Passion fruit seed oil: extraction and subsequent transesterification reaction

2.1. Materials

In the present work, ethanol (99.75%, JT Baker) and n-hexano (98.5%, Anidrol) were used as solvents and the oil was obtained from the seeds of passion fruit (purchased in the local market of Umuarama, PR). Methyl heptadecanoate (> 99%, Sigma-Aldrich), 5α-cholestane (> 99%, Sigma-Aldrich), a FAME (fatty acid methyl esters) mixture (Supelco), heptane and the derivatization reagent Boron trifluoride-methanol solution (14%, Sigma Aldrich) were used for the characterization of the oil, and N,O bis (trimethylsilyl) trifluoroacetamide (1% TMCS, Fluka). Novozym® 435 lipase (Candida Antarctica immobilized), heptane (Anidrol) and methyl heptadecanoate (Sigma-Aldrich, 99.9% purity) were used to determine the ethyl ester contents in the samples, and N-Methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) was used to determine the mono-, di- and triglyceride contents.

2.2. Preparation of the raw material

The fruits were cut in half in the horizontal direction, and their seeds were removed manually. The seeds were then washed under running water, the surface water was removed, and the seeds were put in the oven (Marconi, MA 035) at 70 ºC to obtain samples with a moisture content of 2.34 ± 0.69%. After drying, the aryl was removed completely and the material was triturated and passed through granulometric grading to separate the 0.638-mm fraction for the experiments.

2.3. Oil extraction

UAE was conducted using ethanol as a solvent. In each experimental run, the flask containing the passion fruit seeds and ethanol was placed in the center of an ultrasound bath (Ultronique, Q 5.9/40A), previously heated to the experimental temperature, and ultrasonic irradiation was immediately initiated (power of 165 W and frequency of 25 kHz). To avoid the loss in solvent, the flask was connected to the condenser which was held at 10 °C.

In order to determine the effect of the process variables and the experimental conditions that maximize the removal of the oil from the seeds, the following levels for each variable were evaluated: (a) times (X₁) of 10, 30, and 50 min; (b) temperatures (X₂) of 30, 45, and 60 ºC; and solvent-to- seed ratios (X₃) of 2, 3, and 4 (mL·g^-1), which were combined by means of a Box-Behnken design. The experimental range for process variables was defined based on previous studies: time (Pereira et al., 2017Pereira MG, Hamerski F, Andrade EF, Scheer AP, Corazza ML. 2017. Assessment of subcritical propane, ultrasound-assisted and Soxhlet extraction of oil from sweet passion fruit (Passiflora alata Curtis) seeds. J. Supercrit. Fluids 128, 338-348. https://doi.org/10.1016/j.supflu.2017.03.021 ), temperature (Rodrigues et al., 2017Rodrigues GM, Mello BTF, Garcia VAS, Silva C. 2017. Ultrasound-assisted extraction of oil from macauba pulp using alcoholic solventes. J. Food Process Eng. 40, 1-8. https://doi.org/10.1111/jfpe.12530 ; Rosa et al., 2019Rosa ACS, Stevanato N, Iwassa I, Garcia VAS, Silva C. 2019. Obtaining oil from macauba kernels by ultrasound-assisted extraction using ethyl acetate as the solvent. Braz. J. Food Technol. 22, e2018195. https://doi.org/10.1590/1981-6723.19518 ; Xu et al., 2016Xu G, Liang C, Huang P, Liu Q, Xu Y, Ding C, Li T. 2016. Optimization of rice lipid production from ultrasound-assisted extraction by response surface methodology. J. Cereal Sci. 70, 23-28. https://doi.org/10.1016/j.jcs.2016.05.007 ) and solvent-to-seed ratios (Oliveira et al. 2014Oliveira RC, Guedes TA, Gimenes ML, Barros STD. 2014. Effect of process variables on the oil extraction from passion fruit seeds by conventional and non-conventional techniques. Acta Sci. Technol. 36, 87-91. https://doi.org/10.4025/actascitechnol.v36i1.15217 ).

Analysis of variance was carried out to evaluate the effect of the variables on the oil yield with a 95% confidence interval using Statistica 8.0 software (StatSoft^TM, Inc.). In addition, the experimental data were fitted to a second-order polynomial model, which was used to determine the condition of maximum oil extraction. In this condition, the effect of ultrasound on the removal of oil from passion fruit seeds was evaluated, with extractions performed without ultrasound in an orbital shaker (Marconi, MA 839/A) at 40 rpm as described above.

For comparative purposes, the traditional extraction was performed in a Soxhlet extractor using the n-hexane a at a sample ratio of 30 (mL·g^-1) for 480 min.

For each extraction method used, after the duration of the experimental run, the seeds were separated and the solvent was removed until constant weight was achieved, thereby obtaining the oil. The oil yield was determined as the ratio between the mass of oil extracted and the mass of seeds used.

2.4. Oil characterization

The methodology reported by Gonzalez et al., (2013)Gonzalez SL, Sychoski MM, Navarro-Díaz HJ, Callejas N, Saibene M, Vieitez I, Jachmanián I, Silva C, Hense H, Oliveira JV. 2013. Continuous catalyst-free production of biodiesel through transesterification of soybean fried oil in supercritical methanol and ethanol. Energ. Fuel. 27, 5253-5259. https://doi.org/10.1021/ef400869y was used in the preparation of the samples to determine the fatty acid composition. In this methodology, the samples were derivatized with BF3-methanol and subsequently methyl heptadecanoate diluted in heptane was added to quantify the compounds. The analysis was performed in a gas chromatograph (GC-MS QP2010 SE, Shimadzu) equipped with Rtx - Wax (Shimadzu, 30 m x 0.32 mm id x 0.25 µm) capillary column and flame ionization detector, using the chromatographic conditions reported by Stevanato and Silva (2019)Stevanato N, Silva C. 2019. Radish seed oil: Ultrasound-assisted extraction using ethanol as solvent and assessment of its potential for ester production. Ind. Crop. Prod. 132, 283-291. https://doi.org/10.1016/j.indcrop.2019.02.032 . The components present in the samples were identified through comparison with authentic standards of fatty acid methyl esters.

In the determination of phytosterol and tocopherol contents, 5α-cholestane as internal standard and thereafter 40 µL of N,O-bis (trimethylsilyl) trifluoroacetamide were added to 40 mg of sample. The samples were maintained for 60 min at 60 °C and after dilution in heptane to obtain a volume of 1000 µL of solution, they were analyzed on an GC-MS QP2010 SE (Shimadzu) with SH-RTx-5MS column (Shimadzu, 5% phenyl-methylsiloxane, 30 m × 0.25 mm id x 0.25 μm). The column temperature was initially programmed at 50 °C, increasing to 230 °C at a rate of 10 °C·min^-1 and then to 280 °C at a rate of 15 °C·min^-1, which was held for 22 min. Helium was used as the carrier gas at a 1.0 mL·min^-1 flow rate with split ratio of 10 and the amount of injected sample was 1 μL. Mass spectra were recorded at 70 eV with mass range from m/z 50 to 550 amu. Compounds were identified through the comparison of spectrum data to those presented in the NIST14.lb and NIST14.lbs spectral libraries.

2.5. Sequential reaction

The UAE product (oil + ethanol) was added again in an Erlenmeyer flask and received the enzymatic catalyst in the proportion of 40% (relative to oil mass) as defined by Stevanato and Silva (2019)Stevanato N, Silva C. 2019. Radish seed oil: Ultrasound-assisted extraction using ethanol as solvent and assessment of its potential for ester production. Ind. Crop. Prod. 132, 283-291. https://doi.org/10.1016/j.indcrop.2019.02.032 . The enzyme was activated for 1 h at 40 °C prior to use. The flask was incubated in orbital agitation (Marconi MA 830/A) at 150 rpm by applying reaction times of 12 and 24 h. The experiments were carried out under the temperature of maximum oil yield determined for the UAE (60 ºC). At the end of each reaction, the enzymes and seeds were separated by filtration and the excess ethanol in the samples was evaporated until reaching constant weight.

2.6. Characterization of the reaction product

The analysis of the chemical composition of the reaction product was carried out in a gas chromatograph (Shimadzu, GC-2010 Plus) equipped with a flame ionization detector and capillary columns SH-Rtx-Wax™ (Shimadzu, 30 m × 0.32 mm × 0.25 μm) and ZB-5HT inferno™ (Zebron, 10 m × 0.32 mm × 0.10 μm) to determine the content of methyl esters and acylglycerols, respectively. The chromatographic conditions for both analyses were previously established (Stevanato and Silva, 2019Stevanato N, Silva C. 2019. Radish seed oil: Ultrasound-assisted extraction using ethanol as solvent and assessment of its potential for ester production. Ind. Crop. Prod. 132, 283-291. https://doi.org/10.1016/j.indcrop.2019.02.032 ).

To quantify the content ethyl esters, the injected sample was prepared with 100 µL of a solution of oil in heptane (C = 30 mg·mL^-1), 80 µL of internal standard (methyl heptadecanoate, C = 12.5 mg·mL^-1) and 820 µL of heptane. For simultaneous determination of the contents of glycerol, mono-, di- and triglycerides, MSTFA was used as a silylating agent. Glycerol, monolein, diolein and triolein were used as external standard to construct the calibration curve (R² > 0.99).

The free fatty acid (FFA) content in the reaction product was analyzed in a gas chromatograph (Shimadzu, GC-2010 Plus) and the samples were prepared and analyzed according to the methodology for determining phytosterols and tocopherols as described in section 2.4, using methyl heptadecanoate as an internal standard.

2.7. Analysis of data

The data were subjected to ANOVA using Excel® 2010 software and the Tukey test (p=0.05) to evaluate differences between the results. The experiments and analyses were performed in duplicate, so four observations were used to calculate each mean value.

3.1. Ultrasound-assisted extraction

3.1.1. Effect of experimental variables

 ⌅

The experimental results for the oil yield obtained from passion fruit seeds in each experimental run of the experimental design are presented in Table 1. Table 2 shows the effect of the independent variables on oil yield, considering the linear, quadratic, and interaction effects among the variables.

Equation 1 shows the correlation between oil yield and the experimental variables obtained from regression analysis data (Table 2). From the F-test, the validation of the predictive equation was verified, with values of 101.15 and 3.73 for F_CAL and F_TAB, respectively. Thus, the equation was capable of representing the experimental data for the range of variables investigated, because F_CALC > F_TAB (calculated from the ANOVA table and tabulated, respectively).

 (1)

From the data presented in Table 1, it can be verified that high yields (20.82 and 21.12%) were obtained at the highest level (60 ºC) of the temperature variables (runs 4 and 12), which corroborates the effect of this variable presented in Table 2, which indicates that in the experimental range evaluated, the oil removal from passion fruit seeds was favored by the increase in temperature.

Table 1.  Experimental conditions applied and oil yield obtained in experiments to assess the effects of the operating variables using a Box-Behnken design.

Run	X1 1	X2 2	X3 3	Oil yield (%)
1	10	30	1:3	12.69
2	50	30	1:3	16.39
3	10	60	1:3	18.79
4	50	60	1:3	20.82
5	10	45	1:2	12.75
6	50	45	1:2	16.06
7	10	45	1:4	16.16
8	50	45	1:4	20.09
9	30	30	1:2	12.32
10	30	60	1:2	17.48
11	30	30	1:4	17.37
12	30	60	1:4	21.25
13-16	30	45	1:3	18.064 ±0.08

¹ X₁: time (min); ²X₂: temperature (ºC); ³X₃: solvent to seed ratio (mL·g^-1); ⁴Average of four experiments.

Table 2.  Model coefficients and p-value of the model for the extraction of passion fruit seed oil by UAE.

Variables	Effect a	p-value b	Coefficient c
Mean/Interaction	16.85	< 0.0001	18.06
X1 (L)	3.24	0.0003	1.62
X1 (Q)	0.86	0.0026	-0.86
X2 (L)	4.89	0.0001	2.44
X2 (Q)	0.02	0.6470	-0.02
X3 (L)	4.06	0.0002	2.03
X3 (Q)	0.93	0.0023	-0.93
X1 x X2	-0.83	0.0103	-0.42
X1 x X3	0.31	0.0685	0.15
X2 x X3	-0.64	0.0175	-0.32

^aEffect of the independent variable on the dependent variable; ^b statistical significance p < 0.05; ^c coefficients of second-order polynomial model (Equations 1 and 2).

Baümler et al., (2016)Baümler ER, Carrin ME, Carelli AA. 2016. Extraction of sunflower oil using ethanol as solvent. J. Food. Eng. 178, 190-197. https://doi.org/10.1016/j.jfoodeng.2016.01.020 determined the equilibrium constant (K) of the oil extraction of sunflower collets and reported a decrease in this parameter with increasing temperature and a greater oil extraction capacity. According to Stamenković et al., (2018Stamenković OS, Djalović IG, Kostić MD, Mitrović PM, Veljković VB. 2018. Optimization and kinetic modeling of oil extraction from white mustard (Sinapis alba L.) seeds. Ind. Crop. Prod. 121, 132-141. https://doi.org/10.1016/j.indcrop.2018.05.001 ) and Zhong et al., (2018)Zhong J, Wang Y, Yang R, Liu X, Yang Q, Qin X. 2018. The application of ultrasound and microwave to increase oil extraction from Moringa oleifera seeds. Ind. Crop. Prod. 120, 1-10. https://doi.org/10.1016/j.indcrop.2018.04.028 the increase in temperature may cause increased solubility of the oil in the solvent and diffusivity of the oil from the sample to the solvent, since the properties of ethanol are drastically affected by the increase in temperature. According to Granero et al., (2014), the increase in temperature from 20 to 50 °C results in a reduction in the density and dynamic viscosity of ethanol from 789.48 to 763.22 kg·m^-3 and 1.24 to 0.72 mPa s, respectively. Pereira et al., (2017)Pereira MG, Hamerski F, Andrade EF, Scheer AP, Corazza ML. 2017. Assessment of subcritical propane, ultrasound-assisted and Soxhlet extraction of oil from sweet passion fruit (Passiflora alata Curtis) seeds. J. Supercrit. Fluids 128, 338-348. https://doi.org/10.1016/j.supflu.2017.03.021 reported higher solubility of passion fruit oil in ethanol with increasing temperature and obtained better oil extraction at 60 ºC.

The increase in oil yield with the increase in the solvent to seed ratio is due to the higher dissolution capacity, which leads to changes in the thermodynamic and mass transfer properties of the extraction process. Stamenković et al., (2018)Stamenković OS, Djalović IG, Kostić MD, Mitrović PM, Veljković VB. 2018. Optimization and kinetic modeling of oil extraction from white mustard (Sinapis alba L.) seeds. Ind. Crop. Prod. 121, 132-141. https://doi.org/10.1016/j.indcrop.2018.05.001 performed thermodynamic analyses of oil extraction of white mustard (Sinapis alba L.) seeds, respectively, and found that the spontaneity of the process was favored by increases in the volume of solvent used. Meziane and Kadi (2008)Meziane S, Kadi H. 2008. Kinetics and Thermodynamics of Oil Extraction from Olive Cake. J. Am. Oil Chem.’Soc. 85, 391-396. https://doi.org/10.1007/s11746-008-1205-2 and Toda et al., (2016)Toda TA, Sawada MM, Rodrigues CEC. 2016. Kinetics of soybean oil extraction using ethanol as solvent: Experimental data and modeling. Food Bioprod. Process. 98, 1-10. https://doi.org/10.1016/j.fbp.2015.12.003 reported higher values for the mass transfer coefficients with the use of higher amounts of solvent.

In addition, during ultrasonic treatment, the collapse between the cavitation bubbles generated high-speed solvent microjets that caused damage to the plant matrix cell wall, increasing the contact area between the matrix and solvent and, consequently, increasing the efficiency of oil extraction (Vinatoru et al., 2017Vinatoru M, Mason TJ, Calinescu I. 2017. Ultrasonically assisted extraction (UAE) and microwave assisted extraction (MAE) of functional compounds from plant materials. Trac-Trend Anal. Chem. 97, 159-178. https://doi.org/10.1016/j.trac.2017.09.002 ). Other authors have reported an effect similar to that observed in this study for the experimental interval evaluated; for example, Mabayo et al., (2018)Mabayo VIF, Aranas JRC, Cagas VJB, Cagas DPA, Ido AL, Arazo RO. 2018. Optimization of oil yield from Hevea brasiliensis seeds through ultrasonic-assisted solvent extraction via response surface methodology. Sustain. Environ. Res. 28, 39-46. https://doi.org/10.1016/j.serj.2017.08.001 reported that an increase in the ratio of n-hexane to rubber seeds from 2 to 4 (mL·g^-1) led to an increase in the oil yield from 21.3 to 30.7%, respectively.

On analyzing the interaction between the variables solvent-to-seed ratio and time, it was verified that they had no effect on oil yield. This can be explained on the basis of the results of Meziane and Kadi (2008)Meziane S, Kadi H. 2008. Kinetics and Thermodynamics of Oil Extraction from Olive Cake. J. Am. Oil Chem.’Soc. 85, 391-396. https://doi.org/10.1007/s11746-008-1205-2 , who determined the mass transfer coefficients for the extraction stages characterized by washing and diffusion, respectively, using a different solvent- to-sample ratio and obtaining higher mass transfer coefficients with increasing amounts of solvent used, with higher coefficients in the initial stage.

Extraction occurs in two phases: the first corresponds to rapid extraction, with the removal of the surface constituents of the particle, followed by a slower second phase called diffusion, in which the inner components of the matrix are removed (Chanioti and Tzia, 2018Chanioti S, Tzia C. 2018. Processing parameters on the extraction of olive pomace oiland its bioactive compounds: a kinetic and thermodynamic study. J. Am. Oil Chem. Soc. 95, 371-382. https://doi.org/10.1002/aocs.12021 ; Stamenković et al., 2018Stamenković OS, Djalović IG, Kostić MD, Mitrović PM, Veljković VB. 2018. Optimization and kinetic modeling of oil extraction from white mustard (Sinapis alba L.) seeds. Ind. Crop. Prod. 121, 132-141. https://doi.org/10.1016/j.indcrop.2018.05.001 ; Toda et al., 2016Toda TA, Sawada MM, Rodrigues CEC. 2016. Kinetics of soybean oil extraction using ethanol as solvent: Experimental data and modeling. Food Bioprod. Process. 98, 1-10. https://doi.org/10.1016/j.fbp.2015.12.003 ). In the initial stage, there is a greater extraction of the oil. This effect is observed in the comparison of the results presented in Table 1 for runs 5-6 and 7-8, for example, where it is possible to observe the removal of ~80% of the oil obtained in 50 min (runs 6 and 8) in an extraction time of only 10 min (runs 5 and 7). However, the use of longer extraction times contributes to further rupture of the cell walls, resulting in greater penetration of the solvent (Balachandran et al., 2006Balachandran S, Kentish S, Mawson R, Ashokkumar, M. 2006. Ultrasonic enhancement of the supercritical extraction from ginger. Ultrason. Sonochem. 13, 471-479. https://doi.org/10.1016/j.ultsonch.2005.11.006 ; Kazemi et al., 2016Kazemi M, Karim R, Mirhosseini H, Hamid AA. 2016. Optimization of pulsed ultrasonic-assisted technique for extraction of phenolics from pomegranate peel of Malas variety: punicalagin and hydroxybenzoic acids. Food Chem. 206, 156-166. https://doi.org/10.1016/j.foodchem.2016.03.017 ) and consequently an increase in oil yield (p < 0.05).

Other studies report the best removal of the oil by subjecting the sample to a longer ultrasound treatment time, among which we can mention the works of Mello et al., (2015)Mello BTF, Garcia VAS, Silva C. 2015. Ultrasound-Assisted Extraction of Oil from Chia (Salvia hispânica L.) Seeds: Optimization Extraction and Fatty Acid Profile. J. Food Process Eng. 40, 1-8. https://doi.org/10.1111/jfpe.12298 and Rodrigues et al., (2017)Rodrigues GM, Mello BTF, Garcia VAS, Silva C. 2017. Ultrasound-assisted extraction of oil from macauba pulp using alcoholic solventes. J. Food Process Eng. 40, 1-8. https://doi.org/10.1111/jfpe.12530 , who obtained higher oil yields with increases in the time of treatment with ultrasound from 20 to 60 min.

The effect of time is more pronounced at lower temperatures, as can be seen in the comparison of the data from runs 1-2 and 3-4, since the equilibrium is reached more rapidly with the rise in temperature.

3.2. Effect of ultrasound on extraction

Extraction performed without the use of ultrasound, provided an oil yield of 15.82 ± 0.8% under the conditions of maximum oil yield obtained by Equation 1, which represents 75% of the yield obtained with ultrasound. The advantages and characteristics of ultrasound have already been discussed and justify the result obtained.

3.3. Comparison between UAE and classical extraction

Table 3 shows the results of the comparison between traditional extraction and UAE in terms of oil yield, fatty acid composition, and phytosterol and tocopherol contents.

In the traditional extraction (in Soxhlet), the passion fruit seeds used in this work presented oil yields of 29.02 ± 1.61%. This result is close to that reported in the works of Oliveira et al., (2013)Oliveira RC, Barros STD, Gimenes ML. 2013. The extraction of passion fruit oil with green solvents. J. Food Eng. 117, 458-463. https://doi.org/10.1016/j.jfoodeng.2012.12.004 , Pereira et al., (2017)Pereira MG, Hamerski F, Andrade EF, Scheer AP, Corazza ML. 2017. Assessment of subcritical propane, ultrasound-assisted and Soxhlet extraction of oil from sweet passion fruit (Passiflora alata Curtis) seeds. J. Supercrit. Fluids 128, 338-348. https://doi.org/10.1016/j.supflu.2017.03.021 and Santana et al., (2017)Santana FC, Torres LRO, Shinagawa FB, Silva AMO, Yoshime LT, Melo ILP, Marcellini PS, Mancini-Filho J. 2017. Optimization of the antioxidant polyphenolic compounds extraction of yellow passion fruit seeds (Passiflora edulis Sims) by response surface methodology. Int. J. Food Sci. Tech. 54, 3552-3561. https://doi.org/10.1007/s13197-017-2813-3 , who presented oil yields of 26.4, 28.33, and 28.87%, respectively.

UAE provided 73.7% of the yield obtained from traditional extraction; however, with this method the oil is obtained in a shorter time and mainly using a smaller volume of solvent, since a solvent-to-seed ratio of 4 (mL·g^-1) is required, while in traditional extraction a solvent-to-seed ratio of 30 (mL·g^-1) is used. In UAE, the cavitation of the medium favors mass transfer and thus a smaller volume of solvent is necessary; whereas in the classic method the extraction process is governed by mass diffusion, requiring a high consumption of solvent (Menezes et al., 2018Menezes ML, Johann G, Diório A, Pereira NC, Silva EA. 2018. Phenomenological determination of mass transfer parameters of oil extraction from grape biomass waste. J. Clean. Prod. 176, 130-139. https://doi.org/10.1016/j.jclepro.2017.12.128 ).

The results presented in Table 3 show that there is no difference between the fatty acid compositions of the oils obtained with the techniques used. Passion fruit seed oil contains a high concentration of PUFAs (59.28 to 61.19), with linoleic acid forming the major part (59.13 to 61.07%). Another acid that stands out is oleic acid (17.45%). Other studies have also reported that these were the main fatty acids found in passion fruit seeds (Pereira et al., 2018Pereira MG, Maciel GM, Haminiuk CWI, Bach F, Harmeski F, Scheer AP, Corazza ML. 2018. Effect of Extraction Process on Composition, Antioxidant and Antibacterial Activity of Oil from Yellow Passion Fruit (Passiflora edulis Var. Flavicarpa) Seeds. Waste Biomass Valori. 1-15. https://doi.org/10.1007/s12649-018-0269-y ; Silva and Jorge, 2017Silva AC, Jorge N. 2017. Bioactive compounds of oils extracted from fruits seeds obtained from agroindustrial waste. Eur. J. Lipid. Sci. Tech. 119, 1-5. https://doi.org/10.1002/ejlt.201600024 ).

As can be seen in Table 3, stigmasterol and δ-tocopherol are the predominant phytosterols and tocopherols, respectively, in the composition of passion fruit seed oil.

In the extraction of tocopherols, the evaluated methods provided similar levels of these compounds. The values obtained (33.94 to 44.64 mg per 100 g of oil) were higher than those reported by Pereira et al., (2017)Pereira MG, Hamerski F, Andrade EF, Scheer AP, Corazza ML. 2017. Assessment of subcritical propane, ultrasound-assisted and Soxhlet extraction of oil from sweet passion fruit (Passiflora alata Curtis) seeds. J. Supercrit. Fluids 128, 338-348. https://doi.org/10.1016/j.supflu.2017.03.021 in the extraction using pressurized propane (9.87 to 11.14 mg per 100 g of oil) and UAE with ethanol (10.10 mg per 100 g of oil). However, they were lower than the value of 49.9 mg per 100 g of oil as reported by Malacrida and Jorge (2012)Malacrida CR, Jorge N. 2012. Yellow passion fruit seed oil (Passiflora edulis f. flavicarpa): physical and chemical characteristics. Braz. Arch. Biol. Technol. 55, 127-134. https://doi.org/10.1590/S1516-89132012000100016 with the traditional extraction using petroleum ether.

The UAE provided an extract with a higher content in phytosterols of 166.64 mg per 100 g of oil. The content obtained in this study was lower than that obtained by Piombo et al., (2006)Piombo G, Barouh N, Barea B, Boulanger R, Brat P, Pina M, Villeneuve P. 2006. Characterization of the seed oils from kiwi (Actinidia chinensis), passion fruit (Passiflora edulis) and guava (Psidium guajava). OCL 13, 195-199. https://doi.org/10.1051/ocl.2006.0026 and Silva and Jorge (2017)Silva AC, Jorge N. 2017. Bioactive compounds of oils extracted from fruits seeds obtained from agroindustrial waste. Eur. J. Lipid. Sci. Tech. 119, 1-5. https://doi.org/10.1002/ejlt.201600024 , who reported values of 209.74 and 274.67 mg per 100 g of oil, respectively.

The differences found in relation to the literature are related to the nature of the raw material used, as well as the solvent extractor and the efficiency of the extraction method used.

3.4. Sequential reaction

Table 4 shows the results of the chemical composition of the samples obtained after conducting the reaction step in 12 and 24 h. The analysis of the data must be carried out by observing which compound is of most interest in the product, which must be directed to the separation step aiming at the concentration of the target compound. It should be noted that in addition to the compounds identified (Table 4), these samples contain phospholipids, waxes and sugars (Baümler et al., 2016Baümler ER, Carrin ME, Carelli AA. 2016. Extraction of sunflower oil using ethanol as solvent. J. Food. Eng. 178, 190-197. https://doi.org/10.1016/j.jfoodeng.2016.01.020 ), compounds identified in vegetable oils obtained using ethanol as a solvent.

From the data presented in Table 4, it appears that in 12 h of reaction the product obtained contains ethyl esters, diglycerides (DG) and monoglycerides (MG) in higher concentrations. The high triglyceride conversion in the reaction transesterification products after 24 h of reaction was observed, obtaining a sample with a predominance in ethyl esters and MG under these conditions.

Ethyl esters can be directed to use as biofuels (Gonzalez et al., 2013Gonzalez SL, Sychoski MM, Navarro-Díaz HJ, Callejas N, Saibene M, Vieitez I, Jachmanián I, Silva C, Hense H, Oliveira JV. 2013. Continuous catalyst-free production of biodiesel through transesterification of soybean fried oil in supercritical methanol and ethanol. Energ. Fuel. 27, 5253-5259. https://doi.org/10.1021/ef400869y ; Stevanato and Silva, 2019Stevanato N, Silva C. 2019. Radish seed oil: Ultrasound-assisted extraction using ethanol as solvent and assessment of its potential for ester production. Ind. Crop. Prod. 132, 283-291. https://doi.org/10.1016/j.indcrop.2019.02.032 ), in compliance with current legislation. Bitonto et al., (2019)Bitonto Luigi, Menegatti S, Pastore C. 2019. Process intensification for the production of the ethyl esters of volatile fatty acids using aluminium chloride hexahydrate as a catalyst. J. Clean. Prod. 239, 118122. https://doi.org/10.1016/j.jclepro.2019.118122 reported that these compounds are considered non-hazardous organic compounds, in addition to biofuels, and have industrial applications such as solvent, fragrances and cosmetics.

MG and DG have applications as emulsifiers in the food and pharmaceutical industry (Hartel et al., 2018Hartel RW, von Elbe JH, Hofberger R. 2018. Fats, oils and emulsifiers. In: Confectionery Science and Technology, Springer, Cham., pp.85-124. https://doi.org/10.1007/978-3-319-61742-8_4 ). These emulsifiers are frequently used in bakery products, frozen desserts, and sauces/dressings (Nicholson et al., 2019Nicholson RA, Marangoni AG. 2019. Diglycerides. In: Melton L, Shahidi F, Varelis P, Encyclopedia of Food Chemistry 1, 70-73.). According to Ferreti et al., (2018)Ferretti CA, Spotti ML, Di Cosimo, JI. 2018. Diglyceride-rich oils from glycerolysis of edible vegetable oils. Catal. Tod. 302, 233-241. https://doi.org/10.1016/j.cattod.2017.04.008 diglycerides are widely used for foods such as mayonnaise, salad dressings and in the confectionery industry, as they combine a hydrophilic head and a hydrophobic tail in the molecule that help hydrophilic and lipophilic substances to mix.

Thus, it was found that the sequential process provided a high-quality oil, eliminating the solvent removal step, which favors its application in different products and because it is a food residue, the oil obtained from the seeds Passion fruit can be applied to food products that require emulsifiers during their production process.