Utilization and characterization of flaxseed oil in ultrasonically emulsified mango beverage

Abstract

The present study described the formation of stable emulsion of flaxseed oil (0%, 0.25%, 0.50%, 0.75% v/v) in ready to serve Mango beverages using 20 kHz ultrasound of power input 21 W, 32 W, 43 W for varying times 3, 5, 7 min to create emulsion droplets size 100–800 nm. Oil was extracted by ultrasound-assisted extraction and solvent extraction method by varying solvents, time, ultrasonic intensity etc. and physio-chemical characterization was conducted. Fatty acid profiling revealed that linolenic acid and linoleic acid are major fatty acids present in extracted oil. Effect of flaxseed oil in fruit-based beverage was evaluated in terms of turbidity, pH, acidity, color, antioxidant activity and carotenoids content. Pectin stabilizes emulsion droplets by generating electrostatic repulsion (ζ potential − 25 mV) and the emulsions were stable upto 18 days at (4 ± 2) °C. The rheological behaviour in terms of viscosity of the emulsion stayed unaffected with sonication time upon storage. The carotenoids and antioxidant activity significantly increased from 151.37 to 292.24 µg/mL and 26.99% to 61.43% respectively at 0 to 0.75% added oil in the beverage and enhanced stability by preventing lipid oxidation. Organoleptic score of 0.25% and 0.50% of the flaxseed oil in the beverage was found to be acceptable.

Introduction

Flaxseeds (Linum Usitatissimum L.) are considered as the hub of nutrients containing a good amount of fats, proteins, carbohydrates, minerals, vitamins, fibers and other bioactive compounds such as omega-3 fatty acids, carotenoids, polyphenols, lignans etc. (Zhang et al. 2008). They are a good source of essential fatty acids (EFA) which cannot be synthesized in our body and must be included in our diet from external sources. The functional compounds present in oilseeds have been reported to reduce the risk of many diseases including cancer, diabetes, cardiovascular diseases, arthritis, osteoporosis and other chronic diseases like ulcers, attention-deficit/ hyperactivity disorder, lupus, eating disorder, and panic attacks (Chishty and Monika 2016). Oilseeds are versatile food ingredients due to their high potential to be used as a whole, by extracting oil, utilizing the defatted meal and oilseed hulls also a good source of dietary fiber and antioxidants (Zhang et al. 2008). Flaxseed steroids, phytosterols and phytostanols, having excellent properties reducing the risks of cancers (Bradford and Awad 2010; Grattan 2013). Nowadays consumers are very health conscious and demand natural nutrient rich food products which urge manufacturers to develop functional food as they are in high demand.

The choice of oil extraction technique depends on the maximum yield, purity and nature of the target compound. The traditional techniques for the extraction of fat are hydraulic pressing, soxhlet extraction, and maceration with alcohol (Wang and Weller 2006). The major challenge in thermal technique is deterioration of bioactive high-value compounds, results lower yield and poor quality target compound. Therefore maintaining quality of oil, appropriate cost scanning and selective methods are needed which offers higher yield with good quality product.

The rupturing of cell wall facilitates movement of the active constituents from inside to outside of plant material thereby increasing the yield in lesser time. Ultrasound-assisted extraction comprises high-frequency sound waves which are propagated by compression and rarefactions. The negative pressure caused by this expansion becomes more than the tensile strength of the liquid, formation of the vapor bubbles resulting in cavitation bubbles. High-velocity inter-particle collisions occur due to the collapse of cavitation bubbles resulting in the fast-moving stream of solvent, target compound through the cavity at the liquid–solid interface with minimal loss of the bioactive compounds at the room temperature extraction (Goula, 2013).

The sono-emulsification is a two-step technique comprises 1st step of continuous mechanism, a blend of interfacial waves and unsteadiness directs to the break out of diffused phase droplets into the uninterrupted phase, followed by second step consist of separation of droplets during that acoustic cavitations nearer to the boundary. The scrupulous consequences (disturbance and mixing) of shock waves responsible for the production of extremely tiny droplet size. Mechanical shaking, shock waves, and severe shear forces created by low-frequency high-power ultrasound produced sono-emulsification.

Incorporation of flaxseed into different food products is a recent trend in research & development sector which include omega-3 enriched energy bars with flaxseeds, flaxseed chutney powder, flaxseed cookies, muffins, buns, bread etc. Most of the bioactive compounds are present in the flaxseed oil and thus incorporation of flaxseed oil in the preparation of ready to serve beverages is an emerging trend and yet to be studied its industrial application due to great nutritional significance and high market demand. Flaxseed oil incorporated into milk via ultrasonic treatment resulted novel emulsion with unchanged organoleptic attributes, higher functionality than control raw milk in terms of fatty acid profile and other nutritional aspects (Shanmugam and Ashok kumar 2014).

The objectives of the study were to optimize the flaxseed oil extraction by different methods, characterization and to assess the oil emulsion stability and functionality in ultrasonically prepared mango beverages.

Material and methods

Raw material

Flaxseed variety AL-2063 was obtained from Punjab Agricultural University, Ludhiana. Seed powder was made by using grinder (Philips, HL7505, 500 W, India) and powder was stored in PE zip packet and stored in refrigerator before use. Mangoes (Magnifera indica) Chaunsa variety for beverage were taken from local market. All the solvents and reagents were used of analytical grade and purchased from SD Fine Chemicals, Mumbai, India.

Extraction of fat

Soxhlet apparatus

Solvent extraction of fat was carried out using 25 g of sample and 250 mL of selected solvent n-hexane, petroleum ether or petroleum benzene for varied time 6, 8, 12 and 16 h (AOAC, 1990).

$$\text{Percent Fat content}\left(\frac{\text{g}}{100\text{g}}\right)=\frac{\text{Weight of the ether extract}\left(\text{g}\right)}{\text{Weight of the sample taken}\left(\text{g}\right)}\times 100$$ 1

Ultrasound Assisted Extraction (UAE) method

The ultrasound-assisted extraction of flax seed oil used according to the method described by Omar et al. (2014) and with a little modification. 25g of flaxseed powder was mixed with 100mL solvent such as n-hexane, petroleum ether or petroleum benzene in a 200 mL plastic beaker in Sonicator (QSonica -Model Q-500, Newtown, CT. USA) with 1.5-inch flat tip probe was used and 500W and 20 kHz were the two parameters for maximum oil extraction was applied. Calorimetric conversion of these powers could be done, and ultrasonic output ranged between 10 and 50W according to the method described by Li et al. 2004. The samples were extracted under continuous ultrasonic waves at 20 kHz at different levels of power output 43W, 53W, 66W, 79W, and 93W corresponding to 50, 60, 70, 80 and 90% respectively. During extraction, the temperature was controlled at a desired level with a maximum variation of (±1) ^oC.

Preparation of ultrasonically emulsified flaxseed oil enriched mango beverage

Mangoes were washed with clean water followed by peeling and pulp was homogenized. Total Soluble Solid (TSS) and acidity of the homogenized pulp were observed with refractometer and by titration method. Water, sugar, and citric acid were added to get TSS 15 °B, mango pulp 20% and 0.75% acidity Pectin 0.5%. After the preparation of the beverage, flaxseed oil at concentrations of 0%, 0.25%, 0.50%, 0.75% was incorporated according to FDA's generally regarded as safe (GRAS) notification number GRN-000256. Prepared beverages were treated for ultrasonication to develop emulsions, the sonicator horn was positioned at a depth of 0.3±0.1 cm. Emulsions were obtained using a 20-kHz, 500-W ultrasonic horn (1.5-inch diameter) at the power input of 21W, 32W, and 43W and at a different processing times 3, 5 and 7 minutes. Then the flaxseed oil enriched beverages were filled in 300 mL glass bottles, sealed, pasteurized and stored. The analysis and storage studies were performed on both control and emulsified samples.

Quality evaluation of extracted oil

The antioxidant activity of oil was assessed according to Gorinstein et al. (2004) method using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay measuring absorbance at 515 nm.

The peroxide value of extracted oil was estimated according to AOAC method (AOAC, 1990) using potassium thiosulphate titration method. The peroxide value was calculated with following formula in terms of (milli equivalents peroxide/1000 g sample).

A drop of oil sample was placed on the prism fitted in the instrument. The adjustment of the dispersion screw was made to ensure that no color ting appears in the dark and illuminated halves of refractometer. The dark line was made to fall exactly on the cross wires followed by measurement of the refractive index on the scale.

Iodine value taking 0.2gm of oil, saponification value taking 2 gm of oil, acid value taking 1 g of oil were estimated using AOAC (1990) method.

Droplet size and zeta potential of flaxseed oil incorporated mango beverages were measured using Zetasizer (NanoZS-90 model, Malvern Instruments) (Jain et al., 2015). Emulsions were diluted 100 times with distilled water.

Gas Chromatography-Mass Spectrometry (GC–MS)

The fatty acids profiles of flaxseed oils extracted in two methods were assessed by Gas Chromatography-Mass Spectrometry (GC–MS) by derivatization to fatty acid methyl esters. 1μL volume of prepared aliquot was injected in a Thermo Trace 1300 Gas Chromatograph coupled with a MS TSQ 8000 Detector following the standard protocol of the instrument. The mass spectra of the samples were analyzed by comparing the data with NIST 2.0 Mass Spectral Library (Popa et al. 2012).

Scanning Electron Microscopy (SEM)

The morphology of the flaxseed powder and defatted meal were examined with a scanning electron microscope (Scanning Electron Microscope JEOL JSM-6100, Digital SEM, Tokyo, Japan) at magnification 1000-4000X (Ghoshal et al. 2013).

Quality evaluation of prepared beverage

Quality analysis of prepared beverages was done in fresh and during storage for 18 days.

Turbidity and Cream stability

Turbidity was measured by centrifugation of the beverage samples at 4200 rpm for 15 min followed by collection of supernatants. The absorbance of the supernatants was read at 660 nm using UV–vis spectrophotometer (Model 1900, Shimadzu, Kyoto Japan).

The stability of mango beverage indicates the emulsion stability of final product against phase partition, for about 7 days at refrigeration conditions (4 ± 2 °C). Cream stability of emulsions was determined from the flax seed oil incorporated mango beverages were visually checked for phase partition and oiling-off or creaming on day 1 and upon storage until 18 days at 4 ± 2 °C. The samples were collected and kept in 20-mL sealed graduated tubes and in 100-mL sampling tube. The emulsion stability against creaming was observed by evaluating the volume of the lipid-rich layer on top (V_L) and the of total emulsion volume (V_E) in the tube. Creammg index (CI) was determined using following formula.

$$CI=100\times \frac{V_L}{V_E}$$

In the ‘unstable emulsion’ the oil phase broken up in 3 h when stored at room temperature where as in ‘stable emulsion’ system the oil phase does not break at least stored for 7 days at 4 ± 2 °C (Shanmugam and Ashok kumar, 2015).

Carotenoid content

The carotenoid content of beverage was determined according to Zhou et al. (2009) modified method. 1 mL of mango juice was mixed with 10 ml chloroform/methanol (2:1v/v) in a separating funnel and continuously shaken for better carotene extraction in organic layer till colorless. Then 5 g of sodium sulphate anhydrous was added for dehydration, followed by filtered using Buchner funnel with Whatman no 1 filter paper and collected volume was made up to 50 mL with chloroform/methanol (2:1v/v) and absorbance was measured at 450 nm using UV visible spectrophotometer. From standard graph β-carotene content was determined in mg/100 mL of beverage.

Color

L*, a* and b* values for control and flaxseed oil enriched beverages were measured by using Hunter colorimeter, Colorflex Spectrophotometer (Hunter Associate Lab, Reston, Virginia USA) (Ghoshal et al. 2013).

Acidity

The acidity of the drink was determined using AOAC (1990). The acidity of the drink was determined by taking 10 mL of juice/beverage sample in a conical flask. Few drops of phenolphthalein indicator were added. After thorough mixing by shaking well, it was titrated with 0.1 N sodium hydroxide solution till pink end point. Acidity was calculated using following formula.

$$\text{Titratable acidity}\left(\raisebox{1ex}{$\text{g}$}\!\left/ \!\raisebox{-1ex}{$\text{mL}$}\right.\right)=\text{mL of NaOH}\times \text{Normality of NaOH}\times 0.009$$

Sensory evaluation

Mango beverages containing different concentrations of oil were coded randomly with three digit code and evaluated using 9 point hedonic scale for degree of liking ranging from like extremely to dislike extremely (Larmond 1970). 20 Trained panel members (10 male and 10 female) of average age 30 were asked to mark the appropriate category on the scale for every sample in terms of appearance, color, taste, aroma, and overall acceptability. Samples were marked in 3-digit code randomly. Panellists were asked to drink a few sip of water before testing of another sample to clean the palate.

Statistical analysis

Experiments were conducted in triplicates and data was statistically evaluated using CPCS-1 software. One-way ANOVA with least significant test was used to find significant (< 0.5) difference among data.

Results and discussion

The physio-chemical parameters and proximate analysis results of flaxseed AL 2063 are given in Table 1. The traditional technique of solvent extraction is based on the correct choice of solvent and the use of heat to increase the solubility and to improve mass transfer. n-hexane is one of the popular solvents used for this purpose followed by petroleum ether, and petroleum benzene at different extraction timings for 6 h, 8 h, 12 h and 16 h (Table 2). It can be clearly seen from Fig. 1 that sharp increase of yield from 6 to 12 h followed by a constant value from 12 to 16 h for all three solvents used for extraction. Petroleum ether was found to be the best as comparatively higher yield was obtained by extracting with this solvent. The highest yield of 43.44% was obtained after 12 h of extraction with petroleum ether followed by petroleum benzene (43.13%) and n-hexane (42.56%). Flaxseed to the solvent ratio (1:10) was kept constant and temperature of the extraction was kept slightly higher than the boiling point of the respective solvent. During solvent extraction yield was measured at different interval and it was found that after 6 h and 8 h roughly 80% and 88% oil were extracted and extraction was completed after 12 h. From 12 to 16 h, very minute insignificant decline (p > 0.05) in the yield of the oil was found.

Table 1.

Physio-chemical parameters of flaxseed AL-2063

S. No	Physical parameters	Value	Proximate composition	Value (%)
1	Length (cm)	0.50 ± 0.04	Moisture	7.98 ± 0.45
2	Breadth (cm)	0.27 ± 0.02	Crude fat	43.44 ± 2.17
3	Height (cm)	0.12 ± 0.02	Protein	14.37 ± 1.74
4	1000 Kernel Weight (g)	7.763 ± 0.24	Crude fiber	7.33 ± 1.25
5	Bulk Density (g/mL)	0.694 ± 0.09	Ash	3.21 ± 0.55
6	Color L*	40.70 ± 1.98	Carbohydrates	23.67 ± 2.11
	a*	8.72 ± 0.75
	b*	16.90 ± 1.67

Results are expressed as mean ± standard deviation (n = 10)

Table 2.

Yield of oil (%) using the Soxhlet and Ultrasound extraction method

S. No	Solvent	Soxhlet Extraction Time (h.)				Ultrasound extraction method, Power (W)
		6	8	12	16	43	53	66
1	N-Hexane	32.15a ± 0.52	37.11b ± 0.58	42.56c ± 0.49	42.34c ± 0.41	30.29a ± 0.45	31.72a ± 0.45	31.17a ± 0.45
2	Petroleum Ether	37.34b ± 0.41	40.76c ± 0.42	43.44d ± 0.41	43.31d ± 0.58	31.93a ± 0.54	35.02b ± 0.51	29.89a ± 0.50
3	Petroleum Benzene	33.49a ± 0.56	36.47b ± 0.39	43.13c ± 0.63	43.01c ± 0.41	31.38a ± 0.49	30.31a ± 0.47	28.97b ± 0.46

Results are expressed as mean ± standard deviation (n = 4); Values with different alphabets a,b,c.. in a row are significantly different at p < 0.05

Fig. 1.

Yield of oil (%) using the a Soxhlet extraction b Ultrasound Extraction Method

Because diffusion of the solvent through the oil cake and solubility of the oil was maximum after 12 h. In ultrasonic extraction It can be observed that the size of the emulsion droplets (ranging from 100 to 800 nm) decreased with an increase in Ultrasonic power input for all processed samples. Increasing the power input results in an increase in the size of acoustic cavitation bubbles and hence their collapse intensity generating stronger shear forces in the surrounding environment and production of finer emulsion droplets (Leong et al. 2011). Similarly, for each power input, the size of the emulsion droplets decreased with an increase in sonication time but zeta potential was almost constant (−25 mV) with power input also after 18 days of storage at 4 ± 2 °C (Shanmugam and Ashokkumar 2014).

Longer extraction time and lower efficiency compelled for newer improved methods of non-thermal extraction. The process time and solvent consumption in the extraction of phytochemicals from the plant source can be reduced by using high ultrasound power. From 43 to 53 W there was an increase in the yield of oil with n-hexane and petroleum ether while from 53 to 66 W sharp decline in the yield was observed for petroleum ether with negligible effect with n-hexane. For petroleum benzene, the yield of oil declined with an increase in ultrasonic intensity from 43 to 66 W. After 45 min. of extraction, the highest yield of oil was obtained at 53 W power with petroleum ether followed by n-hexane and petroleum benzene. Thus optimum power at which maximum extraction occurs, deviating from which showed a negative impact on the yield of oil. Five levels of sonication intensity i.e., 43 W, 53 W, 66 W, 79 W, 93 W were investigated in our study presented no linear but dynamic relation between yield of oil and power intensity of the sonication. The yield of oil increased from 31.93% at 43 W to 35.02% at 53 W followed by a sharp decrease to 29.89% at 66 W and become linear afterwards to 30.01% at 93 W as shown in Table 2. 43 W to 53 W was suitable for extraction, due to maximum solubility of the solvents, but above 53 W might be degradation of oil occur or evaporation of volatile fatty acids, which might cause lesser yield of oil.

Extraction efficiency when compared with Soxhlet extraction method (Table 2 and Figs. 1 and 2), showed that the highest yield of oil obtained in the case of ultrasound-assisted extraction as 80.61% of the oil yield with Soxhlet extraction which is approximately in accordance to Zhang et al., 2008. An increase of 7.11% from 43 to 53 W can be detailed on the basis of bubbles formation during the process of ultrasonication. The rate of generation and destruction of the bubbles increased with the increase in the amplitude/output power of ultrasonic waves. As a result of high pressure and rise in temperature inside the bubbles, violent shock waves were generated. These waves were responsible for the enhanced penetration of the respective solvents into their inner cells due to higher speed jets. This caused the disruption of cell walls and better recovery of the target compound (Hemwimol et al. 2006).

Fig. 2.

Effect of a ultrasonic intensity b time on oil extraction (solvent-petroleum ether)

From Fig. 2, it can be seen that the yield of flaxseed oil increased with extraction time. The increase was observed from 30–45 min at 53 W with petroleum ether which is slightly decreased afterwards. After 30 min of extraction, the yield of oil was 26.0% which increased by 9% after 45 min followed by a slight decline of 1.1% after 60 min after which negligible change was observed till 90 min of process. Ultrasonic waves disrupt the cell and create large contact area between solvent and seeds resulting in higher extraction. This process occurs rapidly in initial stages resulting in a greater rise in the yield at the beginning. Similar results were obtained by Balachandran et al. (2006) and Zhao et al. (2007). Balachandran et al. (2006) reported that the ultrasonic waves show a considerable effect even during the later stages through improvement to the internal diffusivity but these effects were smaller in comparison to the initial stages. Major effect of ultrasonic waves on the mass transfer rate is mainly seen in the penetration stage of solvent. The ultrasound treatment was more effective in the first 30 min.

Physicochemical analysis of extracted oil with different techniques

Quality of oil extracted with different solvents was also evaluated in terms of several physio-chemical tests (Table 3). Acid value, saponification value, peroxide value, Iodine value etc. increased till 12 h of extraction then the values decreased irrespective of solvents in solvent extraction method. Whereas antioxidant values were not significantly different from each other only petroleum ether extracted oil exhibited maximum antioxidant activity. On the contrary the trend of ultrasonically extracted flaxseed oils is different.

Table 3.

Physicochemical analysis of Soxhlet and Ultrasonically extracted oil

Extraction conditions	Acid value (mgKOH/g)	Saponification number (mgKOH/g)	Peroxide value (mEq/Kg)	Iodine value (g/100 g)	Refractive index	Antioxidant value (%)
Soxhlet extraction
n-hexane @ 8 h	1.37a ± 0.10	135.21a ± 2.01	1.6a ± 0.2	62.65a ± 2.01	1.584a ± 0.2	65.45a ± 2.36
n-hexane @12 h	1.41b ± 0.12	130.23b ± 3.21	1.8a ± 0.3	39.01b ± 1.25	1.582a ± 0.3	64.35a ± 2.54
n-hexane @16 h	1.38a ± 0.16	122.94c ± 3.15	1.7a ± 0.2	38.95b ± 1.96	1.584a ± 0.1	65.19a ± 2.18
Petroleum ether @ 8 h	1.35a ± 0.12	110.56d ± 2.26	1.7a ± 0.1	62.37a ± 2.15	1.583a ± 0.2	73.98b ± 3.57
Petroleum ether @ 12 h	1.32a ± 0.11	102.65d ± 2.25	1.6a ± 0.4	38.23b ± 1.24	1.584a ± 0.2	72.36b ± 3.12
Petroleum ether @ 16 h	1.10c ± 0.13	95.65e ± 3.42	1.7a ± 0.1	38.78b ± 1.85	1.585a ± 0.3	72.58b ± 3.54
Petroleum benzene @ 8 h	1.32a ± 0.15	105.63d ± 2.01	1.8a ± 0.5	61.70a ± 2.19	1.585a ± 0.1	69.31a ± 2.25
Petroleum benzene @ 12 h	1.31a ± 0.14	103.96d ± 2.35	1.7a ± 0.1	37.92b ± 1.25	1.583a ± 0.2	68.45a ± 2.54
Petroleum benzene @ 16 h	1.21d ± 0.16	101.38d ± 2.45	1.8a ± 0.1	38.09b ± 1.37	1.584a ± 0.1	67.87a ± 2.87
Ultrasound Assisted extraction
Amplitude(50) @ 30 min	1.58e ± 0.12	150.42e ± 2.25	1.6a ± 0.3	56.45c ± 2.54	1.582a ± 0.3	68.21a ± 1.89
Amplitude(50) @ 45 min	1.58e ± 0.14	134.97a ± 2.36	1.7a ± 0.2	53.87c ± 2.36	1.583a ± 0.2	68.31a ± 2.25
Amplitude(50) @ 60 min	1.45b ± 0.13	120.82c ± 2.54	1.7a ± 0.4	52.47c ± 1.15	1.581a ± 0.3	70.28b ± 2.37
Amplitude(60) @ 30 min	1.41b ± 0.12	105.10d ± 2.87	1.7a ± 0.5	61.85a ± 2.87	1.581a ± 0.1	77.91c ± 3.11
Amplitude(60) @ 45 min	1.34a ± 0.15	95.35e ± 3.54	1.6a ± 0.1	59.78c ± 1.25	1.577a ± 0.2	76.44c ± 2.87
Amplitude(60) @ 60 min	1.10 c ± 0.16	77.37f ± 2.21	1.8a ± 0.4	58.45c ± 2.54	1.583a ± 0.3	76.44c ± 2.45
Amplitude(70) @ 30 min	1.39b ± 0.13	90.28e ± 2.25	1.7a ± 0.4	60.25a ± 2.58	1.582a ± 0.2	73.51b ± 3.11
Amplitude(70) @ 45 min	1.34a ± 0.12	80.93f ± 3.25	1.6a ± 0.3	58.64c ± 2.47	1.581a ± 0.2	72.20b ± 2.18
Amplitude(70) @ 60 min	0.82 g ± 0.13	82.28f ± 2.47	1.8a ± 0.2	54.21c ± 2.66	1.583a ± 0.1	72.59b ± 2.85

Results are expressed as mean ± standard deviation (n = 4); Values with different alphabets a,b,c.. in a row are significantly different at p < 0.05

The acid value is an important factor signifying the storability of oils. Rancidity of oil occurs due to the presence of free fatty acids which are highly susceptible to oxidation. The amount of free fatty acid present in the oil gives an indication of age and quality of fat. The acid value of the oil extracted by ultrasonic extraction presented acid value ranging from 0.82 to 1.58 mg KOH/g and highest and lowest value was obtained at the ultrasonic intensity of 43 W for 30 min and 66 W for 60 min respectively. The results are in accordance with Popa et al. (2012) where acid value of linseed oil was found to be 0.80 mg KOH/g of oil.

There was a decrease in the saponification value with an increase in the extraction time at constant ultrasonic intensity as shown in Table 3. There is slight variation in the saponification value from that reported by Popa et al. (2012) who reported approximately 180 mg KOH/g oil saponification value for control flaxseed oil. The Saponification value gives an idea about the ester equivalents per unit mass of the oil. Longer the chain of fatty acid less is the value.

The refractive index of flaxseed oil was found to be 1.58 ± 0.3 for all samples no significant change of values with solvent or extraction time. The refractive index of oils could be helpful in judging the purity of oil (Kochhar 2002).

UAE gave the iodine value ranging between 152.47 g/100 g to 161.85 g/100 g. The lowest value was obtained at 43 W power for 60 min and the highest was obtained at 53 W for 30 min. The amount of iodine absorbed by oils is directly proportional to the degree of unsaturation.

Peroxide value is a very important parameter in determination of lipid quality. It determines the primary product of oxidative degradation of oil indicates as rancidity. Higher peroxide value shows a negative effect on the storage stability of the oil. Peroxide value gives the amount of oxygen bound in unsaturated fatty acid in mEq O₂/kg of oil. Peroxide value ranged from 1.6 mEq/kg to 1.82 mEq/kg for power 43 W for 30 min to 66 W for 60 min respectively. The results are in accordance with the findings reported by Popa et al. (2012).

Flaxseeds possess antioxidant activity due to the presence of several functional compounds having major health benefits of functional compounds due to their high antioxidant activities. It was observed in flaxseed oil that it contained a high amount of natural antioxidants which works as oxygen radical scavengers preventing the oil from auto degradation. The antioxidant activity of oil extracted with Ultrasound assisted method ranged from 68.21% to 77.91% (Table 3).

Flaxseed is mainly grown as an oil crop. Fatty acid profile of flaxseed oil is excellent in case of omega- 3 fatty acids. The fatty acid profile of oil was done by using Gas Chromatography-Mass spectro photometry (Fig. 3). Figure 3 illustrated the fatty acid profile chromatograms and mass spectra of fatty acid methyl esters obtained from oil extracted with ultrasonic assisted extraction. The major fatty acids found in extracted oil were linolenic acid, linoleic, palmitic, stearic and oleic acids (Table 4). Fumaric acid was absent in the oil extracted with ultrasound-assisted extraction method. The ratio of omega- 3 to omega- 6 fatty acids is an important property determining the quality and health benefits of any oil. It is considered as one of the best dietary source of omega- 3 fatty acids of the vegetarian region due to the highest content of linolenic acid i.e. omega- 3 fatty acid. Omega-6 fatty acid content of flaxseed was found to be comparatively less than other vegetable oils which were similar to findings of Gogus and Smith (2010). It has also been reported that the ratio of omega-6 to omega-3 fatty acids less than one is considered to posses several health benefits. They play a significant role in the anti-inflammatory properties of the cell membranes (Dunstan et al. 2007; Smith et al. 2010). As seen from the Table 4, this ratio being less than one justifies its potential for health benefits. The relationship between omega 3 and omega-6 fatty acids is approximately 4:1 in flaxseeds oil. In a similar study by Popa et al. (2012), it was concluded that saturates composed an average of 11.01% of the total fatty acids and of 88.97% unsaturated acid. Several other authors (Bayrak et al., 2010) have already reported that the major saturated fatty acid in flaxseed oil was palmitic (16:0) and stearic acid (C18:0) and the major unsaturated fatty acid in the flaxseed oils samples were linolenic (18:3), followed by oleic (18:1) and linoleic acid (18:2).

Fig. 3.

Gas Chromatogram of a Soxhlet extracted oil and b Ultrasound-assisted extracted oil

Table 4.

Fatty acid profile of Soxhlet extracted oil

S. No	Soxhlet extracted oil			Ultrasound assisted extracted oil
	Retention time (min)	Fatty acid	Area %	Retention time (min)	Fatty acid	Area %
1	14.76	Linolenic	62.37 ± 1.56	14.89	Linolenic	67.44 ± 1.74
2	14.62	Linoleic	3.04 ± 0.12	14.64	Linoleic	3.95 ± 0.16
3	12.54	Palmitic	3.15 ± 0.19	12.35	Palmitic	12.29 ± 0.38
4	15.14	Stearic	2.32 ± 0.09	15.19	Stearic	12.80 ± 0.24
5	20.55	Oleic	5.29 ± 0.24	10.99	Oleic	1.5 ± 0.09
6	19.30	Fumaric	10.32 ± 0.58

Surface morphology of whole and defatted flaxseeds

Whole flaxseed powder and defatted flaxseed powder were analyzed through SEM to reveal the difference in the surface morphology of flaxseed powder before and after the extraction process at magnifications 750 × and 1500x. A significant amount of oil was present on the surface in whole flaxseed powder as shown in Fig. 4. However, in Fig. 4 no oil is visible on the surface. The process of oil extraction has made a significant impact on the surface and microstructure of the sample. Images of defatted meal at different magnifications show a porous structure which is due to the effect of cavitations occurred during ultra-sonication for accelerating the oil extraction process. Figure  4 shows the flaxseed endosperm with outer covering revealing that surface structure of the flaxseeds was more uniform and continuous before extraction process which became ruptured and porous later after the extraction process was over. Zhang et al., (2008) have also reported the similar results that application of ultrasound during extraction of oil exposes a substantial effect on the microstructure due to an accelerated extraction process in comparison to conventional process as that structure was comparatively less porous and deformed. Arbelaiz et al. (2006) have also reported the similar outcomes in their study on mechanical properties of flaxseed fibres.

Fig. 4.

SEM of a whole flaxseed powder b defatted flaxseed powder Porous structure, non-uniform Oily layer on surface, unruptured surface

Quality of flaxseed oil enriched beverage

Fruit-based beverages are the most convenient carriers of bioactive compounds or other high-value nutrients. Due to an excellent lipid profile of flaxseed oil, the study was carried out to utilize mango based beverage as a vehicle for delivery of functional compounds like omega- 3 fatty acids and other antioxidant constituents. The most general technique that has been used in the past few years to deliver functional compounds in liquid foods is a high shear mixture and piston homogenizer. Low-frequency high-intensity ultrasound waves are now a day's gaining popularity to perform this function. Mechanical vibrations and intensive shear forces when applied on a liquid food or aqueous base containing lipid-based nutrients causes acoustic cavitations which form collisions (Bhaskaracharya et al. 2009; Chandrapala et al. 2012). In order to evaluate the effect of flaxseed oil in fruit-based beverage, in which the flaxseed oil was incorporated at different levels of 0.25%, 0.5%, and 0.75% (Fig. 5a).

Fig. 5.

a Flaxseed oil enriched functional mango beverages (0–0.75% oil), b Sensory analysis of flaxseed oil enriched functional beverages

Effect of processing variables

To optimize the processing conditions during ultrasonication process ultrasonic power and ultrasonic time were considered as variables. Ultrasonic power of 21 W, 31 W and 43 W were evaluated for the processing time of 3 min, 5 min, and 7 min. As shown in Table 5, beverage containing 0.25% oil processed at an ultrasonic power of 21 W for 3 min resulted in an unstable emulsion, oil phase and aqueous phase get separated within three hours at room temperature (CI is 0.9 min). Stable emulsion does not separate at least up to seven days at 4 °C was reported by Shanmugam and Ashokkumar, 2015. With increased processing time for 5 min at 21 W power get separated after 3^rd day whereas the emulsion processed for 7 min was stable up to 7 days. Ultrasonic intensity was very significant at 31 W and 43 W powers for 5 and 7 min was found to be stable for up to 18 days (CI values were 4.68 and 9.8 respectively). It can be concluded that the processing time 3 min was insufficient for the formation of stable emulsion (CI values were less than 1). The successful stable emulsions can be created with minimum ultrasonic power of 31 W for 5 min with a reasonable shelf life up to 18 days (CI of 4.6).

Table 5.

Effect of ultrasonication variables on emulsion stability of beverage

Variables	Emulsion stability	Creaming index (%)
Ultrasonic Power (21 W) for 3 min	Unstable	0.9
Ultrasonic Power (21 W) for 5 min	Emulsion breakdown at 3rd day	0.78
Ultrasonic Power (21 W) for 7 min	Emulsion breakdown at 7th day	0.98
Ultrasonic Power (31 W) for 3 min	Emulsion breakdown at 7th day	0.67
Ultrasonic Power (31 W) for 5 min	No emulsion breakdown till 18th day	4.6
Ultrasonic Power (31 W) for 7 min	No emulsion breakdown till 18th day	6.8
Ultrasonic Power (43 W) for 3 min	Emulsion breakdown at 8th day	0.78
Ultrasonic Power (43 W) for 5 min	No emulsion breakdown till 18th day	7.6
Ultrasonic Power (43 W) for 7 min	No emulsion breakdown till 18th day	9.8

Physicochemical analysis of beverage

The visual appearance of such cloudy drinks is an important parameter for better consumer acceptance. Generally, emulsions are made stable using stabilizers like gums as hydrophobically modified starches. Thus, measurement of turbidity reflects the homogenization of ultrasound processed oil in juice emulsion. But there was no significant difference in the turbidity values with increasing oil content from 0 to 0.75%. It has been reported that particle size is the most important factor which affects the turbidity of the emulsion. The relative turbidity of the beverage ranged between 101.19 and 103.31. In a similar study, Shanmugam and Ashok kumar (2015) found that the size reduction in case of cloud particle was comparatively lower in comparison to sono-emulsification of flaxseed oil, thus size reduction in mango based beverage didn’t considerably altered the turbidity and cloudy behavior of the drink.

The major pigment present in flaxseed oil is ß-carotene. Thus, the concentration of carotenoids in the prepared beverage was measured in terms of ß-carotene. The Carotenoids content significantly increased to 292.24 µg/mL at 0.75% added oil from 151.37 µg/mL in control. Carotenoids plays very important role in enhancing quality of food product for its photosensitizers singlet oxygen quencher’s function. Confirmation of the presence of carotenoids was done from the typical peaks in carotenoid finger print region (400–500 nm) during UV visible spectrophotometric analysis. ß-carotene is the most effective pro-vitamin A, which prevents night blindness. Carotenoids also act as pro-oxidants because of their instability in heat and light. Shanmugam and Ashokkumar (2015) found that the amount of β-carotene enhanced by 1.5 mg/100 mL of carrot juice (19%) irrespective of sonication time. According to Abid et al. (2014) such increase is attributed to the mechanical disruption occurred during the ultrasonic processing of cell wall, which increase the release of free carotenoids in the juice. However in present case, this increase was due to the combined effect of elevation of level of flaxseed oil incorporation and carotene release caused during sonication process.

The antioxidant activity was measured in terms of DPPH radical scavenging activity. As shown in Table 6, there was a sharp increase in the antioxidant activity from 26.99 to 61.43% at 0% to 0.75% of added flaxseed oil in the beverage. The antioxidant activity of the control was due to the presence of radical scavengers in mango pulp. Flaxseed is also a rich source of polyphenols containing 10–100 times more than most of the other edible plants. Other phenolic acids such as P-coumaric acid and vanillin are also present in minor quantities in flaxseed. These compounds have multiple modes of action to act as antioxidants by inhibiting the formation of free radicals, chelating the transitional metal ions and modulation of the activity of endogenious pre-oxidative enzymes.

Table 6.

Physiochemical analysis of flaxseed oil enriched functional beverage

Properties	0%	0.25%	0.50%	0.75%
Turbidity	100.02a ± 1.55	103.31a ± 1.41	100.29a ± 1.07	101.19a ± 1.78
Antioxidant activity (%)	26.99d ± 0.78	44.62c ± 0.49	52.63b ± 0.62	61.43a ± 0.61
Carotenoid content (µg/mL)	151.37d ± 2.51	251.52c ± 2.83	280.13b ± 3.41	292.24a ± 3.02
Particle size (nm)	250 nm	150-230 nm	600-640 nm	787-800 nm
pH	4.8a ± 0.1	4.7a ± 0.2	4.6a ± 0.3	4.6a ± 0.2
Acidity (%)	0.69 a ± 0.08	0.71 a ± 0.07	0.73 a ± 0.06	0.76 a ± 0.07
Color
L*	31.16c ± 0.45	40.02b ± 0.39	41.71a ± 0.47	41.12a ± 0.38
a*	0.90c ± 0.52	4.51b ± 0.41	5.74a ± 0.36	6.01a ± 0.47
b*	33.49d ± 0.58	44.74c ± 0.47	48.77b ± 0.38	50.11a ± 0.41

Results are expressed as mean ± standard deviation (n = 4); Values with different alphabets a,b,c.. in a row are significantly different at p < 0.05

When dealing with fruit-based beverages it is very important to evaluate the pH and acidity. pH was 4.8 for the control sample with no added flaxseed oil which decreased to 4.7 at 0.25% added oil, 4.6 at 0.5% added oil and 4.5 at 0.75% of the added oil in the beverage. Moreover, in resemblance to pH measurement, there was slight variation in the titratable acidity of the beverage as given in the Table 6. The titratable acidity for the control sample was measured as 0.69% which increased to 0.71% at an addition of 0.25% oil, 0.73% in beverage containing 0.5% oil and ultimately reached 0.76% in beverage containing 0.75% oil. The variation in the pH and acidity with the effect of oil incorporation was very slight and insignificant (p > 0.05) in fresh beverages as well as after storage for 18 days at (4±2) °C (Table  6).

The estimation of the color of prepared beverage was done in terms of L*, a*, and b* value. L* value signifying the lightness or darkness of the sample ranged from 31.16 to 41.12 at different levels of added flaxseed oil in the beverage. The a* value illustrating the redness of the product was found to vary between 0.90 and 6.00. Higher is the positive b* value more is the yellowish color b* value was found between 33.49 and 50.11. It was observed that with increase in the level of flaxseed oil incorporation, the color of the drink became lighter. This effect occurred after sonication process. The color of the sample containing 0.75% level of flaxseeds was light yellow. The brightest yellow color was seen in control sample (Fig. 5a).

The highest hedonic score for appearance, color, aroma, and overall acceptability were given to the control beverage and the order is 0.25% > 0.5% > 0.75% for oil containing beverage. Addition of 0.25% oil had a low significant effect on taste of the beverage but had poor hedonic score for beverage containing 0.75% oil because of typical flaxseed aftertaste in the beverage. The beverage containing 0.50% oil was considered good. From overall acceptability response, it was concluded that 0.25% and 0.50% oil levels could be considered for development of such functional beverages. The overall acceptability was highest in control (8.6) followed by 0.25% oil incorporated beverage (7.5) and the lowest score was obtained in beverage containing 0.75% oil. There was a linear decrease in the hedonic scores of other parameters with an increase in flaxseed oil level (Fig. 5b).

Conclusion

The optimum conditions found during the investigation of extraction of flaxseed oil were an ultrasonic power of 53 W for 45 min using petroleum ether. Fatty acid profile revealed that flaxseed AL 2063 oil has balanced ratio of omega 6 to omega 3 fatty acids. Linolenic acid and linoleic acid were major polyunsaturated fatty acids found in extracted oil. Though rise in the concentration of flaxseed oil in the mango beverage resulted in better bioactive profile but, addition of oil above 0.5% resulted in decline in the organoleptic scores. Thus, incorporation of 0.5% oil extracted using ultrasonic-assisted extraction method in ready to serve beverage was most acceptable.

Declaration

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Authors' contribution

Mr. Sukhwant did experiment and wrote the ME desertation, Mr. Rajan help him to execute and write and Dr. Gargi supervised the work and edited the thesis and manuscript.

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