     

Effect of high pressure treatment on the physicochemical and functional properties of egg yolk

The effects of high-pressure (HP) treatment at 100–500 MPa on some physicochemical and functional properties of egg yolk (EY) were investigated. Protein solubility, viscosity, surface hydrophobicity (H₀), free sulfhydryl (SH) content, differential scanning calorimetry characteristics, emulsifying activities and emulsifying stability were evaluated. HP-treatment resulted in protein aggregation, as evidenced by gradual decrease in protein solubility and significantly increased in viscosity. HP-treatment at 100–500 MPa induced a gradual decrease in H₀ and SH content, possibly due to protein unfolding and subsequent aggregation/re-association of unfolded proteins. Emulsifying activity index (EAI) was slightly decreased between 100 and 300 MPa and when the pressure is above 400 MPa, EAI was significantly (P < 0.05) decreased relative to the untreated EY. HP-treatment at 100 MPa significantly (P < 0.05) increased the ESI values of EY, while a significant (P < 0.05) decrease was observed when the pressure was above 200 MPa. It was also investigated that there are significant correlations between physicochemical properties of EY, and the differences in the modification of EY protein by HP treatment at different pressure levels may be attributed to the differences in aggregation and unfolding/refolding extents of proteins.     

Freeze-thaw stability of oil-in-water emulsions prepared with native and thermally-denatured soybean isolates

Freeze-thaw stability of oil-in-water emulsions prepared with native or thermally-denatured soy isolates (NSI and DSI, respectively) as the sole emulsifier and sunflower oil (? = 0.25) has been examined at various protein concentrations (0.5, 1.0 and 2.0% w/v), comparatively with sodium caseinate (SC). The freeze-thaw stability was assessed by measurements of particle size, oiling off and gravitational separation after isothermal storage at −20 °C for 24 h and further thawing. The oil phase remained in liquid state and the amount of ice formed was similar (>97%) whatever the sample type and protein concentration. At 0.5%, NSI and DSI emulsions where highly unstable, exhibiting a coagulated cream layer with appreciable oiling off (>25%), whereas those prepared with SC were more stable, due to their initial lower flocculation degree (FD %) and particle size. For all emulsions, the increase of protein concentration (0.5–2.0% w/v) improves the freeze-thaw stability as a consequence of a decrease of initial FD %. At 2.0%, where is enough protein to cover the interface, a lower coalescence stability of NSI emulsion respect to those prepared with NSI was observed after freeze-thawing. This result can be attributed to the high tendency to aggregation of native soy globulins at subzero temperatures. Notwithstanding this, unlike the SC emulsions, the formation of new flocs in soy isolates-stabilized emulsions during freeze-thawing cannot be totally controlled.     

Effects of flaxseed oil concentration on the performance of a soy protein isolate-based emulsion-type film

Biodegradable edible films have the potential to either replace or reduce the amount of synthetic packaging utilized by the food industry. The overall goal of this research was to investigate the effect of flax seed oil concentration (1–10%) on the mechanical, moisture barrier and swelling properties of soy protein isolate (SPI) (5.0% w/w SPI, 40% w/w glycerol) emulsion-based films. Film forming solutions showed a bimodal oil droplet distribution with peak sizes occurring at < 10 and ~ 100 μm. As the oil content increased, the size distribution shifted towards smaller droplet sizes. An equal size ratio was noted at the 5.0% oil content level. All film forming solutions were pseudoplastic in nature, where viscosity increased from 18 to 58 mPa (at 1 s^− 1) as a function of oil content (3% to 10%). Tensile strength of formed films reached a maximum at 5.35 MPa at the 5% w/w oil level, whereas tensile elongation increased from 11.3% to 22.2% with increasing oil content. Puncture strength and deformation, as well as water vapour permeability was relatively independent of the oil content. Moisture content and swelling properties of formed films were found to both decrease from 22.8% to 18.7%, and from 3114% to 1209%, respectively as the oil content was raised from 1 to 10%, and films became darker, redder and more yellow in colour as the percentage of flax seed oil increased.