LD was extracted using three batches of adipose tissues of swine collected from local slaughter houses. Dried fruit seeds of mee (Madhuca longifolia) were collected from three different locations in the North Central Province of Sri Lanka. Avocado fruits were collected from two locations in West Malaysia. Samples of palm oil and palm stearin were obtained as generous gifts from the Malaysian Palm Oil Board (MPOB). Samples of CB were purchased from Malaysian Cocoa Board while samples of SBO were purchased from a supermarket in Malaysia (Sime Darby Food Marketing Sdn Bhd, Selangor, Malaysia). All chemicals used in this experiment were of analytical or HPLC grade obtained from BDH Laboratories (Poole, England) and Merck (Darmstadt, Germany).

Avocado fruits were cut open, and minced manually into fine pieces after removing the seeds. The minced pieces of the avocado mesocarp were dried in a tray type dryer (Memmert Model UFB 400, GmbH + Co. KG, Germany) for 24 h at 60 °C and subsequently ground into powder using a Warring blender Model 32BL80 (Dynamic Corporation of America, New Hartford, CT).

Fat extractions from finely ground samples of mee seeds and dried avocado were carried out via the soxhlet extraction method using petroleum ether (at 40–60 °C) for 8 h (AOAC, 2007). The extracted fats were used for the preparation of binary, tertiary and quaternary blends after expelling solvent under reduced pressure using a rotary evaporator system.

Firstly, all fats were heated at 70 °C until completely melted to erase the memory crystals present in the matrix systems. All fats were weighed and mixed at different ratios in triplicate. The formulated blends were as follow: binary: MF:PS (99:1); ternary: Avo:PS:CB (84:7:9); quaternary: PO:PS:SBO:CB (38:5:52:5).

Shortening was produced on a laboratory scale following a method described by Danthine et al. (2005) with some modification. Initially, 500 g of each fat blend was melted at 70 °C for 10 min to erase crystal memory. A pre-cooling stage was then introduced at 50 °C for 15 min before the blend was crystallized under shear at 20 °C and 125 rpm for 45 min. This procedure was performed in a 1L jacketed glass reactor connected to a water-bath (Lauda, Germany). The resulting slurry was transferred to a freezer at −20 °C for 45 min to complete the crystallization. The shortening was then stored in a thermostatic cabinet at 25 °C ± 1° C for 24 h prior to use for analysis. The same procedure was repeated for all fat blends and LD.

The TAG compositions of the samples were determined according to the method described by Yanty et al. (2014) using a Waters Model 510 liquid chromatography equipped with a differential refractometer Model 410 as the detector (Waters Associates, Milford, MA). The analysis of TAG was performed on a Merck Lichrosphere RP-18 column (5 μm) (12.5 cm × 4 mm i.d.; Merck, Darmstadt, Germany) which was maintained at 30 °C. The mobile phase was a mixture of acetone:acetonitrile (63.5:36.5) and the flow rate was 1.5 mL/min. The injector volume was 10 μL of 5% (w/w) oil in chloroform. Each sample was chromatographed three times, and the data were reported as peak area percentages. The identification of the peaks of the samples was made using a set of TAG standards purchased from Sigma-Aldrich (Deisehofen, Germany) as well as the TAG profiles of LD, PS, and Avo reported previously.

The determination of the hardness of the shortening was carried out using a TA.HD Plus Model texture analyzer (Stable Micro System, Surrey, United Kingdom) according to the method described by Kanagaratnam et al. (1995). Before analysis, samples were melted at 70 °C for 10 min, followed by chilling at −20 °C for 90 min, and then tempered at 25 °C for 24 h. Hardness of the samples was then determined as follows: A 5-mm cylinder (P/5) was used as probe with a 5-kg load cell. A 5 g surface trigger was attached to the probe. During the test, the probe was released to penetrate the sample to a depth of 5 mm with a pre-test speed of 1 mm/s, test speed of 2 mm/s, and post-speed of 1 mm/s. The measurements were performed in triplicate for each sample. The hardness of the shortening was indicated by the maximum force detected during compression. The positive area denotes the compression force while the negative area denotes the adhesiveness force.

The consistency of the shortening was determined in terms of penetration yield value (g/cm²) (deMan et al., 1991) using a cone penetrometer (Stanhope-Seta, Surrey, England) with a 40° angle, where the weight of the cone assembly was 79.03 g. The penetrating cone was placed just above the surface of the sample before it was released. The penetration time was 5 s and penetration depth was read directly from the instrument in 0.1 mm unit. Yield values were calculated using Equation 1:

where, K = constant (5840 for 40° cone angle), W = weight of the cone assembly (79.03), P = mean of penetration depth from two replicates (mm).

PLM (Olympus, Model BH-2, Tokyo, Japan) was used to determine the microstructure of the shortening (Litwinenkoa et al., 2002). The microscopical examination was conducted after placing a drop of shortening, which was removed immediately from the incubator (25 °C ± 1.0 °C) onto a glass slide covered by a glass slip and viewed under PLM connected to a video color camera (Leica Q500mc Qwin Vol 0.02, Leica Cambridge Ltd., Cambridge, UK).

The polymorphic forms of the shortening fat crystals were determined using a wide angle X-ray diffraction (WAXD) machine (D8 Advance Bruker AXS, Karlsruhe, Germany). The power used was 40 Kv, 40 mA with the source of beam from Cu Kα1 X-ray beam (λ = 0.15406 Å). The samples were scanned from 15°2θ to 25°2θ, increasing with a step size of 0.025°/0.1 sec (Ribeiro et al., 2009). Short spacing on the X-ray film was measured with an Evaluation Diffract plus software. The short spacings of the β′ form were at 4.2 and 3.8 Å while that of the β form was at 4.6 Å (D‘Souza et al., 1991).

All analyses were carried out in triplicate and the results were expressed as mean value ± standard deviation. Data were statistically analyzed by one-way analysis of variance (ANOVA), by using Tukey’s Test of MINITAB (version 15) statistical package at 0.05 probability level.

The TAG distribution pattern of the three formulated shortenings and LD shortenings were compared as shown in Table 1. According to Litwinenkoa et al. (2002), TAG composition plays an important role in determining the physical and functional properties of shortening. LD had POL, POO, PPO, and StPO as the most dominant TAG molecules. The most dominant TAG molecule of binary and ternary shortening was POO while that of quaternary shortening was PPO. Although there were wide variations between the formulated plant-shortening and LD with regard to proportions of several TAG molecules, some similarities were still seen in some respects. For instance, there were no significant (p > 0.05) differences among the three formulated shortenings and LD with regard to the proportion of PPSt. Likewise, no significant (p > 0.05) differences were noticed between ternary shortening and LD with regard to amounts of LLL, OOL, and POO. Quaternary shortening and LD displayed the smallest difference with regard to the proportion of de-saturated TAG contents. This was because only small differences were seen between these two with regard to the proportions of TAG molecules such as PLL, OOL, OOO, and StOSt.

The hardness values of the formulated shortening and LD shortening were compared as shown in Table 2. Hardness is the resistance to deformation, which is a critical factor in determining the functionality and consumer acceptance of semi-solid fat systems (de Man et al., 1991). There is a possibility that fat products with similar SFC profiles could tend to display similar hardness characteristics. According to Table 2, the hardness value of LD shortening was 25.67 g while those of the binary, ternary, and quaternary shortening mixtures were 26.19 g, 28.35 g and 26.66 g, respectively. According to statistical analysis, there was no significant (p > 0.05) difference among the hardness values of LD and those of the three formulated plant-based shortenings. Previously, Yanty et al. (2014) reported that the binary mixture of MF:PS (99:1) and LD had the smallest differences in their SFC profile at 5 and 25 °C. Likewise, the LD and ternary fat mixture of Avo:PS:CB (84:7:9) were found to display roughly similar SFC values at 5, 25 and 35 °C (Yanty et al., 2016). In another study, the LD and quaternary fat mixture of PO:PS:SBO:CB (38:5:52:5) was also found to display similarities in the SFC at 5 and 25 °C (Yanty, 2016). These similarities in the SFC profiles of formulated plant-based mixtures and LD in various temperature regions could be attributed to the observed similarities in their hardness values. It has been stated that the hardness characteristic of plastic fats has a good correlation with their SFC profiles and could be the result of a structurally stronger network produced with the increasing solid content (Narine and Humphrey, 2004).

Compression force is of great interest with regard to the texture of plastic shortening. Researchers previously commented that the compression force of shortening is closely related to its SFC as well as the constituent TAG molecular groups (Liu et al., 2010). According to Table 2, the highest compression force was recorded for the ternary mixture shortening (190.34 g/s), followed by binary (190.00 g/s), LD (187.10 g/s) and quaternary (186.74 g/s) shortening mixtures. Although the compression force of quaternary and LD shortening were found to be lower than those of binary and ternary shortening, there were no significant (p > 0.05) differences among them statistically. As seen before, the observed similarities between the compression force values of LD and the formulated shortening mixtures could be attributed to the similarities in their SFC profiles at various temperatures. According to Marangoni (2002), however, polymorphism and microstructure are another two factors which might have some influence on compression force.

Another important property related to the texture of shortening is adhesiveness. According to Table 2, the adhesiveness value of binary, ternary, quaternary shortening and LD were 135.86, 137.00, 82.46 and 123.88 g/s, respectively. The adhesiveness values of binary and ternary were similar to that of LD while the adhesiveness value of quaternary shortening was significantly (p < 0.05) different from any other shortening type investigated. This could probably be due to the fact that quaternary shortening had a higher proportion of tri-unsaturated TAG while ternary shortening contained a higher proportion of tri-saturated TAG (Table 1). According to Kanagaratnam et al. (2013), the free liquid present inside shortening could also contribute to the increase in adhesiveness values of shortening.

The data presented in Table 2 shows the yield values of the shortening. ‘Yield value’ is the most used parameter to evaluate the consistency of fats. Consistency denotes those aspects of the food texture that relate to flow and deformation. The highest yield value belonged to the ternary shortening mixture (326.26 gf/cm²), followed by LD (323.43 gf/cm²), binary (320.89 gf/cm²) and quaternary (319.20 gf/cm²) shortening mixtures. However, no significant (p > 0.05) difference was noticed among the different shortening types. The consistency of the quaternary mixture shortening was the lowest when compared to any other shortening probably due to the highest amount of tri-unsaturated TAGs (34.89%) (Table 1). This observation was in accordance with Silva et al. (2008) who also found that interesterifed fat mixtures with higher amounts of tri-unsaturated TAGs had lower consistency values. According to Haighton’s (1959) classification, the yield value of fats between 200-800 gf/cm² was categorized as plastic fats that can be spreadable. Since the three formulated plant-based shortenings of this study had yield values between 219.20 and 226.26 g/cm², they also could be classified as plastic and spreadable products.

The crystal morphology of binary, ternary, and quaternary shortenings and LD shortenings are compared as shown in Figures 1a, 1b, 1c and 1d, respectively. Crystal size property is important for final product consistency and acceptability since smaller crystals lead to firmer fat products, while larger crystals might produce a sandy feeling in the mouth. In an early report from the fifties, Hoerr and Waugh (1950) mentioned that native LD consisted of large crystals while chemically-rearranged LD displayed small crystals. According to Figure 1a, the crystal network of LD shortening was composed of a high amount of larger crystals with a lesser amount of smaller crystals in a lacy network. This was in accordance with the findings reported by Silva et al. (2008) as well as Campos et al. (2002) who reported that pure LD had a granular crystal structure composed of large crystals. According to a number of other reports, the crystal morphology of fats is also influenced by crystallization temperature, cooling rate and velocity of agitation (Campos et al., 2002). Out of the three formulated plant-based shortenings, only binary (Figure 1b) displayed a crystal pattern somewhat similar to that of LD shortening. The crystal pattern of ternary shortening (Figure 1c) was found to contain smaller well-organized spherulites with a greater number of needle-shaped crystals. According to Figure 1d, quaternary shortening showed the greatest amount of tiny spherulites, which were more tightly packed with less space between adjacent crystals when compared to those found in binary, ternary and LD shortening. This could probably be due to TAG composition where quaternary shortening had a higher proportion of tri-unsaturated TAGs (34.89%) when compared to the other shortening types investigated (Table 1). This difference might also be due to the gap between the crystallization temperature and melting point of the shortening. According to Gamboa and Gioielli (2006), the crystal morphology of fats could be influenced by their melting points as for instance a greater number of smaller crystals are formed when the crystallization temperature is far from the melting point of the fat. According to our previous report (Yanty, 2006), the melting point of quaternary shortening was higher (41.25 °C) than those of binary (37.25 °C), ternary (40.50 °C) and LD (27.5 °C) shortening.

The diffractograms of formulated plant-based shortening and LD shortening are compared as in Figure 2. LD displayed both β’ and β-form polymorphs, of which the β’ form was found to be dominant. This could probably be due to the presence of the monounsaturated TAG (USS) molecular species of LD such as PPL, PPO, StOP, and StOSt. The data presented in Table 2 show that these were important components of the TAG molecular species of LD, contributing to up to 26.6% of the total. According to Timms (1984), the development of the β’ crystal polymorph in LD is mostly influenced by TAG molecules such as StPO (12.52%). Apart from the influence of molecular composition, crystallization conditions also play an important role. According to Campos et al. (2002), LD crystallized in either β’ in rapid crystallization or both β’ and β forms in slow crystallization. Apart from this, the heat treatment applied to LD may have an effect on the polymorphic forms (Herrera et al., 1998). In this case, LD was exposed to a change in temperature from 20 °C to −20 °C. With the decrease in crystallization temperature, the rate of nucleation became rapid, leading to the formation of a large number of nuclei, which resulted in smaller crystals. The tendency of these smaller crystal formations has lead LD to take the β’-form of polymorph. In the meantime, all formulated plant-based shortening also displayed both the β polymorphic form at 4.6 Å and the β’ polymorphic form at 3.8 Å and 4.2 Å. This kind of crystal form is consistent with small spherulites observed previously in the formulated plant-based shortening. According to O’Brien (2004), fats with the β’-form polymorph are softer and display good aeration and creaming properties. Because of this reason, the β’-form polymorph is preferred for the production of fat-rich foods such as cakes, biscuits and other confectionary products (Oh et al., 2005).