Hass avocado (Persea americana Mill.) oil enriched in phenolic compounds and tocopherols by expeller-pressing the unpeeled microwave dried fruit

Accepted Manuscript Hass avocado (Persea americana Mill.) oil enriched in phenolic compounds and tocopherols by expeller-pressing the unpeeled microwave dried fruit Isabelle Santana, Vanessa Naciuk Castelo-Branco, Barbara Mello Guimarães, Laís de Oliveira Silva, Vanessa Oliveira Di Sarli Peixoto, Lourdes Maria Corrêa Cabral, Suely Pereira Freitas, Alexandre Guedes Torres PII: DOI: Reference:

S0308-8146(19)30328-0 https://doi.org/10.1016/j.foodchem.2019.02.014 FOCH 24325

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

10 September 2018 6 February 2019 8 February 2019

Please cite this article as: Santana, I., Castelo-Branco, V.N., Guimarães, B.M., de Oliveira Silva, L., Peixoto, V.O.D., Cabral, L.M.C., Freitas, S.P., Torres, A.G., Hass avocado (Persea americana Mill.) oil enriched in phenolic compounds and tocopherols by expeller-pressing the unpeeled microwave dried fruit, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hass avocado (Persea americana Mill.) oil enriched in phenolic compounds and tocopherols by expeller-pressing the unpeeled microwave dried fruit

Isabelle Santanaa,*, Vanessa Naciuk Castelo-Brancob, Barbara Mello Guimarãesc, Laís de Oliveira Silvad, Vanessa Oliveira Di Sarli Peixotod, Lourdes Maria Corrêa Cabrale, Suely Pereira Freitasc, Alexandre Guedes Torresd,*

a

Institute of Nutrition, Rio de Janeiro State University

b

School of Pharmacy, Federal Fluminense University

c

Chemical Engineering Laboratory, Federal University of Rio de Janeiro

d

Laboratory of Food Science and Nutritional Biochemistry, Institute of Chemistry, Federal

University of Rio de Janeiro e

Embrapa Food Technology, Rio de Janeiro

*Corresponding authors: A.G. Torres: [email protected]; Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, CT, Bloco A, 528A, 21941-909, Rio de Janeiro, RJ, Brasil. Fax: +55 21 3938-8213

1

ABSTRACT This study investigated how the quality of avocado oil is affected by the fruit ripening stage and peeling, and the drying process used. Expeller pressed avocado oils were obtained from unripe or ripe pitted avocados after drying peeled or unpeeled pulps by convection oven, microwave or freeze-drying. Oils from the unpeeled microwave dried pulp (from unripe or ripe avocados) showed the highest induction period (54.2 – 83.6 h) and antioxidant capacity (4.07 – 5.26 mmol TE/kg), and high amounts (mg/100 g) of α-tocopherol (11.6 – 21.0), β-carotene (0.49 – 0.65) and chlorophyll (44.3 – 54.0), and unsaponifiable matter (2.48 – 2.99 g/100 g). Pulp drying process and avocado (un-)peeling were the major contributors to the induction period (R2= 0.61; p= 0.0139) and antioxidant capacity (R2= 0.62; p= 0.011), and the oils from microwave dried unpeeled pulp were those that presented the best performance. The phenolic composition of these oils improved with ripening and keeping the peel during the pressing process.

Keywords: Hass avocado oil; drying technology; oxidative stability; ripening; phenolic compounds

2

1. Introduction Avocado oil stands out as one of the fruit’s derived products with the highest market values, with appreciable flavor and color, and with varied positive health effects (Duarte, Chaves, Borges, & Mendonça, 2016). The pressed oil can be consumed pure and added cold to salads, or used in food preparations, or in formulations for pharmaceutical and cosmetic applications. In these last cases, the unsaponifiable matter, containing carotenoids, chlorophylls and tocopherols, and the phenolic compounds are of major interest, for their antioxidant and anti-inflammatory properties (Zhang, Huber & Rao, 2013; Kosínska, Karamác, Estrella, Hernandéz, Bartolomé, & Dykes, 2012). Avocado oil oxidative stability determines its quality during storage and heat processing, eventually affecting flavor, color, and nutrient and bioactives composition. Oil stability on its side is affected by the original avocado oil composition, and may be affected by processing factors, such as pulp drying and oil extraction technologies (Krumreich, Borges, Mendonça, Jansen-Alves, & Zambiazi, 2018; Santana, dos Reis, Torres, Cabral, & Freitas, 2015; Costagli, & Betti, 2015). However, there is scarce information concerning the influence of the avocado fruit characteristics, for instance ripening stage, on the quality of the extracted oil (Villa-Rodríguez, Molina-Corral, Ayala-Zavala, Olivas, & González-Aguilar, 2011). Avocado matures when attached to the tree, increasing in size and weight in parallel to exponential lipid accumulation in the mesocarp and losing approximately 30 % of the fruit’s water (Ozdemir & Topuz, 2004). Oleic acid content increases at higher rates compared to the other fatty acids, although large differences in fatty acid composition can be observed even for the same cultivar depending on the growing areas and harvesting season (Carvalho, Bernal, Velásquez, & Cartagena, 2015; Duque, Londoño-Londoño, Álvarez, Paz, & Salazar, 2012). Moreover, plant secondary metabolites also accumulate, increasing the contents of unsaponifiable matter and phenolic compounds (Zhang et al., 2013; Kosínska et al., 2012). Commercially, avocado fruit ripening starts after harvesting, and generally net lipid synthesis does not occur after that (Meyer & Terry, 2008). As a climacteric fruit, postharvest 3

ripening induces physiological changes culminating in full avocado ripening after 3 to 8 days (Wang, Zheng, Khuong, & Lovatt, 2012; Ozdemir et al., 2004), when at 21 to 25 °C. Regarding Hass avocado, ripening modifies the skin color from green to purple/black, due to the accumulation of anthocyanins (Cox, Mcghie, White, & Woolf, 2004) and chlorophyll degradation (VillaRodríguez et al., 2011; Ashton et al., 2006). Therefore, oils extracted from avocados with different ripening stages could present distinct composition. Nevertheless, the extraction of oil from unripe avocados has been poorly addressed so far (Ashton et al, 2006; Mostert, Botha, Plessis & Duodu, 2007). Besides eliminating the demanded time for ripening, processing the unripe avocados may limit postharvest issues linked to microbiological and biochemical agents leading to unwanted changes in chemical and physical characteristics. Furthermore, the contents of avocado fruit secondary metabolites, several of which present antioxidant activity and potential bioactivity, might be enhanced in the oil from unripe avocados, but this was not investigated previously. Another factor that could enhance avocado oil contents of natural antioxidants and bioactives is keeping the fruits’ peel or other parts, besides the pulp, during oil extraction. Antioxidant capacity and contents of phenolic compounds are much higher in avocado peel than in the fruit’s pulp (Kosínska et al., 2012; Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011; Rodríguez-Carpena, Morcuende, & Estévez, 2011; Wang, Bostic & Gu, 2010). Even though presenting low lipid amounts, using the peel for extraction might reduce extra waste disposal, while allowing the potential enhancement of antioxidants in the extracted oil. Our aim was to investigate the effects of Hass avocado ripening stage and peeling on the composition and quality of the oil obtained by expeller pressing, in combination with the effects of the fruit drying techniques adopted, either in a convection oven with forced air circulation (60 °C), in a microwave-oven or by freeze-drying.

2. Materials and methods 2.1. Reagents and standards 4

Tocopherols (α-, β-, γ- and δ-) and phenolic compounds standards (quercetin, 3,4dihydroxyphenylacetic, 3,4-dihydroxybenzoic, gallic, vanillic, syringic, p-coumaric, m-coumaric, ferulic, ellagic, trans-cinnamic acids), fatty acid methyl esters (37 FAME mix), 6-hydroxy-2,5,7,8 tetramethylchromane-2-carboxylic acid (trolox) and 2,2'-azino-bis (3-ethylbenzothiazoline)-6sulfonic acid (ABTS) were purchased from Sigma-Aldrich (São Paulo, Brazil). β-Carotene was isolated from carrot extract by open column chromatography and chlorophylls a and b were obtained from spinach, as described by Silva, Castelo-Branco, de Carvalho, Monteiro, Perrone, & Torres, 2017. All pigment standards showed purity grades > 95%, determined by HPLC-PDA. All solvents used were of chromatography grade (Tedia, Rio de Janeiro, Brazil)

ultrapure water

(Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.

2.2. Raw material Avocado (Persea americana Mill. cv. Hass) fruits grown in Carandaí (Minas Gerais, Brazil) were purchased from a central wholesale distributor of fruits and vegetables (CADEG, Rio de Janeiro, Brazil). The fruits were acquired in the unripe stage after 1 day of harvest during winter (July), having reached the horticultural maturity defined by the fruit dry matter content (25.7  0.4 g/100 g). Avocado fruits were stored in a cold chamber at 4 C immediately after purchase. Ripening was carried out in a darkroom at 20  2 °C, and the ideal point of ripeness was based on the skin color change from green to purplish/black (>75 % of the skin) and pulp softening by hand (Cox et al., 2004; Osuna-García et al., 2010), which occurred in 6 to 7 days. Fruits were daily turned approximately 90° around their longer axis to keep air exposure uniform and to avoid deformations resulting from fruit softening during ripening. Avocados were sanitized with chlorine solution (100 mg/kg) prior to processing.

2.3. Hass avocado pulp processing and oil extraction

5

Before oil extraction, the pulps from unripe (two days after harvest) and ripe avocados were classified into four groups: 1) unripe and peeled; 2) unripe and unpeeled; 3) ripe and peeled and 4) ripe and unpeeled. All avocados were pitted before processing. Resulting pulps were crushed in a pilot scale cutter (Geiger; São Paulo, Brazil) to obtain small irregular pieces (up to 7 mm), in the case of unripe avocado pulps, or a homogeneous creamy mass when ripe avocado pulp was processed, and in this case peel fragments with up to 4 mm were dispersed in this mass. Subsequently, avocado pulp was dehydrated by convection oven with forced air circulation at 60 °C, freeze-drying or microwave-oven drying, as detailed below. For convection oven drying (60 °C), fruit pulp was spread on stainless steel trays (80 × 100 cm) in layers up to 10 mm thick, and placed in a forced air circulation oven, until reaching equilibrium moisture, which took 18.0  0.3 hours. For freeze-drying, pulp was dried in a freezedryer (Edwards Pirani 501, West Sussex, United Kingdom) at 40 °C for 8 to 10 h. For microwaveoven drying, pulp was spread in the rotating plate and dried for 19.0  0.4 minutes, pausing after 10 minutes to stir the sample, in a domestic microwave oven (Brastemp, 2450 MHz, 1140 W; Brazil) set at 80 % power. At the end of the drying processes, equilibrium moisture was between 5.4 and 11.7 g/100 g (Supplementary Table 1). Oil temperature exiting the expeller, measured by a thermopar coupled to a digital thermometer varied between 40 and 45 °C, thus characterizing the samples as cold pressed. All drying processes were performed in triplicate and dried pulps were vacuum packaged in dark containers and stored at 18 °C until oil extraction. Crude oil extraction was conducted in triplicate in a continuous expeller (Oekotec, CA59G, Germany) at room temperature (25 ± 2 °C). Oil temperature exiting the expeller, measured by a thermo-par coupled to a digital thermometer varied between 40 and 45 °C, thus characterizing the samples as cold pressed. Crude oils were decanted and the liquid portion was separated from the sludge, resulting in the following oil groups: 1) oil from unripe and peeled avocado, oven dried at 60 °C; 2) oil from unripe and unpeeled avocado, oven dried at 60 °C; 3) oil from ripe and peeled avocado, oven dried at 60 °C; 4) oil from ripe and unpeeled avocado, oven dried at 60 °C; 5) oil 6

from unripe and peeled freeze-dried avocado; 6) oil from unripe and unpeeled freeze-dried avocado; 7) oil from ripe and peeled freeze-dried avocado; 8) oil from ripe and unpeeled freeze-dried avocado; 9) oil from unripe and peeled microwave-dried avocado; 10) oil from unripe and unpeeled microwave-dried avocado; 11) oil from ripe and peeled microwave-dried avocado; and 12) oil from ripe and unpeeled microwave-dried avocado. All crude oils were stored at 18 °C, in the dark, in nitrogen atmosphere until analysis.

2.4 Assessment of avocado oil quality 2.4.1. Fresh oil quality The quality of fresh crude avocado oils was determined by conventional quality indices such as acid (method Cd3d-63), peroxide (method Cd8-53) and iodine (method Cd1c-85) values and refractive index (method CC7-25) as described by AOCS (2012). Refractive index was determined in a digital refractometer (Atago® PAL-BX/RI) at 25 °C. Temperature correction to 40 °C was provided by Equation 1: 𝑅1 = 𝑅2 + 𝐾(𝑇2 − 𝑇1)

(Equation 1)

where, R1 = refractive index adjusted to 40 °C; R2 = refractive index read in the apparatus; K = 3.885 x 10-4; T2 = reading temperature; T1 = adjustment temperature (40 °C).

2.4.2. Oxidative stability and Total Antioxidant Capacity (TAC) Oxidative stability was determined by Rancimat 743 (Metrohm AG, Herisau, Switzerland) where 3 g of sample were oxidized at 110 °C with an air flow of 20 L/h. Electrical conductivity (μS) was measured by electrodes immersed in 50 mL of distilled water and the induction period (IP) was determined by the instrument’s software package as the time (h) elapsed until a sharp increase in water conductivity due to the building-up of oxidation products. TAC was determined spectrophotometrically (UV-1800, Shimadzu, Japan) by TEAC assay exactly as previously 7

described (Castelo-Branco, & Torres, 2012), and results were expressed as mmol of trolox equivalents/kg oil (mmol TE/kg).

2.5. Analysis of avocado oil chemical components 2.5.1. Fatty acid composition by GC-FID Fatty acid methyl esters (FAME) obtained after direct transesterification of samples (Lepage, & Roy, 1986) were analyzed using a gas chromatograph (GC-2010, Shimadzu, Japan), an Omegawax-320 (30 m × 0.32 mm, 0.25 μm; Supelco, Co., EUA) column, flame ionization detector (FID), and with split injection at 1:30 split ratio. Column temperature was programmed as follows: 160 °C constant for 2 min, followed by a gradient of 2.5 °C/min up to 190 °C, kept constant for 5 min, followed by a temperature gradient of 3.5 °C/min up to 220 °C, remaining constant for 15 min. The injector and detector were operated at 260 °C and 280 °C, respectively, and He was used as carrier gas. Peak identities in samples’ chromatograms were determined by comparison of relative retention times with those of a commercial mixture of fatty acid methyl esters standards (37 FAMEmix; Sigma-Aldrich; São Paulo, Brazil). Quantification was performed by internal normalization after correction of peak areas by their respective theoretical correction factors (Wolf, Bayard, & Fabien, 1995). Fatty acid content in the samples was expressed as g/100 g of total fatty acids.

2.5.2. Unsaponifiable matter Samples (2.5 g) were cold-saponified with saturated KOH and ethanol 95% (v/v) for 30 minutes. The supernatant was collected and first washed with cyclohexane and aqueous ethanol 50% (v/v), followed by washing with ethanol 50% (v/v) and distilled water. The solvent was evaporated until dryness, and the remaining mass weighed to the nearest 0.1 mg. Results were expressed as g/100 g oil, adapted from Hartman, Viana, & Freitas (1994).

2.5.3. Analysis of tocopherols, β-carotene and chlorophyll by HPLC-PDA 8

The contents of tocopherols (α, β, γ and δ), β-carotene and chlorophylls in avocado oils were determined simultaneously by normal-phase HPLC with a photon-diode array detector (PDA), as previously described by Silva et al (2016). Briefly, the oil was dissolved in n-hexane, centrifuged (10,000 ×g, 5 min) and the supernatant was filtered through a PTFE syringe filter (0.45 m) and injected (20 L) in the HPLC system (Shimadzu, Kyoto, Japan) equipped with a quaternary pump (LC-20AT), system controller (CBM-20A), degasser DGU-20A5, and SPD-M20A PDA detector. Chromatographic separation was achieved using a normal-phase silica column (ZORBAX Rx-Sil, 5 µm, 4.6 mm × 250 mm, Agilent Technologies, USA) with isocratic elution (n-hexane:2-propanol; 99:1, v/v) at 1.0 mL/min. Tocopherol isoforms, β-carotene and chlorophyll a were detected at 295, 450 and 665 nm, respectively. The samples chromatographic peaks identities were determined based on standards’ retention times, and UV/Vis spectra, and confirmed by perfect coelution and spectra matching with standards. Contents were calculated by using calibration curves ranging from 0.5 to 3.0 g/mL for tocols (R2 > 0.9979; p < 0.0001; LODmax= 0.091 g/mL; LOQmax= 0.30 g/mL), 0.5 to 5.0 g/mL for β-carotene (R2 = 0.9965; p < 0.0001; LODmax= 0.32 g/mL; LOQmax= 1.08 g/mL) and 6.25 to 100.0 µg/mL for chlorophylls (R2 = 0.9983; p < 0.0001; LODmax= 5.4 g/mL; LOQmax= 17.81 g/mL). Results were expressed in mg/100 g oil for each tocopherol, βcarotene or chlorophyll.

2.5.4. Analysis of phenolic compounds by HPLC-PDA Phenolic compounds were extracted from avocado oil by liquid-liquid extraction with aqueous methanol 80% (v/v) after dissolving the oil in n-hexane (2:1, w/v). Extraction was repeated twice. The upper-phases from the three extractions were combined, and the solvent was evaporated. The residue was reconstituted with 3 mL of methanol and analyzed by HPLC-PDA. The LC system used was the same described in 2.6.3 and chromatographic separations were achieved into a reversed-phase column (C18, 4.6 mm i.d × 150 mm, 5 μm particle size; Kromasil®) and the mobile phase consisted of a gradient of 0.3% aqueous formic acid (eluent A), methanol (eluent B) and 9

acetonitrile (eluent C) at flow rate of 1.0 mL/min as follow: 24% (B) at 8 min, 28% (B) at 18 min, 33% (B) at 30 min, 65% (B) at 60 min, followed by re-equilibration for 15 minutes. Eluent C was kept constant at 1% during analysis. Phenolic compounds were monitored by the PDA detector from = 190 to 370 nm. Chromatographic peaks in samples were identified as phenolic compounds by analytical comparisons with commercial standards behavior, as follows: retention time, UVspectra analysis, perfect co-elution with phenolic standards, and perfect overlapping of the co-eluted peaks’ UV-spectra. Quantitative analysis was based on external calibration curves with concentrations ranging from 1.0 to 20.0 g/mL (R2 > 0.9965, p <0.0001; LODmax= 0.42 g/mL, LOQmax= 1.41 g/mL). Only the oil samples with the highest oxidative stability and TAC values were analyzed.

2.5. Statistical analyses All results are presented as mean  standard deviation of triplicate processes. Multivariate analysis of variance (MANOVA) followed by Fischer LSD post-hoc test was used to compare the composition of the oils obtained, according to the three investigated factors: avocado peeling (peeled or unpeeled) and ripening stage (mature-unripe or mature-ripe), and pulp drying processes applied (convection oven, microwave-oven or freeze-drying). To investigate the effects of processing factors (avocado ripeness, fruit peeling, and pulp drying process) on parameters of fresh avocado oil quality, data was analyzed in a Multi-factor categorical design matrix, with a desirability function, in order to determine the best combination of factors that enhances avocado oil quality. Pearson’s correlation analysis was used to investigate associations between continuous variables of oil composition and to select variables to insert into a general linear model (GLM) matrix. The GLM, which allowed including continuous and categorical factors in the models, was used to investigate the major determinants of oil quality variability. The dependent variable in the model was induction period (IP) of oils and independent variables were α-tocopherol, β-carotene and chlorophyll contents, as continuous variables; and ripening stage, peeled/unpeeled and pulp 10

drying process, as categorical factors. All analyses were performed using Statgraphics v. 18 (Manugistics, EUA) and P values < 0.05 were considered as statistically significant.

3. Results and discussion

3.1 Oil yield, avocado oil quality, oxidative stability and antioxidant capacity were affected by the fruit ripening stage, peeling, and pulp drying technology Avocado oil yield was, in general, lower for the fruits dried by freeze-drying, especially the unripe avocado (Supplementary Table 1). Additionally, processing the unripe fruit markedly reduced oil yield independently of the fruit-drying process used, except for the microwave-dried pulp, that was not affected by avocado ripening. The highest values of oil yield were observed for oven-dried unpeeled ripe avocados, which showed yield values roughly 20% higher (p< 0.05; t-test) than those of unripe microwave-dried fruits, either peeled or not (Supplementary Table 1). The fruit ripening stage and peeling, and the drying process used had influence on the quality of pressed oils concerning acid, peroxide and iodine values and refractive index (Table 1). Even so, all oils presented acid and peroxide values below the maximum limits recommended for pressed oils (Codex standard for named vegetable oils, amended 2013) (≤ 4.0 mg KOH/g oil and 15 mEq O2/kg oil, respectively), indicating that no extensive hydrolytic or oxidative degradation happened during avocado pulp processing and/or oil extraction. In this sense, pressed oils from microwave dried pulps presented the lowest acid and peroxide values in both ripening stages, both from peeled and unpeeled fruits. In parallel, iodine values varied from 71.1 to 75.6, only being influenced by peeling. Refractive indexes ranged from 1.458 to 1.465, at 40 °C, for all samples, which is in agreement with Mexican standards for avocado oil (NMX-F-052-SCFI-2008). Oxidative stability (expressed as IP, in hours), and TAC (by TEAC assay), were influenced by ripening, fruit peeling and pulp drying process used (Figure 1). Oils from microwave dried pulp 11

exhibited the highest oxidative stability (Figure 1A) and TAC values (Figure 1B). Additionally, keeping the avocado peel during expeller pressing clearly improved these parameters for all extracted oils, especially those from the microwave dried pulps, for which IP and TAC were approximately 1.6-fold higher in the oils from unpeeled avocados in relation to their paired peeled samples. In the present study, IP varied from 7.03 h in the oil from ripe peeled oven-dried avocados to 83.6 h in the unripe and unpeeled microwave dried ones. Sensibly lower IP values were previously reported, for instance 2.8 h (at 110 °C and 20 L/h) for solvent-extracted and refined avocado oils (Knothe, 2013), and 3.93 and 5.24 for centrifuged oils from Hass and Fuerte avocados (Ferrari, 2015). However, there is scarce information regarding avocado oils’ IP by Rancimat, and therefore, more data is needed before attempting to rank the samples according to this parameter. But, except for the ripe peeled avocados dried in the oven or freeze-dried, all oils presented IP above 10 hours, which is generally considered a reasonable threshold for highly stable oils such as those from macadamia (up to 37.4 h; 110 °C) (Souza, Antoniassi, Freitas, & Bizzo, 2007), olive (6 to 11 hours, 120 °C) and palm (7 to 12 hours, 120 °C) (Metrohm Application Bulletin 240/02). Taken together, the results of oxidative stability and PV agree well with the upper temperature limit that was achieved during oil extraction ( 45 °C), indicating that the avocado oils processed and analyzed herein are cold-pressed crude oils. Similarly to IP, there is limited data of avocado oils’ TAC, and specifically using the TEAC assay to the best of our knowledge there have been no reports, and Krumreich et al. (2018) used DPPH assay for pressed Breda avocado oil dried at 60 °C, but results are not directly comparable. But by comparing the TEAC values obtained herein, it is fair to say that pressed avocado oil, especially those samples obtained by using the processes with best results, are among the oils with the highest antioxidant activity values (Castelo-Branco, Santana, Di-Sarli, Freitas, & Torres, 2016; Pellegrini et al., 2003).

12

3.2 Avocado oil fatty acid composition was only marginally affected by fruit ripening stage and peeling, and was not affected by pulp drying technology As expected, all avocado oils showed 18:1n-9 as the major fatty acid (> 44%), followed by palmitic (16:0), palmitoleic (16:1n-7) and linoleic (18:2n-6) acids (Table 2). Only 18:2n-6 content varied slightly, though significantly between oil samples, and showed the following descending order, regardless the pulp drying technology used: ripe, unpeeled > unripe, unpeeled > unripe, peeled > ripe, peeled. Therefore, it might be stated that avocado pulp drying technologies tested herein did not affect avocado oil fatty acid composition, and that the fruit ripening stage and peeling affected it only marginally. Oleic acid content was lower than the values established by Mexican standards for avocado oil (56 to 74%) (NMX-F-052-SCFI-2008), and some previous reports (Knothe, 2013; Haiyan, Bedgood Jr., Bishop, Prenzler, & Robards, 2007), although similar values to those herein reported have been found elsewhere (Carvalho et al., 2015; Meyer and Terry, 2008). Lower amounts of 18:1n-9 are related to premature harvest but also depend on environmental factors (Donetti & Terry, 2014; Landahl, Meyer, & Terry, 2009). Because the avocados used in the present study were harvested when horticulturally mature, as confirmed by dry matter content (> 21.5%), other factors than fruit maturity stage at harvest, such as environmental ones (Carvalho et al., 2015), might have led to reduced contents of oleic acid in the oil. Thus, technical parameters adopted in specific countries’ legislations for avocado oil quality and identity might not be universally adopted, raising the need for local regulations and acceptable levels of technical standards whenever feasible.

3.3 Avocado oil minor components: unsaponifiable matter, tocopherol, β-carotene and chlorophyll Unsaponifiable matter varied from 0.89 g% in oils from ripe, unpeeled freeze-dried avocado to 3.89 g% in unripe unpeeled oven-dried avocado (Table 3). For each raw-material, oils obtained from freeze-dried pulps showed the lowest contents of unsaponifiable matter, independently of fruit ripening stage or peeling. Additionally, for the oils from unripe and peeled avocados, drying under 13

microwaves promoted higher contents of unsaponifiables than in oven-drying, whereas the opposite was observed for ripe unpeeled oils. It is not easy to explain these results by considering the current understanding of the effects of avocado ripening on the contents of unsaponifiables in the fruit pulp and peel, and merits future investigations. Among the components of the unsaponifiable fraction analyzed, chlorophyll was the major one, followed by total tocopherols and β-carotene (Table 3). -Tocopherol was the major tocol in all avocado oil samples. β-Tocopherol was present only in the oil from unripe oven-dried avocado, while δ-tocopherol was present in most of the unpeeled samples, with the exception of those dried under microwaves, suggesting that the peel altered tocol isoforms profile. This was especially the case for δ-tocopherol, which is more effective in protecting vegetable oils against lipid oxidation than α-tocopherol. Additionally, pressing unpeeled avocado markedly increased α-tocopherol contents in the oils, except for the oil from microwave dried ripe avocado. Concerning the drying technique used, oils from microwave dried pulps showed highest contents of α-tocopherol, whereas those from oven dried and freeze-dried pulps resulted in oils with only slight differences between them. β-Carotene was also higher in the unpeeled samples, but reduced from unripe to ripe, and with all microwave dried samples showing the highest contents. Therefore, oils from ripe avocados and microwave dried pulps will have technological benefits from the α-tocopherol antioxidant activitytechnological properties, whereas oils from unripe avocados will benefit from β-carotene properties as natural pigment and singlet oxygen scavenger. Total chlorophyll contents were higher in oils extracted from unpeeled avocados, especially when obtained from the unripe avocado fruits, because chlorophyll is hydrolyzed by chlorophyllases during ripening (Ashton et al., 2006). Besides that, the drying methods showing the best performances in preserving chlorophyll were microwave and oven drying, consistently with the higher stability of other minor components in avocado oil shown by these technologies. Although freeze-drying is generally considered as a gold-standard fruit drying method for vitamin preservation, because it occurs at moderate temperatures, in the present work this was not 14

the case for Hass avocado’s minor components during drying for oil extraction. Freeze-drying the Hass avocado pulp resulted in lower contents of tocopherols, chlorophyll and total unsaponifiables in the oil (Table 2). From our data it is not possible to determine what factors have promoted the loss of these compounds, but we may speculate that enzymes that remained active during freezedrying might have caused these losses. Freshly freeze-dried avocado pulps had light-green color that was preserved during freezing under vacuum packaging. However, after opening the package the pulp rapidly turned brown, as typically occurs during enzymatic browning. Although polyphenoloxidase is the major enzyme causing these browning reactions, peroxidase and lipoxygenase activity might parallel and contribute to these transformations, promoting the oxidation of other compounds, such as lipids and tocopherols, which in turn may oxidize other fruit components. These enzymatic activities have been shown in avocado (Jacobo-Velázquez, Hernández-Brenes, Cisneros-Zevallos, & Benavides, 2010; Vanini, Kwiatkowski, & Clemente, 2010) and would possibly resist the mild processing conditions during freeze-drying. In a previous work, freeze-dried guava powder contained less soluble phenolic compounds than the oven-dried powder (Nunes et al., 2016). These results indicate that preservation of fruits components during freeze-drying might be compound- and fruit-specific, and this deserves proper direct investigations in future studies.

3.4 Identifying the main determinants of avocado oil quality To identify the main factors that influenced the quality of avocado oils, we used a multifactor categorical experimental design for data analysis and GLM to model avocado oil oxidative stability. The multi-factor design showed that unpeeling the avocados significantly (p< 0.05) improved all the response variables investigated herein (-carotene, -tocopherol, chlorophyll-a, oxidative stability, and antioxidant capacity) (Supplementary Figure 2). Additionally, both oxidative stability and antioxidant capacity of avocado oil were improved by microwave-drying the fruit pulp, compared to oven- or freeze-drying (Supplementary Figure 2). A desirability function 15

was only marginally significant (p< 0.1) for (adj. R²) chlorophyll-a (0.8845), -tocopherol (0.8924), oxidative stability (0.9035), and antioxidant capacity (0.9247). GLM is a multivariate statistical method that combines multivariate regression and analysis of variance, therefore it allows considering simultaneously the contribution of categorical and continuous independent factors, such as ripening stages and contents of chemical compounds, as determinants for a dependent variable. In the present investigation we were interested in investigating the determinants of avocado oil oxidative stability (IP, h). Continuous variables that correlated (Pearson’s) with IP were included in the GLM matrix as independent factors. In addition to these continuous factors, avocado ripening stage, fruit peeling and drying process were also included as independent categorical factors. Only the categorical variables fruit peeling and drying process were retained as significant predictors of avocado oils’ oxidative stability (p= 0.0312 and p= 0.0193, respectively). Taken together, these analyzes confirmed the general impression captured from Figure 1 and Table 3, which was that extracting avocado oils from unpeeled and microwave dried pulps, regardless the avocado ripening stage, resulted in the oils with the best quality, considering oxidative stability and contents of minor components. Heating avocado oils over 40 °C reduced its nutritional value and oxidative stability (Santos, Alicieo, Pereira, Ramis-Ramos, & Mendonça, 2014). However, comparisons between microwavedrying and freeze-drying showed that process duration was very important to the resulting avocado oil quality. In the case of convection oven, the oxidative atmosphere (high temperature and forced air circulation) was potentially a decisive factor to decrease avocado oil quality. Concerning the drying process and independently of other factors, avocado oil stability tended to be improved by the fast process of microwave-drying. Krumreich et al. (2018) showed that longer drying processing decreased oil quality, even when occurring at lower temperatures, more than high temperature drying for shorter periods. Although this seems counterintuitive, there are two factors that might explain these results. Because drying occurs before oil extraction, the food matrix components serve 16

as physical and chemical barrier, protecting the oil from degradation that could be accelerated by heating. Additionally, concerning the longer drying process tested (freeze-drying), although chemical pathways of oil degradation would be inhibited at lower temperatures, these conditions would allow biochemical pathways of oil degradation, for instance by endogenous enzyme activities of polyphenoloxidase, peroxidase and/or lipoxygenase. Another contributing factor to the extractability of minor avocado components (from the unsaponifiable fraction) into the oil might have been the avocado pulp microstructure resulting from each of the drying technologies used, although this was not examined in the present work. Therefore, concerning pulp processing before oil extraction, it seems important to keep as low as possible the processing duration and its temperature. We confirmed that microwave oven drying can be successfully applied for drying avocados before pressing to obtain high quality oils, as previously shown (Santana et al., 2015), but oven drying at 60 ºC should also be considered, preferably for the unpeeled fruit, because it leads to increased contents of minor components, especially in oils extracted from ripe avocados. Although the GLM model confirmed the overall conclusion towards the importance of the fruit peeling and pulp drying process used to determine avocado oil composition, quality and stability, unexpectedly tocopherols were not retained as independent factors in the model. In addition, although the model was sound, and consistent with the general conclusions revealed by the data (Figure 1 and Table 3), the fraction of IP variability explained by the independent factors was below 75%, which would be the advisable threshold value. Possibly, phenolic antioxidants in avocado pulp and peels, might have varied according to the factors investigated (presence or absence of peel, ripening stage, and pulp drying process), and this might have been a factor missing in the GLM. It has been reported that avocado peels are rich in phenolic compounds (RodríguezCarpena et al., 2011), and therefore we decided to determine phenolic compounds profile of selected oils, as complementary data. Avocado oils obtained by the seemingly best drying technology used, which was using microwaves, were analyzed (Table 4). 17

3.5 Phenolic compounds profile in avocado oils from the best pulp drying process used: microwave oven Avocado ripening stage and peeling influenced the phenolic composition of the pressed oils (Table 4; Supplementary Figure 1). Fruit ripening increased the total phenolic content approximately 3- and 13-fold in the oils extracted with or without the peel, respectively. Similarly, unpeeling avocado increased approximately 7- and 1.4-fold the total phenolic contents in oils from unripe and ripe avocados, respectively. In the oils from unripe and ripe peeled avocados, two and five phenolic compounds were identified, respectively. In contrast, six and seven phenolic compounds were identified in the unripe and ripe unpeeled samples, respectively. Taken together, these results point out to the importance of processing unpeeled avocados to enhance the contents of phenolic antioxidants and the stability of avocado oils. Interestingly, although oils extracted from unpeeled avocados showed high contents of p-coumaric, gallic and 3,4 di-OH-p-phenylacetic acids, oils from ripe avocados presented high contents of these phenolic compounds independently of peeling. Therefore, it seems that avocado ripening induced accumulation of these phenolic compounds in the fruit.

4. Conclusion Avocado peeling and pulp drying process were the main factors that influenced the final quality of avocado oils, especially concerning oxidative stability and antioxidant capacity, and content of minor components in the oil. Oil extraction from microwave dried and unpeeled Hass avocados resulted in high quality and stable avocado oil, with high antioxidant activity, besides valuable nutritional properties because of the amounts of α-tocopherol, β-carotene, and phenolic compounds, although it reduced oil yield by roughly 20%, compared to oven-drying unpeeled ripe avocado. Interestingly, by combining the technological factors investigated herein (avocado ripening stage and peeling, and the pulp drying process applied) it is possible to produce high 18

quality avocado oil, tailored for the desired application and depending on the available resources and the potential market.

5. Acknowledgements The authors would like to thank the financial support granted by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance code 001), Brazil. I.S. and L.O.S. were recipients of CNPq scholarships, and V.O.Di-S. was a recipient of a FAPERJ scholarship. A.G.T., L.M.C.C. and S.P.F. are recipients of CNPq fellowships.

6. References AOCS. (2012). American Oil Chemists´ Society. Official methods and recommended practices of the American Oil Chemists` Society. 6th ed. Champaign, USA, AOCS. [AOCS Official method Cd3d – 63, Cd8 – 53, Cd1c – 85]. Ashton, O. B. O., Wong, M., Mcghie, T. K., Vather, R., Wang, Y., Requejo-Jackman, C., Ramankutty, P., & Woolf, A. B. (2006). Pigments in Avocado Tissue and Oil. Journal of Agricultural and Food Chemistry, 54, 10151−10158. Carvalho, C. P., Bernal, J., Velásquez, M. A., & Cartagena, J. R. (2015). Fatty acid content of avocados (Persea americana Mill. cv. Hass) in relation to orchard altitude and fruit maturity stage. Agronomía Colombiana, 33(2), 220-227. Castelo-Branco, V. N., & Torres, A. G. (2012). Generalized linear model describes determinants of total antioxidant capacity of refined vegetable oils. European Journal of Lipid Science and Technology, 114, 332-342. Castelo-Branco, V. N., Santana, I., di-Sarli, V. O., & Torres, A. G. (2016). Antioxidant capacity is a surrogate measure of the quality and stability of vegetable oils. European Journal of Lipid Science and Technology, 118(2), 224-235. Codex standard for named vegetable oils. (Revision 2009; Amendment 2013). Codex standards for fat and oils from vegetable sources. CODEX-STAN 210. Available at: http://www.fao.org/docrep/004/y2774e/y2774e04.htm#bm4.1. Accessed in 01/08/2018. Costagli, G., & Betti, M. (2015). Avocado oil extraction processes: method for cold-pressed high-quality edible oil production versus traditional production. Journal of Agricultural Engineering, XLVI:467, 115-122.

19

Cox, K. A., Mcghie, T. K., White, A., & Woolf, A. B. (2004). Skin color and pigment changes during ripening of ‘Hass’ avocado fruit. Postharvest Biology and Technology, 31, 287-294. Donetti, M., & L.A. Terry. (2014). Biochemical markers defining growing area and ripening stage of imported avocado fruit cv. Hass. Journal of Food Composition and Analysis, 34, 90-98.

Duque, A. M. R., Londoño-Londoño, J., Álvarez, D. G., Paz, Y. B., & Salazar, B. L. C. (2012). Comparación del aceite de aguacate variedad Hass cultivado en Colombia, obtenido por fluidos supercríticos y métodos convencionales: una perspectiva desde la calidad. Revista Lasallista Investigación, 9, 151-161. Ferrari, R. A. (2015). Scientific Note: Physicochemical characterization of avocado oil extracted by centrifugation and of the process byproducts. Brazilian Journal of Food Technology, 18(1), 79-84. Haiyan, Z., Bedgood Jr., D. R., Bishop, A. G., Prenzler, P. D., & Robards, K. (2007). Endogenous biophenol, fatty acid and volatile profiles of selected oils. Food Chemistry, 100, 1544–1551. Hartman, L., Viana, H. S., & Freitas, S. P. (1994). Modified method for the determination of unsaponifiable matter in oils and fats. Analyst, 119, 1793-1795. Jacobo-Velázquez, D.A., Hernández-Brenes, C., Cisneros-Zevallos, L., & Benavides, J. (2010). Partial purification and enzymatic characterization of avocado (Persea americana Mill, cv. Hass) lipoxygenase. Food Research International, 43, 1079–1085. Knothe, G. (2013). Avocado and olive oil methyl esters. Biomass and Bioenergy, 58, 143-148. Kosínska, A., Karamác, M., Estrella, I., Hernández, T., Bartolomé, B., Dykes, G. A. (2012). Phenolic compound profiles and antioxidant capacity of Persea americana Mill. peels and seeds of two varieties. Journal of Agricultural and Food Chemistry, 60, 4613−4619. Krumreich, F. D., Borges, C. D., Mendonça, C. R. B., Jansen-Alves, C., & Zambiazi, R. C. (2018). Bioactive compounds and quality parameters of avocado oil obtained by different processes. Food Chemistry, 257, 376–381. Landahl, S., Meyer, M. D., & Terry, L. A. (2009). Spatial and temporal analysis of textural and biochemical changes of imported avocado cv. Hass during fruit ripening. Journal of Agriculture and Food Chemistry, 57, 7039–7047. Lepage, G., & Roy, C. C. (1986). Direct transesterification of all classes of lipids in a one-step reaction. Journal of Lipid Research, 27, 114-20. Metrohm Application Bulletin 240/02. Oxidation stability of oils and fats – Rancimat method. Available at: . Accessed in: 29/07/18. Meyer, M. D., & Terry, L. A. (2008). Development of a rapid method for the sequential extraction and subsequent quantification of fatty acids and sugars from avocado mesocarp tissue. Journal of Agricultural and Food Chemistry, 56(16), 7439–7445.

20

Mostert, M. E., Botha, B. M., Plessis, L. M. D., & Duodu, K. G. (2007). Effect of fruit ripeness and method of fruit drying on the extractability of avocado oil with hexane and supercritical carbon dioxide. Journal of the Science and Food Agriculture, 87, 2880–2885. NMX-F-052-SCFI-2008. (2008). Norma Mexicana. Aceite de aguacate—especificaciones, 10. Available at: http://aniame.com/mx/wp-content/uploads/Normatividad/CTNNIAGS/NMX-F052-SCFI-2008.pdf. Accessed in 02/08/18. Nunes J. C., Lago M. G., Castelo-Branco V. N., Oliveira F. R., Torres A. G., Perrone D., Monteiro M. (2016). Effect of drying method on volatile compounds, phenolic profile and antioxidant capacity of guava powders. Food Chemistry, 197(A):881–890. Osuna-García, J. A., Doyon, G., Salazar-García, S., Goenaga, R., González-Durán, I. J. L. (2010). Effect of harvest date and ripening degree on quality and shelf life of Hass avocado in Mexico. Fruits, 65:367–375. Ozdemir, F., & Topuz, A. (2004). Changes in dry matter, oil content and fatty acids composition of avocado during harvesting time and post-harvesting ripening period. Food Chemistry, 86, 79–83. Pellegrini, N., Serafini, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M., & Brighenti, F. (2003). Total antioxidant capacity of plant foods, beverages, and oils consumed in Italy assessed by three diferente in vitro assays. The Journal of Nutrition, 133, 2812-2819. Rodríguez-Carpena, J. G., Morcuende, D., Andrade, M. J., Kylli, P, & Estévez, M. (2011). Avocado (Persea americana Mill.) phenolics, in vitro antioxidant and antimicrobial activities, and inhibition of lipid and protein oxidation in porcine patties. Journal of Agricultural and Food Chemistry, 59, 5625–5635. Rodríguez-Carpena, J. G., Morcuende, D., & Estévez, M. (2011). Avocado by-products as inhibitors of color deterioration and lipid and protein oxidation in raw porcine patties subjected to chilled storage. Meat Science, 89, 166–173. Santana, I., dos Reis, L.M.F., Torres, A.G, Cabral, L.M.C., & Freitas, S.P. (2015). Avocado (Persea americana Mill.) oil produced by microwave drying and expeller pressing exhibits low acidity and high oxidative stability. European Journal of Lipid Science and Technology, 117, 999–1007. Santos, M. A. Z, Alicieo, T. V. R., Pereira, C. M. P., Ramis-Ramos, G., & Mendonça, C. R. B. (2014). Profile of bioactive compounds in avocado pulp oil: influence of the drying processes and extraction methods. Journal of the American Oil Chemists’ Society, 91, 19–27. Silva, L. O., Castelo-Branco, V. N., de Carvalho, A. G. A., Monteiro, M. C., Perrone, D., & Torres, A. G. (2017). Ethanol extraction renders a phenolic compounds-enriched and highly stable jussara fruit (Euterpe edulis M.) oil. European Journal of Food Science and Technology, 119, 1700200. Souza, D. F. S., Antoniassi, A., Freitas, S. C., & Bizzo, H. R. (2007). Estabilidade oxidativa dos óleos de macadâmia e de pistache. Boletim Centro de Pesquisa e Processamento de Alimentos, 25(1), 141-156. Vanini, L.S., Kwiatkowski, A., & Clemente, C. (2010). Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill.). Food Science and Technology, 30 (2), 525-531. 21

Villa-Rodríguez, J. A., Molina-Corral, F. J., Ayala-Zavala, J. F., Olivas, G. I., & GonzálezAguilar, G. A. (2011). Effect of maturity stage on the content of fatty acids and antioxidant activity of ‘Hass’ avocado. Food Research International, 44, 1231–1237. Wang, M., Zheng, Y., Khuong, T., & Lovatt, C. J. (2012). Effect of harvest date on the nutritional quality and antioxidant capacity in ‘Hass’ avocado during storage. Food Chemistry, 135, 694–698. Wang, W., Bostic, T. R., & Gu, L. (2010). Antioxidant capacities, procyanidins and pigments in avocados of different strains and cultivars. Food Chemistry, 122, 1193–1198. Wolf, R. L., Bayard, C. C., & Fabien, R. J. (1995). Evaluation of sequential methods for determination of butterfat fatty acid composition with emphasis on trans-18:1 acids. Application to the study of seasonal variation on french butter. Journal of American Oil Chemist’ Society, 72(12), 1471-83. Zhang, Z., Huber, D. J., Rao, J. (2013). Antioxidant systems of ripening avocado (Persea americana Mill.) fruit following treatment at the preclimacteric stage with aqueous 1methylcyclopropene. Postharvest Biology and Technology, 76, 58–64.

22

Tables captions

Table 1. Acid value (mg KOH/g), iodine value, refractive index (at 40 °C) and peroxide value (mEq O2/kg oil) of pitted unripe and ripe avocado (Persea americana Mill. cv. Hass) oils obtained from three pulp drying technologies. Table 2. Major fatty acids (>1 g/100 g fatty acids; min-max*) of pitted unripe and ripe avocado (Persea americana Mill. cv. Hass) oils obtained by pressing the fruit pulps dried by varied methods. Table 3. Minor components of pitted ripe and unripe avocado (Persea americana Mill. cv. Hass) oils: unsaponifiable matter, tocopherols, β-carotene and chlorophyll a.

Table 4. General linear models of Hass avocado oil induction period (IP, h) and antioxidant capacity (TEAC assay), according to major factors tested during processing: fruit peeling (peeled and unpeeled) and pulp drying technology (oven drying at 60 °C, microwave-oven or freeze-drying).

Table 5. Phenolic acids and quercetin composition (mg/100 g) in pressed avocado oil obtained from microwave-oven dried pulps, varying in ripening stage and peeling.

Supplementary Table 1.

Avocado residual moisture after drying and oil yield obtained by

expeller-pressing dried pitted fruit, unripe or ripe and peeled or unpeeled.

23

Figures Captions

Figure 1. Oxidative stability (A) and total antioxidant capacity (B) of pitted unripe and ripe avocado (Persea americana Mill. cv. Hass) oils obtained from three pulp drying technologies.

Supplementary Figure 1. Representative HPLC-PDA chromatogram of phenolic acids and quercetin of pressed avocado oil obtained from microwave-oven dried pulps, varying in ripening stage and peeling. (1) gallic acid; (2) 3,4-dihydroxybenzoic acid; (3) 3,4dihydroxyphenilacetic acid; (4) 5-caffeoylquinic acid; (5) p-hydroxybenzoic acid; (6) 4hydroxyphenilacetic acid; (7) vanillic acid; (8) caffeic acid; (9) syringic acid; (10) 2,4dihydroxybenzoic acid; (11) p-coumaric acid; (12) ferulic acid; (13) m-coumaric acid; (14) rutine; (15) benzoic acid; (16) quercetin. Chromatogram line colors corresponding to :

, 254 nm;  , 280 nm;  , 320 nm.

Supplementary Figure 2. Results of the Multi-factor categorical experimental design used to assess the effects on avocado oil quality attributes of the experimental factors: Hass avocado fruit ripening, fruit peeling and pulp drying process. Each graph shows a quality attribute used as response variable in the experimental design matrix.

24

Table 1. Acid value (mg KOH/g), iodine value, refractive index (at 40 °C) and peroxide value (mEq O2/kg oil) of pitted unripe and ripe avocado (Persea americana Mill. cv. Hass) oils obtained from three pulp drying technologies.

ass avocado characteristics and pulp drying chnology used

Expeller-pressed Hass avocado quality indices Acid value (mg KOH/g)

Iodine value

Refractive index

Peroxide value

(mEq O2/kg oi 25

tted unripe avocado Peeled

Unpeeled

Oven drying (60 °C)

1.09 ± 0.03b

72.7 ± 1.77b

1.4598 ± 0.0002d,*

8.04 ± 0.06a,*

Microwave-oven

0.47 ± 0.02f

72.7 ± 0.44b

1.4601 ± 0.0006c,*

2.83 ± 0.05f,*

Freeze-drying

1.27 ± 0.14a

73.0 ± 1.81b

1.4581 ± 0.0002f,*

5.05 ± 0.21d,*

Oven drying (60 °C)

0.80 ± 0.08c

74.8 ± 1.37a

1.4602 ± 0.0002b,*

5.10 ± 0.43c*

Microwave-oven

0.53 ± 0.06e

74.8 ± 0.14a

1.4609 ± 0.0006a,*

2.90 ± 0.10e,*

Freeze-drying

0.67 ± 0.10d

75.6 ± 0.73a

1.4596 ± 0.0012e,*

5.14 ± 0.06b,*

Oven drying (60 °C)

0.97 ± 0.11B

71.1 ± 1.53B

1.4596 ± 0.0007D

10.7 ± 0.21A

Microwave-oven

0.47 ± 0.02F

73.9 ± 0.70B

1.4596 ± 0.0003C

4.96 ± 0.30E

Freeze-drying

1.28 ± 0.03A

72.8 ± 0.88B

1.4586 ± 0.0002F

7.78 ± 0.12B

Oven drying (60 °C)

0.69 ± 0.04D

75.5 ± 1.47A

1.4601 ± 0.0001B

6.33 ± 0.51C

Microwave-oven

0.57 ± 0.03E

74.4 ± 0.47A

1.4602 ± 0.0001A

4.49 ± 0.16F

Freeze-drying

0.84 ± 0.12C

75.6 ± 0.45A

1.4590 ± 0.0010E

5.68 ± 0.60D

tted ripe avocado Peeled

Unpeeled

Results are expressed as mean ± standard deviation of triplicate processes. Different superscript lowercase letters in the same column indicate significant differences between oils from unripe avocado. Different superscript capital letters in the same column indicate significant differences between oils from pitted ripe avocado. * Significantly different from ripe avocado (peeled vs. peeled and unpeeled vs. unpeeled; for each drying process). MANOVA followed by Fischer’s LSD test (p ≤ 0.05).

Table 2. Major fatty acids (>1 g/100 g fatty acids) of pitted unripe and ripe avocado (Persea americana Mill. cv. Hass) oils obtained by pressing the fruit pulps dried by varied methods. Fatty acid

Pitted unripe avocado

composition

Peeled

Unpeeled

Pitted ripe avocado Peeled

Unpeeled

Oven drying (60 °C)/ Microwave / Freeze-drying Fatty acids and groups of fatty acids 22.0  0.77

21.2  0.59

21.9  0.75

21.8  0.29

22.1  1.12

21.7  0.82

22.2  0.99

22.2  0.42

16:1 n-7

12.5  1.05

11.4  0.71

11.7  0.75

12.3  0.69

18:1 n-9

45.2  0.71

46.7  0.62

46.6  0.94

45.6  1.18

64.7  2.60

65.2  2.29

66.1  1.17

65.4  2.92

9.74  0.29c

11.0  0.33b

10.0  0.26d

11.4  0.35a

11.4  0.74

12.9  0.63

12.0  0.58

12.6  0.96

16:0 Total SFA

Total MUFA 18:2 n-6 Total PUFA

Meaningful fatty acids ratios

26

UFA/SFA

3.43  0.06

3.60  0.02

3.52  0.15

3.52  0.11

PUFA/SFA

0.51  0.01

0.59  0.01

0.54  0.00

0.57  0.03

18:1 n-9/18:2 n-6

4.59  0.15

4.21  0.18

4.51  0.14

4.00  0.18

18:2 n-6/18:3 n-3

19.36  0.33

18.87  0.50

19.05  0.33

18.85  0.51

* Values were presented as mean  standard deviation of means from the three drying processes used, and this samples’ grouping was adopted because fruit pulp drying technology did not affect fatty acid composition. Values in the same row with different superscript letters are significantly different (MANOVA, with Fischer’s LSD post-hoc test; p ≤ 0.05). MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids; UFA: unsaturated fatty acids.

Table 3. Minor components of pitted ripe and unripe avocado (Persea americana Mill. cv. Hass) oils: unsaponifiable matter, tocopherols, β-carotene, and chlorophyll a. Hass avocado characteristics and pulp drying technology used

Unsaponifiable

β-caroten

Tocopherols (mg/100 g)

matter (g/100 g)

α

β

δ

(mg/100

Oven drying (60 °C)

2.35 ± 0.15 b,*

4.49 ± 0.49 d,*

2.41 ± 0.12

ND

0.30 ± 0.02

Microwave-oven

3.83 ± 0.06 a,*

10.4 ± 1.00 c,*

ND

ND

0.42 ± 0.01

2.24 ± 0.19

c,*

6.17 ± 0.03

d,*

ND

ND

0.37 ± 0.02

Oven drying (60 °C)

3.89 ± 0.15

a,*

17.1 ± 0.18

b,*

5.32 ± 0.08

3.23 ± 0.23

0.67 ± 0.02

Microwave-oven

2.99 ± 0.17 b,*

21.0 ± 0.12 a,*

ND

ND

0.65 ± 0.04

d,*

b,*

ND

9.15 ± 0.12

0.83 ± 0.02

3.44 ± 0.42 A

7.42 ± 0.96 D

ND

ND

0.24 ± 0.0

B

A

ND

ND

0.33 ± 0.0

Pitted unripe avocado

Peeled

Freeze-drying

Unpeeled

Freeze-drying

1.51 ± 0.13

15.2 ± 1.02

Pitted ripe avocado Oven drying (60 °C) Peeled

Unpeeled

Microwave-oven

2.37 ± 0.41

12.8 ± 2.25

Freeze-drying

2.09 ± 0.06 C

8.65 ± 0.16 D

ND

ND

0.28 ± 0.0

B

B

ND

6.96 ± 1.03

0.44 ± 0.0

Oven drying (60 °C)

1.41 ± 0.18

11.7 ± 1.02

Microwave-oven

2.48 ± 0.15 B

11.6 ± 3.13 C

ND

ND

0.49 ± 0.1

Freeze-drying

0.89 ± 0.01 D

12.0 ± 0.21 B

ND

4.40 ± 0.33

0.38 ± 0.0

Results are expressed as mean ± standard deviation of triplicate processes. Different lowercase superscript letters in the same column indicate significant differences between oils from unripe avocado. Different capital superscript letters in the same column indicate significant differences between oils from pitted ripe avocado. *Significantly different from ripe avocado (peeled vs. peeled and unpeeled vs. unpeeled; for each drying process). MANOVA followed by Fischer’s LSD test (p ≤ 0.05).

Table 4. Phenolic acids and quercetin composition (mg/100 g) in pressed avocado oil obtained from microwave-oven dried pulps, varying in ripening stage and peeling. Contents of phenolic compounds (mg/100 g) in oil from avocado pulp dried in 27

microwave-oven Phenolic compounds

Unripe

Ripe

Peeled

Unpeeled

Peeled

Unpeeled

ND

0.45 ± 0.02c

0.52 ± 0.01b

0.94 ± 0.03a

ND

ND

0.51 ± 0.03b

1.39 ± 0.05a

p-OH-benzoic acid

ND

0.30 ± 0.06a

ND

0.17 ± 0.06b

Vanillic acid

ND

0.17 ± 0.01a

ND

0.19 ± 0.01a

0.10 ± 0.00d

0.50 ± 0.01c

1.29 ± 0.03a

0.93 ± 0.14b

ND

0.12 ± 0.00c

0.72 ± 0.06a

0.64 ± 0.16b

0.14 ± 0.01a

0.11 ± 0.00a

0.11 ± 0.01a

0.13 ± 0.03a

0.24 d

1.65 c

3.15 b

4.39 a

Gallic acid 3,4 di-OH-p-phenylacetic acid

p-Coumaric acid Ferullic acid Quercetin Total phenolics contents

Results are expressed as mean ± standard deviation of triplicate processes. Different lowercase superscript letters in the same row indicate significant differences between oils. MANOVA followed by Fischer LSD test (p≤0.05).

Manuscript Title: Hass avocado (Persea americana Mill.) oil was enriched in phenolic compounds and tocopherols by expeller-pressing the unpeeled microwave dried fruit

Highlights 

First known study to report extraction of avocado oil from unripe fruit by expeller-pressing



Fruit unpeeling and microwave drying resulted avocado oils with the highest quality



Avocado peel enhanced phenolic compounds in the oil, regardless of ripening stage

28