A Flaxseed Oil emulgel formulation for Prospective Food Applications Synthesis and Characterization

 

KaushitaBanerjee¹, N.Thiagarajan² and Padma Thiagarajan^1*

¹School of Biosciences and Technology, VIT University, Vellore, India-632014

²Material Science Division, National Aerospace Laboratories, Bangalore, India-560017

 

ABSTRACT:

Plant oil formulations have been effective for the production of encapsulating systems that could be used for the oral delivery of various bioactives to achieve nutritive and therapeutic effects. These kinetically stable formulations prevent the degradation of such actives and also enhance their bioavailability. In this context, synthesis of a flaxseed oil-alkyl polyglucoside based formulation was carried out and its ultrasonication under a set of optimized conditions was employed to reduce the particle size of this emulsion. The ultrasonication conditions were optimized with respect to duty cycle, time, cavitation intensity and amplitude. Physiochemical characterization and FAME analysis of the oil were carried out. The final formulation was characterized for particle size, zeta potential and electrophoretic mobility. FTIR spectra identified different functional groups in the base constituents and also the presence of extensive hydrogen bonding in the formulation. Its SEM analysis showed well dispersed oil globules in water. The rheological studies of the formulations with respect to relevant parameters like hysteresis loop areas, yield stress, consistency and pseudoplasticity indices revealed non-Newtonian thixotropic behavior and shear thinning feature that contribute to good flow characteristics. Thus, it is inferred that this formulation could serve as an excellent base matrix for potential food applications.

 

 

 

 

INTRODUCTION:

Nanotechnology is an emerging industry that has a huge impact on food industry.¹The conglomeration of nanotechnology with the food applications has led to the favorable development of texture, taste, other sensory attributes, coloring strength, process ability and shelf life stability of various food formulations.² Bioactive components, that are a part of the food based products present in the market, claim to provide health benefits for prevention or treatment of diseases. Nevertheless, these products may not sustain the health benefits of the biologically active components for a long time mainly due of their low bioavailability and dose dumping.

 

Flaxseed oil is the richest source of omega-3 fatty acids that is rich in α-linolenic acid (ALA) and can be exploited to fill the ω-3 gap in vegetarian diet.³ It is inexpensive and easily available; but due to its polyunsaturated nature (>75%), it is extremely susceptible to oxidation in presence of oxygen, metal ions and at high temperatures. This leads to the production of toxic hydroperoxides and off-flavor compounds during processing conditions⁴.Previous reports depict that α-linolenic acid (ALA, C18:3, ω-3) has a high susceptibility to oxidation as compared to oleic acid (C18:1). This limits its usage in the form of cooking/frying oil as well as in food fortification. Hence, there is an immediate requirement to produce a formulation of flaxseed oil, that can be supplemented in food products for the delivery of ω-3 fatty acids, without adversely affecting its taste and flavor.⁵

 

In this context, formulations with a combination of plant oil-water-emulsifier in the form of emulsions and/or emulgels are of much interest. In the form of food formulations they can be used as thickeners, flavoring agents, preservatives, etc. Additionally, if a small particle size can be achieved during the emulsification process, then it could confer great stability to the system. It can also enhance its ability to act as delivery matrix for bioactive compounds.^6,7 Such formulations are kinetically stable to gravitational separation because the Brownian motion seen in their small particle sizes masks the gravitational forces that facilitate sedimentation. They hence act as suitable vehicles for encapsulated components and serve effectively as potential delivery matrices.⁸

 

The objective of the present study was to optimize a flaxseed oil-alkyl polyglucoside (APG) based base matrix formulation using optimized ultrasonication conditions to obtain its least particle size. The oil was also subjected to physiochemical and FAME analysis. Characterization studies of the formulation were done by particle size, zeta potential, electrophoretic mobility, FTIR, rheology and SEM. It is envisaged that this flax seed plant oil and APG surfactant based formulation can be utilized for delivery of actives for potential food applications.

 

MATERIALS AND METHODS:

Chemicals:

Flaxseed oil was procured from Falcon’s, Bangalore, India. APG emulsifier was purchased from Yasham Speciality Ingredients Pvt. Ltd. Milli Q water was used for all experiments.

 

Physiochemical parameters of flaxseed oil:

Physiochemical parameters of the oil were carried out using standard test procedures from the previous reports.⁹

 

FAME analysis of flax seed oil:

The oil was esterified using methanol and sodium hydroxide and subjected to GC-MS analysis (JOEL GCMATE II) using a HP 5Ms ultra inert column with very low bleeding characteristics. Pure helium was used as a carrier gas (1mL/minute). The GC inlet temperature was set to 220˚C and the oven temperature was ramped from 50˚C to 250˚C at a rate of 10˚C/minute. The GC interface and ion chamber temperature were both set to 250˚C. Electron Impact ionization (70eV) mode was employed with a scan rate of 50 to 600 amu. Quadruple double focusing mass analyzer and photo multiplier tube were used and NIST library was employed to identify the compounds.³

 

 

Optimization of the formulation:

5 g of oil was weighted into a series of conical flasks and heated to 50˚C. Then, 0.5 to 5 g of emulsifier was weighted and added to the conical flasks for complete dispersion in the oil. Then water preheated at 50˚C was added to the flasks dropwise with continuous stirring until the total volume reached to 50 mL. The best oil: emulsifier ratio was selected based on the non - separation of oil phase or coalescence of emulsifier by observing each of the flasks. The formulations were also observed for liquid crystal formation in the form of maltese cross when viewed under the light microscope at 40X.

 

Thus, the oil: emulsifier ratio of 1:0.5 was selected based on the above observations for the final optimization of the formulation. A pulse rate of 2/minute was chosen to achieve the least possible particle size that was measured after 24 hours of synthesis. Duty cycle variation of 3.33, 6.66, 9.99, 13.33 and 16.66 %, sonication time of 3, 5, 7, 9 and 11 minutes, Cavitation intensity of 150.82, 226.24, 301.65, 377.07 W/cm² and amplitude of sonication 30, 40, 50, 60, 70 % were standardized serially one after the other to arrive at the least particle size of the formulation.¹⁰ In each case, the least significant particle size formed the basis of selection. The final formulation was then synthesized under the above optimized conditions. Particle size, zeta potential and electrophoretic mobility were measured for the final formulation.

 

Characterization studies:

The formulation was suitably diluted for the particle size analysis using a Horiba Scientific SZ 100 instrument equipped with Windows [Z type] version 2.00 software. A scattering angle of 90˚ and a temperature of 25˚C were employed for the measurement. Frequency of particles, as their percentage, was plotted against their diameter in each case, using the software. Zeta potential and electrophoretic mobility was also recorded after suitable dilutions, by the same instrument to ascertain the surface charges around the droplets. FT-IR spectra were recorded for the oil, and their formulation with a spectrophotometer (Bruker Optics, Germany) using attenuated total reflection technique and diamond crystal. A spectral range between 4000 and 500cm^-1 was used. The SEM image was captured using a Carl Zeiss EVO 18 Research instrument.

 

Rheology studies:

The torque and apparent viscosity, at an rpm range of 5 to 200 was recorded for the formulation at room temperature. Shear rates ranging from 1.09/s to 43.95/s were calculated and also used to calculate shear stress using specific “System Factors” of the instrument. Ascending and descending orders of shear rate mode was used and plotted against shear rate, for area of hysteresis loop. The plot of their corresponding logarithmic components was extrapolated to calculate the yield stresses. Similarly, extrapolation of log viscosity against log shear rate gave Consistency Indices (k). The slope gave the Pseudoplasticity Indices (PI).¹¹

 

Statistical analysis:

All experiments were performed in triplicate and values were expressed as Mean ± S.D.¹²

 

RESULTS AND DISCUSSION:

APG’s are nonionic, biocompatible and biodegradable emulsifiers used for efficacious emulsification of various cosmetic, pharmaceutical and food products. It is produced from rapeseed, which is of 100% vegetable origin. It comprises of arachidyl and behenyl alcohols, derived from all naturally found components such as corn, manioc, etc.^13,14 It is free from solvents, antioxidants and preservatives and has excellent emulsifying property at low concentrations (1-3%) and across a wide range of pH (3 to 11). It promotes liquid crystal growth that forms water pockets and stores water for several hours retaining the hydration without any drying effect. It also stabilizes such plant oil based formulations by avoiding their creaming, cracking, coalescence or phase separation and also imparts an optimum viscosity to them.

 

The physiochemical characterization of the oil was according to the standard methods prescribed by AOCS and is reported in Table 1 below.

Table 1: Physiochemical characterization of flaxseed oil

 

FAME analysis of the oil using GC-MS showed the presence of fatty acids as depicted in Table 2. The constituents were further identified and their relative percentages were recorded.

 

Table 2: Fame analysis of flaxseed oil

Physiochemical parameters	Result	Test method
Calorific value(Kcal/100g)	899.73	By calculation
Total fat(g/100g)	99.97	By calculation
Specific gravity	0.927	AOCS Cc 10b-25
Refractive index at 40°C	1.473	AOCS Cc 7-25
Saponification value	188.11	AOCS Cd 3-25
Iodine value	175.36	AOCS Cd 1-25
Unsaponifiable matter	0.73	AOCS Ca 69-40
Acid value	0.51	AOCS Cd 3d-63
Free fatty acid (g/100g)	0.26	AOCS Ca 5a-40

 

The analysis revealed the presence of mainly octanoic and decanoic acids and their methyl esters. Other studies have also revealed such compositions.³These fatty acids are ones that are the most widely distributed in nature (few fats known to contain less than 10%) and exist as glycerol esters. These fatty acids are precursors of most polyunsaturated fatty acids (or PUFA) and form a unique class of food constituents that have a wide range of functions in biological systems.

 

Figure 1: Visual inspection of oil separation and /or aggregation in flaxseed oil on addition of emulsifier and water

 

The synthesis of the optimized formulation was done by choosing a ratio of 1:0.5 of flaxseed oil: emulsifier based on the oil separation and/or aggregation observed in the conical flasks upon the addition of the emulsifier as mentioned above (Figure 1).

 

The selected ratio also showed well dispersed oil and water globules under the optical microscope at 40X magnification (Figure 2).

 

The average particle size of the formulation with different duty cycles (DC), time, cavitation intensity and amplitude was recorded and is shown in Figure 3(a-d).

 

Figure 2: Optical Microscopic image of the formulation (40X)

 

 

Duty cycle of 16.66% showed the least particle size of 425.1 ± 6.498nm and the particle sizes decreased with increase in the duty cycle percentages. This was due to the cavitation energy that increased the applied force for disintegration of larger particles and led to decrease in their sizes. Hence DC of 16.66 % was selected for the synthesis of the formulation to reduce energy and cost.

 

The particle size 430.9±10.340 nm was recorded for ultrasonication time of 5 minutes in comparison to the other times. Sonication time is directly proportional to the shear forces applied onto the particles and leads to their rapid break down with time. Thus, the overall size should decrease with the increase in time. Thus, an ultrasonication time of 5 minutes was selected for the synthesis.

 

Cavitation intensity of 226.24 W/cm² showed the least particle size of 432.5±14.10 nm and the trend in size decreased with increasing intensities from 75.41 to 226.24 W/cm².This was because as the particles hit each other in a system, they increase the shear stress that in turn leads to an increase in the cavitation energy and reduction in the size. The sizes for intensities higher than 226.24 W/cm² were higher and hence this value was selected to save energy during the emulsification process.¹⁰ Likewise, amplitude of 50 % was chosen based on the least particle size of 424.7±8.63 nm. The particle sizes of amplitudes 30, 40 and 50 % were not significantly different from each other and hence 50 % was chosen as per previous reports.¹⁵Thus, a low mean size was responsible for retarding the gravitational settling and subsequent aggregation of the particles.

 

The particle size, zeta potential and the electrophoretic mobility of the final formulation was found to be 426.8 ± 2.560, - 59.5 ± 1.345 mV and 3.08 ± 0.45 µm cm/Vs respectively. The polydispersity index (PDI) of the final formulation was found to be 0.75 ± 0.1. Polydispersity Index (PDI) indicates the relative variance in particle size distribution and is important for determining long term stability of the product. For attaining maximum stability, its value should be between 0 and 1 as seen in the case of above synthesized formulation.¹⁶

 

IR spectroscopy is important for the identification and analysis of plant oils.¹⁷ When compared to classical polyethoxylated surfactants, extensive hydrogen bonding is observed in the formulation with APG due to their higher cloud pointatan absorption band at 3348 cm^-1thatis a finger print for O-H bending vibration of water. These observations strongly show an evidence for the stable formation of the flaxseed oil-APG based formulation as seen in Figure 4(a&b).

 

Figure 4(a): FTIR analysis of flaxseed oil

 

Figure 4(b): FTIR analysis of flaxseed oil APG based formulation

 

The topographical behavior that resulted in the formation of this stable formulation was analyzed with Scanning Electron Microscopy. The emulsifier forms shells around the oil droplets to prevent their coalescence. These droplets, along with the bilayers, also serve as reservoirs for additional water molecules. Hence the product maintains the hydration. Figure 5 depicts the SEM image of the formulation.¹⁶

 

Figure 5: SEM image of the flaxseed oil-APG based formulation

 

Rheology is an important feature of such formulation as it describes the latter’s flow behavior. A well-defined wide and clear hysteresis loop was obtained both in terms of ascending and descending flow curve(Figure 6 a). Shapes, positions and areas of hysteresis loops are dependent on several characteristics of the formulations; the maximum shear rates were used for intervals of time at which the rates are increased and decreased, and on the shear history of the materials. Structures that breakdown upon increasing the shear rate, are built back upon decreasing it. The area of the loop for the flaxseed oil-APG based formulation was found to be 182.41 kPa.s.¹⁸

 

Figure 6(a): Shear rate versus shear stress plot showing the area of hysteresis loop

 

Another important parameter that decides the deformation of the liquid is the Thixotropic index (TI). It is interesting to note that this formulation also shows a time independent viscoplastic behavior. The flow curve plot on the logarithmic scale approached an asymptote, indicating a dynamic yield stress value of 3.658 Pa as the shear rate tended to zero (Figure 6 b). Hence it can be inferred that for this product to flow, the applied stress must exceed 3.658 Pa. This is advantageous in food industry since the formulation would remain in the semisolid form during storage. After shaking and using the product, the thixotropic nature would ensure its reversal back to the thicker form upon cessation of applied force.

 

Figure 6(b): log plot of shear rate against shear stress showing the yield stress on extrapolation

 

Figure 6(c): Log plot of shear rate against viscosity showing the consistency index(k) and the pseudoplasticity index (PI)

 

Pseudoplasticity index (PI) and Consistency index (k) are two vital variables that affect the nature of non-Newtonian fluids, like the one in the present study. In general, PI represents the extent of non-Newtonian behavior of the fluids and its value ranges from 0 to 1.  PI is inversely proportional to consistency of fluids.^19,20 The PI and K were found to be 0.56 and 22.38 respectively. This formulation shows a very good pseudoplasticity index and excellent shear thinning behavior. A unique combination of high K and low PI represent its time independent behavior and ensures good flow of the product (Figure 6 b & c).

 

It is well known that many shear thinning liquids show Newtonian trends at very low and very shear rates. In our formulation, the oil droplets, stabilized by the emulsifier molecules, are highly solvated. Solvation keeps them bound together through hydrogen bonding and this contributes to its viscosity. When high shear rates are applied, the water layers are probably removed from the droplets. Their interactions are thus lowered decreasing the viscosity of the formulation. Further, the emulsifier molecules and oil droplets are randomly oriented in the stable undisturbed formulation. But with increasing shear, they tend to orient themselves along the direction of flow. The points of entanglements between these molecules are also reduced. This leads to high flow and low viscosities. Both these phenomena involve structural breakdown and take considerable time to occur in plastic materials.

 

CONCLUSION:

Formulations based on flaxseed oil and APG emulsifier is proposed as a novel base matrix in this study. The protocol for its formulation is simple. The formulation is inferred to be a pseudoplastic and thixotropic fluid. It is non Newtonian in nature with good shear thinning behavior. These properties are very important for potential food applications. Further studies on its long term stability, material behavior, its ability to deliver actives with its antimicrobial activities are being explored in our laboratory.

 

ACKNOWLEDGMENTS:

The authors thank the Management of VIT University, Vellore and CSIR National Aerospace Laboratories, Bangalore, India for facilitating this study.

 

CONFLICT OF INTEREST:NIL

 

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Accepted on 28.04.2017        © RJPT All right reserved