Fatty acids and triacylglycerols in the mesocarp and kernel oils of maturing Costa Rican Acrocomia aculeata fruits

NFS Journal 14–15 (2019) 6–13

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Original article

Fatty acids and triacylglycerols in the mesocarp and kernel oils of maturing Costa Rican Acrocomia aculeata fruits Veronika M. Lieba, Roland Schexa,b, Patricia Esquivelc, Víctor M. Jiménezd,e, H.-G. Schmarrf,g, ⁎ Reinhold Carlea,h, Christof B. Steingassa, a

Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology and Analysis, University of Hohenheim, Garbenstraße 25, 70599 Stuttgart, Germany Institute of Beverage Research, Analysis and Technology of Plant-based Foods, Geisenheim University, Von-Lade-Straße 1, 65366 Geisenheim, Germany c School of Food Technology, Universidad de Costa Rica, 2060 San Pedro, Costa Rica d CIGRAS/IIA, Universidad de Costa Rica, 2060 San Pedro, Costa Rica e Food Security Center, University of Hohenheim, Wollgrasweg 43, 70599 Stuttgart, Germany f Institute for Viticulture and Oenology, Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Breitenweg 71, 67435 Neustadt an der Weinstraße, Germany g Faculty of Chemistry, University Duisburg-Essen, Universitätsstraße 5, 45141 Essen, Germany h Biological Science Department, King Abdulaziz University, P.O. Box 80257, Jeddah 21589, Saudi Arabia b

ARTICLE INFO

ABSTRACT

Keywords: Total lipids Macauba Fruit ripening Fatty acids Triacylglycerols HPLC linear retention indices

Total lipids, fatty acids, and triacylglycerols in mesocarp and kernels from Costa Rican Acrocomia aculeata fruits were characterized at three maturity stages. C16:0 and C18:1n9 were the most abundant fatty acids in the mesocarp. C12:0, C14:0, C16:0, and C18:1n9 prevailed in the kernel oil. Comprehensive HPLC-DAD-APCI-MS and ESI-MSn analysis revealed 125 triacylglycerols with equivalent carbon numbers ranging between 28 and 52. Mesocarp oils mainly contained unsaturated triacylglycerols composed of long-chain fatty acids. In the kernel, high proportions of saturated triacylglycerols with medium-chain fatty acids were observed. Within the maturity stages assessed, fatty acid and triacylglycerol compositions remained unaffected by ripening. Total lipids in the mesocarp increased from 12.1 to 23.4% with progressing maturity, whereas those in the kernel constantly ranged between 54.1 and 56.1%. For the recovery of A. aculeata oil, utilization of all fruits in a bunch at progressed maturity is possible without affecting the composition of its major lipid fractions.

1. Introduction Acrocomia aculeata (Jacq.) Lodd. ex Mart. (Arecaceae) is a Neotropical palm bearing oleaginous fruits. It is distributed over the tropical and subtropical regions from northern Mexico to southern Paraguay and Argentina. Due to its widespread occurrence, it is named with different colloquial terms, such as macaw, bocaiuva, coyol, macaúba, or mbocayá palm [1,2]. Several synonyms of the species are known, including A. mexicana Karw. ex Mart. or A. vinifera Oerst. Besides A. aculeata, the Acrocomia genus further comprises A. totai Mart., A. hassleri Barb. Rodr., and others [1,3,4]. Due to its high economic and agronomic potential, A. aculeata has been recently promoted as “Neotropical green gold” [3]. Wild-growing palms produce 13.7–25.5 kg fruits per tree [5]. Ciconini et al. [5] have estimated that Acrocomia yields up to 3900 kg mesocarp and kernel oil/ha when cultivated on a commercial scale at a planting density of 1000 palms/ha. The African oil palm (Elaeis guineensis Jacq.) currently represents the most important crop for the

⁎

production of vegetable oil, supplying 77.7 million tons of palm mesocarp and kernel oil in 2017 [6]. In contrast to Elaeis palms, A. aculeata is highly adaptable to different ecosystems [4,7]. Intercropping of the Acrocomia palm has been reported to increase the productivity and efficiency of coffee plantations [8]. Brazil has the largest populations of wild-growing A. aculeata palms and is deemed to provide even more potential cultivation areas [9]. Hence, a vast body of literature has dealt with the characterization of Brazilian fruits [4,5,10–20], whereas information about samples from other provenances is scarce [7,21]. In Costa Rica, 58% of the available land would be suitable for the cultivation of A. aculeata which is frequently dispersed in pasture lands and used for the production of food and animal feed [9,22]. The round-shaped A. aculeata fruits are arranged in bunches. They consist of a thin exocarp enclosing the fibrous, oily mesocarp. The kernel contains the lipid-rich endosperm protected by a hard endocarp. Fruits ripen unevenly within the bunch [2,10]. Complete maturation is indicated by their natural abscission. The effect of ripening on fruit

Corresponding author. E-mail address: [email protected] (C.B. Steingass).

https://doi.org/10.1016/j.nfs.2019.02.002 Received 20 December 2018; Received in revised form 18 February 2019; Accepted 18 February 2019 Available online 20 February 2019 2352-3646/ © 2019 The Authors. Published by Elsevier GmbH on behalf of Society of Nutrition and Food Science e.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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morphology and accumulation of total carbohydrates and total lipids in the mesocarp has already been investigated [18]. Moreover, carotenoids and α-tocopherol have been comprehensively characterized in the meso- and exocarp of three progressing maturity stages of A. aculeata fruits [21]. However, ripening-dependent changes of individual fatty acids or triacylglycerols (TAGs) in both the mesocarp and kernel still remain unknown. Currently, fully ripe fruits are manually harvested by picking them from the ground. To avoid decompartmentalization followed by enzymatic lipid degradation and microbial spoilage caused by the contact with the ground microflora, harvesting of the entire bunch as soon as fruits start to detach is recommended instead [2,10,15]. Postharvest storage and/or the use of chemicals such as fungicides are reported [15]. Edible oils can be extracted from Acrocomia mesocarp and kernel, mainly differing in their chemical composition and technological properties [14]. Harvest and postharvest conditions may influence the quality of the extracted oils [15]. Apart from free fatty acids, phytosterols, mono- and diacylglycerols, TAGs represent the predominant lipid fraction, contributing to 78.5% in the mesocarp and up to 98.6% in the kernel oil [14]. However, TAG fractions mostly affecting technological properties, such as melting and crystallization, of A. aculeata oils have not yet been assessed in detail. This study shall provide a comprehensive characterization of mesocarp and kernel lipids of Costa Rican A. aculeata fruits at three defined maturity stages. Our in-depth profiling of fatty acids and TAGs using gas chromatography (GC), high performance liquid chromatography (HPLC), and mass spectrometry (MS) should gain novel insights into the lipid composition of this underutilized crop. Understanding the alterations in total lipids, fatty acids, and TAGs during ripening should allow the optimization of harvesting time and techniques.

After harvesting, mesocarp and kernels of at least five intact, randomly selected fruits of each palm and maturity stage were manually separated, lyophilized, and milled under liquid nitrogen. Moisture loss during lyophilization was determined gravimetrically. Boiling n-hexane at 67–69 °C was used to extract mesocarp and kernel lipids in a Soxhlet apparatus for 3 h. Total lipids in dry matter (%, w/w) were quantitated gravimetrically. 2.4. Fatty acid analysis Derivatization of fatty acids as FAMEs and their subsequent quantitation were performed according to Lieb et al. [23] using a CP 9001 gas chromatograph (Chrompack, Middelburg, the Netherlands) equipped with a flame ionization detector (FID). Chromatographic separation was achieved using a highly polar ionic-liquid stationary phase (fused silica capillary column; 30 m × 0.25 mm i.d., df = 0.20 μm SLB-IL82; Supelco, Bellefonte, PA, USA). Assuming the same response factors for all FAMEs, relative fatty acid composition was expressed as proportion of the total fatty acid peak area (%). For FAME identification, a Trace GC Ultra and a PolarisQ ion-trap mass spectrometer (both ThermoFisher Scientific, Waltham, MA, USA) were used applying the aforementioned column. The injection volume was 1 μL at a split ratio of 1:50. Injector temperature was set to 250 °C. The temperature program was: isothermal hold at 110 °C for 1 min, constant rise (5 °C/min) to 240 °C, held for 5 min. Helium was used as carrier gas at a constant flow of 1.2 mL/min. Mass spectra were recorded in the electron impact positive ion mode (EI+) at a scan range of m/z 40–350 between 1.8 and 15 min and m/z 40–450 for the final segment, respectively. Source and transfer line temperatures were set to 200 and 250 °C, respectively [23]. Equivalent chain length (ECL) was calculated relative to the FAME standards as ECL = (tRs − tRn)/(tRn+1 − tRn) + n, with tR being the retention times. The subscripts n and n + 1 indicate the linear saturated FAMEs eluting before and after the FAMEs in the sample (s), respectively. The abbreviation n represents the carbon number of the FAME eluting at tRn [24]. The retention times of C19:0 and C25:0 were obtained by linear inter- and extrapolation, respectively. The fractional chain length (FCL) represents the difference between the ECL values of unsaturated and saturated FAMEs with equal carbon numbers [25]. FAMEs in the samples were assigned according to their retention times, mass spectra as well as ECL and FCL values compared to those of authentic standards and literature [24,26,27].

2. Materials and methods 2.1. Nomenclature of fatty acids Caprylic acid (C8:0) is denoted Cy, capric acid (C10:0) C, lauric acid (C12:0) La, myristic acid (C14:0) M, palmitic acid (C16:0) P, palmitoleic acid (C16:1n7) Po, stearic acid (C18:0) S, oleic acid (C18:1n9) O, vaccenic acid (C18:1n7) V, linoleic acid (C18:2n6) L, α-linolenic acid (C18:3n3) Ln. 2.2. Reagents

2.5. Triacylglycerol analysis

Acetonitrile for HPLC-MS analysis, n-hexane, and isopropanol were purchased from Merck (Darmstadt, Germany), formic acid from Th. Geyer (Renningen, Germany). Acetonitrile for HPLC analysis with diode array detection (DAD), methanol, potassium hydroxide, and sodium hydroxide were from VWR International (Fontenay-sous-Bois, France; Leuven, Belgium). Ammonium formate, boron trifluoride–methanol solution (14% BF3 in methanol), cis-11-vaccenic acid methyl ester, and Supelco 37 component fatty acid methyl ester (FAME) mix were obtained from Sigma-Aldrich Chemie (Steinheim, Germany). The triacylglycerol standards, i.e., trinonanoin, triundecanoin, tritridecanoin, tripentadecanoin, and triheptadecanoin were from Larodan (Solna, Sweden). All chemicals were either of analytical or HPLC grade.

Oil samples (50 mg/mL) and authentic TAG standards (0.5 mg/mL) were prepared in acetonitrile, isopropanol, and n-hexane (2:2:1, v/v/v) and filtered through a 0.2 μm PTFE-filter (Ziemer Chromatographie, Langerwehe, Germany) into amber HPLC vials. An Agilent 1100 Series HPLC system, a DAD set to 210 nm, and two C18 columns (both Kinetex™, 250 × 4.6 mm i.d., 5 μm particle size; Phenomenex, Aschaffenburg, Germany) connected in series were used to quantitate TAGs in A. aculeata oils [23]. Identification was performed using the HPLC system coupled on-line to an Esquire 3000+ ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany). The system was equipped with an atmospheric pressure chemical ionization (APCI) or an electrospray ionization (ESI) source, both operated in the positive ion mode. Ionization was enhanced by the admixture of 10 mM ammonium formate and 0.1% formic acid to eluent B (isopropanol). Mass spectra were recorded in the range of m/z 50–1200 at a scan speed of 13,000 (m/z)/s. The APCI parameters were as follows: nebulizing gas pressure: 65 psi (N2); dry gas flow rate: 4.5 L/min (N2); nebulizer temperature: 350 °C; vaporizer temperature: 400 °C; corona current: 2000 nA; capillary voltage: −2779 V. ESI parameters were: nebulizing gas pressure: 70 psi (N2); dry gas flow rate: 12 L/min (N2); nebulizer temperature: 365 °C; capillary voltage: −4000 V; helium gas pressure for collision-induced dissociation (CID): 4.6 × 10−6 mbar; fragmentation amplitude: 1.0 V.

2.3. Sample material and preparation Acrocomia aculeata fruits were collected in April 2016 from three wild-growing palms in Bagaces (Guanacaste, Costa Rica). The maturity stages of the fruits were classified according to their attachment to the bunch and surface color. Unripe fruits with green (A) and ripe fruits with green to brownish exocarp (B) were harvested from the bunch. Fully ripe fruits with mostly brown exocarp (C) were manually collected from the ground immediately after their natural abscission. Exact locations of the palms and further fruit descriptions have been provided elsewhere [21]. 7

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TAGs were labelled using the abbreviated trivial names of their individual fatty acids (Section 2.1) arranged according to their carbon number and degree of unsaturation. The positional distribution of fatty acids between sn-1, sn-2, and sn-3 positions was neglected. Relative TAG compositions were calculated as percentage of total peak area. The equivalent carbon number (ECN) was calculated as ECN = CN − 2 × DB, with CN representing the number of acyl carbons and DB the number of double bonds within the TAG molecule. Experimental linear retention indices (LRIs) relative to the authentic TAG standards were determined as defined by Rigano et al. [28]. Theoretical LRIs were calculated as LRI = 100 × (CN + a1 × DB + a2 × NUFA) according to Nájera et al. [29] with NUFA representing the number of unsaturated fatty acids within the TAG molecule. Multiple linear regression of log k = e1 × CN + e2 × DB + e3 × NUFA, with k representing the capacity factor of unsaturated TAGs enabled the calculation of the variables a1 = e2/e1 and a2 = e3/e1.

saturated FAMEs and their saturated homologue of higher carbon number. In contrast, the FCL of the polyunsaturated fatty acids (PUFAs) C18:2n6 and C18:3n3 were 1.23 and 2.15, respectively. 3.2.1. Mesocarp oils Seven fatty acids including C16:0, C16:1n7, C18:0, C18:1n9, C18:1n7, C18:2n6, and C18:3n3 prevailed in the mesocarp oils. To the best of our knowledge, C18:1n7 is being identified for the first time in A. aculeata, presumably due to the good resolution of unsaturated FAMEs obtained in our work using a highly polar ionic-liquid stationary phase for GC separation. The mesocarp oil contained high proportions of MUFAs (72.0–75.0%). C18:1n9 (64.9–67.7%) and C16:0 (17.0–19.1%) were the most abundant fatty acids among all maturity stages, followed by C18:2n6 in unripe (maturity stage A) and C16:1n7 in ripe fruits (B and C). The profile of mesocarp fatty acids from mature Costa Rican fruits, i.e., stages B and C, was comparable to those reported for Brazilian provenances [12–14,16,17,20]. Moreover, the fatty acid patterns of the A. aculeata mesocarp samples assessed in our work resembled those of the American oil palm (Elaeis oleifera [Kunth] Cortés) [30]. Without statistical evidence, maturation resulted in diminished PUFA and increased MUFA proportions, as has been previously described in ripening E. guineensis mesocarp [31]. Proportions of saturated fatty acids (SFAs) did not alter during ripening. Changes of mesocarp fatty acids were significant for C18:0 and C18:1n7, representing low 1.7–3.3 and 2.2–4.4%, respectively. The proportions of C18:1n9 tended to drop from 67.7 ± 2.8 to 64.9 ± 4.3%, whereas its n7 isomer significantly increased from 2.2 ± 0.1 to 4.4 ± 0.7%. Interestingly, the sum of C18:1 isomers remained almost constant, contributing to 69.9 ± 2.9, 70.5 ± 4.5, and 69.3 ± 3.7% of total fatty acids in unripe, ripe, and fully ripe fruits, respectively (data not shown). These findings may indicate a positional isomerization of oleic acid with progressing maturation. However, further investigations are needed to verify this hypothesis.

2.6. Statistical analysis Analyses of at least five fruits from three different palms were performed in two analytical replicates. Normality and homogeneity of variances were analyzed using Shapiro–Wilk's (p ≤ 0.05) and Levene's test (p ≤ 0.01), respectively. Analysis of variance (ANOVA) followed by Tukey's multiple-range test (p ≤ 0.05) were used to determine significant differences between sample sets with normal distribution and homogeneous variance, the non-parametric Kruskal–Wallis test (p ≤ 0.05) between data without normal distribution. Statistical analyses were performed using SAS 3.5 statistical software (SAS Institute Inc., Cary, NC, USA) and Excel 2013 (Microsoft, Redmond, WA, USA). 3. Results and discussion 3.1. Total lipid content

3.2.2. Kernel oils The most abundant kernel fatty acids were C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C18:1n9, and C18:2n6. The oil was mainly composed of SFAs, totaling to 76.2–77.8%. C12:0 represented the major fatty acid (41.6–42.9%), followed by C18:1n9 (18.7–20.2%) and C14:0 (13.4–13.6%). Fatty acid composition of kernels from mature Costa Rican fruits ranged between the profiles described for Brazilian samples [12–14,17], with C14:0 being the exception. Elevated proportions of the latter were observed in the Costa Rican kernel oils compared to samples originating from Brazil (7.7–12.1%) [12,14,17]. A similar composition of fatty acids with predominance of C12:0, C14:0, and C18:1n9 has been reported for kernel lipids from E. guineensis, E. oleifera, and their interspecific hybrids [30]. As observed for the mesocarp, fatty acid profiles of A. aculeata kernel extracts were almost independent of ripening. Concomitantly with a slight increase of C16:0, the oil showed a statistically insignificant drop of the medium-chain fatty acids C8:0, C10:0, and C12:0 during ripening. This consistent fatty acid composition, in particular within the maturity stages B and C, indicated that the lipid deposition in the endosperm is already completed at an earlier maturity stage.

The total lipid contents of A. aculeata mesocarp tended to increase during maturation from 12.1 ± 4.9% of dry matter (DM) in unripe (maturity stage A) to 21.4 ± 4.2% in ripe (B) and 23.4 ± 6.3% in fully ripe fruits (C), respectively (Table 1). Owing to the accumulation of oil prior to fruit abscission [19], the total lipid contents of the unripe fruits assessed (A) were lower than those reported for mature fruits of 35 Brazilian genotypes (14.7–77.5%) [13]. In contrast, the ripe fruits analyzed herein (B and C) were within the range of diverse ripe Brazilian samples [5,17,20]. Dehydration of A. aculeata mesocarp during ripening as previously reported by Montoya et al. [18] caused the significant increase of total lipids from 3.2 ± 1.4 to 10.6 ± 3.2 g/100 g fresh weight (FW). Overall, total lipid contents in the kernel were remarkably higher than those in the mesocarp, ranging between 54.1 and 56.1% of DM in all maturity stages. Since the dry weight of A. aculeata endosperm remained constant at 44 weeks after anthesis [18], variances in the total lipid contents expressed relative to both dry matter and fresh weight were found to be insignificant among the Costa Rican kernel samples. They were within the wide range of total lipids reported by da Conceição et al. [13] (33.2–61.2%) and even surpassed the values of further Brazilian fruits [11,17].

3.3. Identification of triacylglycerols

3.2. Fatty acid composition

A total of 125 TAGs with ECNs ranging between 28 and 52 was identified in A. aculeata mesocarp and kernel oils using HPLC-DAD-APCIMS and ESI-MSn (Table S1). Only the main fatty acids as revealed by GCFID (each ≥1%; Table 2) were considered for the identification of TAGs. The kernel oil displayed a more complex profile with 101 TAGs (ECN 28–52) compared to 37 TAGs (ECN 36–52) identified in the mesocarp. To the best of the authors' knowledge, this in-depth profiling of A. aculeata TAGs has been presented for the first time. Representative mass spectra of POO obtained by APCI-MS and ESI-MSn are illustrated in Fig. 2. Both

GC-MS chromatograms of mesocarp and kernel FAMEs are presented in Fig. 1. The compositions of fatty acids are compiled in Table 1. ECL values as determined by GC-MS were in good agreement with data provided by others, applying comparable [26,27] or different [24] chromatographic conditions. FCL values increased with the degree of unsaturation of the FAMEs, as has been previously reported [26]. The FCL of all monounsaturated fatty acids (MUFAs) detected ranged between 0.46 and 0.57, indicating their elution between the corresponding 8

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Table 1 Total lipids (% of DM; g/100 g FW) and individual fatty acids (%) in A. aculeata mesocarp and kernels at different maturity stages. Abbr.

Total lipid content (% of DM) Total lipid content (g/100 g FW) Fatty acids (%) C8:0 Cy C10:0 C C12:0 La C14:0 M C16:0 P C16:1n7 Po C18:0 S C18:1n9 O C18:1n7 V C18:2n6 L C18:3n3 Ln a Others SFAs MUFAs PUFAs

ECL

7.99 9.99 12.00 13.99 15.99 16.48 17.99 18.47 18.55 19.22 20.14

FCL

– – – – – 0.49 – 0.49 0.57 1.23 2.15

Mesocarp

Kernel

A

B

C

A

B

C

12.1 ± 4.9a 3.2 ± 1.4b

21.4 ± 4.2a 8.1 ± 2.4ab

23.4 ± 6.3a 10.6 ± 3.2a

56.1 ± 1.6a 44.4 ± 0.7a

54.1 ± 2.5a 42.9 ± 2.6a

54.7 ± 3.3a 43.9 ± 3.1a

0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.1 ± 0.0a 17.0 ± 1.0a 2.0 ± 0.4a 3.3 ± 0.2a 67.7 ± 2.8a 2.2 ± 0.1b 4.4 ± 2.3a 2.6 ± 1.0a 0.8 ± 0.2a 21.0 ± 0.7a 72.0 ± 3.0a 6.9 ± 3.4a

0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.1 ± 0.0a 18.5 ± 1.9a 4.3 ± 1.8a 1.8 ± 0.5b 66.7 ± 5.3a 3.8 ± 0.8a 2.7 ± 1.2a 1.5 ± 0.3a 0.6 ± 0.1a 20.7 ± 1.6a 75.0 ± 2.6a 4.2 ± 1.5a

0.0 ± 0.0a 0.0 ± 0.0a 0.1 ± 0.1a 0.1 ± 0.0a 19.1 ± 1.6a 4.8 ± 1.5a 1.7 ± 0.5b 64.9 ± 4.3a 4.4 ± 0.7a 2.9 ± 0.8a 1.4 ± 0.3a 0.6 ± 0.1a 21.4 ± 1.2a 74.3 ± 2.2a 4.3 ± 1.1a

5.2 ± 0.6a 3.3 ± 0.3a 42.9 ± 0.9a 13.4 ± 0.3a 8.9 ± 0.4a 0.1 ± 0.0a 3.3 ± 0.2a 18.8 ± 1.3a 0.3 ± 0.0a 3.1 ± 0.1a 0.0 ± 0.0a 0.8 ± 0.0a 77.7 ± 1.3a 19.2 ± 1.3a 3.1 ± 0.1a

5.1 ± 0.2a 3.3 ± 0.1a 42.7 ± 0.5a 13.6 ± 0.2a 9.1 ± 0.2a 0.1 ± 0.0a 3.4 ± 0.3a 18.7 ± 0.7a 0.3 ± 0.0a 3.0 ± 0.2a 0.0 ± 0.0a 0.8 ± 0.0a 77.8 ± 1.0a 19.1 ± 0.8a 3.0 ± 0.2a

4.9 ± 0.4a 3.1 ± 0.2a 41.6 ± 0.7a 13.4 ± 0.6a 9.2 ± 0.5a 0.1 ± 0.0a 3.2 ± 0.1a 20.2 ± 0.6a 0.3 ± 0.0a 3.1 ± 0.2a 0.0 ± 0.0a 0.8 ± 0.0a 76.2 ± 0.5a 20.7 ± 0.6a 3.2 ± 0.2a

Values represent means ± standard deviations of each maturity stage (n = 3). Different letters within a row of mesocarp and kernel, respectively, indicate significant differences of means (p < 0.05). DM, dry matter; ECL, equivalent chain length; FCL, fractional chain length; FW, fresh weight; SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids. a Including the minor fatty acids C6:0, C11:0, C13:0, C15:0, C17:0, C17:1n7, C20:0, C20:1n9, C22:0, C23:0, and C24:0 with ECL 6.00, 11.00, 12.99, 14.98, 16.98, 17.44 (FCL 0.46), 19.98, 20.46 (FCL 0.49), 21.97, 22.97, and 23.97, respectively. Table 2 Relative TAG composition (%) in A. aculeata mesocarp oil at different maturity stages.

Fig. 1. GC-MS total ion current (TIC) chromatograms of the most abundant FAMEs from mesocarp (a) and kernel oils (b) of fully ripe fruits.

ionization techniques yielded ammonium adducts [M + NH4]+ in MS1, for instance at m/z 876.8 for POO as depicted in Fig. 2b and c. In addition, APCI-MS produced sodium [M + Na]+ and potassium [M + K]+ adducts as well as low abundant protonated molecules [M + H]+. The intensity of the latter, in particular of those obtained from highly saturated TAGs, was increased by the addition of mobile phase modifiers [32–35]. In our study, the admixture of ammonium to the mobile phase was essential for the comprehensive structural elucidation of TAGs by APCI- and ESI-MS. After the neutral loss of ammonia (NH3; 17 amu) and one fatty acid from the glycerol backbone (Fig. 2a), APCI-MS1 spectra additionally displayed the corresponding diacylglycerol fragment ions, i.e., [M + NH4 − NH3 − RCOOH]+ equaling [M − RCO2]+. Higher background signals applying

TAG

ECN

A

B

C

LnLnLn LLnLn/PoLnLn LLLn PoLLn OLnLn/PoPoLn PLnLn OLLn PoOLn/PoPoPoa PLLn PPoLn OLL OOLn/PoOLa/PLLa/PoPoOa POLn/PPoLa/PPoPoa PPLn OOL PoOO POL PPoO PPL OOO POO PPO SOO Othersb Trisaturated TAGs Disaturated TAGs Diunsaturated TAGs Triunsaturated TAGs

36 38 40 40 40 40 42 42 42 42 44 44 44 44 46 46 46 46 46 48 48 48 50

0.7 ± 0.3a 1.6 ± 1.4a 0.8 ± 0.7a 0.3 ± 0.1a 2.3 ± 0.9a 1.0 ± 0.3a 4.1 ± 2.0a 0.7 ± 0.2a 1.5 ± 0.8a 0.5 ± 0.3a 1.9 ± 1.2a 12.7 ± 0.9a 8.0 ± 0.5a 0.9 ± 0.1a 10.4 ± 1.9a 2.0 ± 0.5b 8.0 ± 1.4a 0.9 ± 0.5b 1.5 ± 0.3a 20.8 ± 5.0a 13.0 ± 3.9a 2.5 ± 0.6a 2.7 ± 1.2a 1.2 ± 0.5a 0.1 ± 0.1a 5.9 ± 0.9a 35.6 ± 2.8a 58.4 ± 3.6a

0.7 ± 0.2a 0.5 ± 0.1a 0.2 ± 0.1a 0.3 ± 0.2a 1.2 ± 0.2b 0.6 ± 0.1a 2.3 ± 0.8a 1.3 ± 0.3a 0.8 ± 0.4a 1.1 ± 0.3a 1.1 ± 0.7a 11.3 ± 1.4ab 7.5 ± 0.7a 1.0 ± 0.1a 9.3 ± 1.8a 4.3 ± 0.8a 6.6 ± 1.5a 2.4 ± 0.8a 1.4 ± 0.4a 23.8 ± 5.1a 16.4 ± 2.6a 3.0 ± 0.6a 1.8 ± 0.9a 1.2 ± 0.1a 0.3 ± 0.1a 6.3 ± 0.8a 37.2 ± 1.2a 56.2 ± 2.0a

0.8 ± 0.3a 0.4 ± 0.2a 0.2 ± 0.1a 0.3 ± 0.2a 0.9 ± 0.2b 0.6 ± 0.2a 1.9 ± 0.8a 1.3 ± 0.3a 0.8 ± 0.4a 1.1 ± 0.4a 1.2 ± 0.5a 10.3 ± 1.2b 7.3 ± 1.0a 1.0 ± 0.2a 9.7 ± 0.4a 4.8 ± 0.7a 7.5 ± 1.1a 2.7 ± 0.7a 1.7 ± 0.5a 22.5 ± 4.8a 17.3 ± 2.2a 3.2 ± 0.5a 1.6 ± 0.6a 1.1 ± 0.1a 0.2 ± 0.1a 6.8 ± 1.1a 38.8 ± 2.3a 54.2 ± 3.5a

Values represent means ± standard deviations (n = 3). Significant differences of means (p ≤ 0.05) within a row are indicated by different letters. For abbreviations, see Section 2.1 and Table 1. Positional distribution of fatty acids was neglected. a Co-eluting TAGs in trace amounts. b Including the minor TAGs PPPo, PPP, PSO, PPS, SSO, and PSS.

APCI hampered the identification of co-eluting TAGs with low-abundant molecular and diacylglycerol fragment ions. Nevertheless, single-stage MS experiments supported by the admixture of ammonium to the mobile 9

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Table 3 Relative TAG composition (%) in A. aculeata kernel oil at different maturity stages

Fig. 2. Mass fragmentation scheme (a) and representative APCI-MS1 (b), ESIMS1 (c), and ESI-MS2 (c') spectra of POO.

phase rendered the unambiguous assignment of TAGs by APCI-MS possible. As can be seen in Fig. 2c, ESI-MS1 spectra exhibited intense signals of the [M + NH4]+ precursor ion. Apart from the latter, low abundant [M + K]+ adducts were occasionally observed. Further structural information was obtained by CID experiments of the ammonium adducts. [M − RCO2]+ fragment ions prevailed in the ESI-MS2 experiment along with low abundant [M + H]+ and occasionally [M + H − H2O]+ (Fig. 2c'). Noteworthy, the ESI-MS2 spectra resembled those of APCI-MS1 (Fig. 2b), as has been previously described by Byrdwell and Neff [33]. ESIMSn enabled the identification of 125, APCI-MS of merely 115 TAGs. The enhanced efficiency of ESI for TAG analysis was in accordance with previous reports [32,33,35]. Apart from the identification of TAGs via their mass spectra, linear retention indices (LRIs) were determined (Table S1). LRIs linearly increased with the respective ECNs. Within an ECN cluster, LRIs of saturated TAGs were lower compared to those of unsaturated TAGs. Our experimental LRIs differed from those reported by Rigano et al. [28]. According to these authors, LRIs are affected by diverse chromatographic conditions, such as column temperature, mobile phase composition, and particle size of the stationary phase. Contrary to the authors' suggestion, interlaboratory comparison of LRIs used for the identification of TAGs was intricate. Similarly, reproducibility of LRIs of diverse drugs has been described to be lower in an inter- than intralaboratory comparison [36]. Using the same chromatographic conditions, in particular saturated TAGs showed an excellent linear correlation (correlation coefficient R2 = 0.9998; n = 47) and small deviations (mean sum of square of errors MSSE = 93) between our experimental and theoretical LRIs. This high accuracy of data enables the prediction of LRIs and the corresponding retention times of unknown saturated TAGs. In contrast, prognosis of LRIs of unsaturated TAGs was more complex. The addition of each double bond to the TAG reduces its LRI by a factor of two, equaling the shortening of the

TAG

ECN

A

B

C

CyCLa/CCC CyLaLa/CCLaa CLaLa CyLaM/CCM CyCP CyLaO/CCOa LaLaLa/CyLaP/CLaMa/ CyMMa/CCPa CyOL LaLaL/CMLa/CyPLa CLaO/CyMOa LaLaM/CMMa/CLaPa/CCSa/ CyLaSa/CyMPa LaLL CyOO/LaMLa/CPLa LaLaO/CyPO LaMM/LaLaP/CMPa/CyPPa/ CyMSa/CLaSa LLL LaOL LaPL/MMLa/CSLa LaMO/CPOa/CySOa LaMP/LaLaS/MMM/CPPa/ CMSa OLL PLL MOL LaOO MPL/LaSL LaPO/MMOa OOL POL MOO PPL/MSLa MPO/LaSO OOO SOL POO PPO/MSOa SOO Othersb Trisaturated TAGs Disaturated TAGs Diunsaturated TAGs Triunsaturated TAGs

30 32 34 34 34 36 36

0.9 1.9 1.4 3.0 1.0 1.2 7.1

± ± ± ± ± ± ±

0.2a 0.7a 0.3ab 0.5a 0.2a 0.1ab 0.6ab

1.0 1.6 1.5 3.2 1.1 1.3 7.5

± ± ± ± ± ± ±

0.0a 0.6a 0.0a 0.1a 0.1a 0.0a 0.2a

0.8 1.8 1.1 2.5 1.0 1.0 6.1

± ± ± ± ± ± ±

0.0a 0.3a 0.0b 0.2a 0.1a 0.1b 0.7b

38 38 38 38

1.0 1.6 1.2 5.2

± ± ± ±

0.1a 0.1ab 0.1a 0.2a

1.1 1.8 1.2 5.4

± ± ± ±

0.1a 0.2a 0.0a 0.2a

0.9 1.3 1.0 4.6

± ± ± ±

0.1a 0.1b 0.1a 1.0a

40 40 40 40

1.2 1.9 4.0 3.0

± ± ± ±

0.1a 0.2a 0.3ab 0.2a

1.3 2.0 4.2 3.0

± ± ± ±

0.1a 0.1a 0.0a 0.1a

1.1 1.7 3.6 2.7

± ± ± ±

0.1a 0.2a 0.5b 0.7a

42 42 42 42 42

2.5 5.0 2.8 2.1 1.5

± ± ± ± ±

0.1a 0.2a 0.0a 0.2a 0.1a

2.3 5.3 2.9 2.2 1.5

± ± ± ± ±

0.4a 0.1a 0.1a 0.1a 0.1a

2.2 4.6 2.6 2.1 1.4

± ± ± ± ±

0.3a 0.6a 0.4a 0.4a 0.3a

44 44 44 44 44 44 46 46 46 46 46 48 48 48 48 50

5.1 ± 0.2a 2.6 ± 0.3a 1.7 ± 0.0a 3.8 ± 0.5a 1.7 ± 0.1a 2.4 ± 0.3a 7.0 ± 0.2a 5.4 ± 0.3a 1.4 ± 0.2a 1.4 ± 0.1a 1.4 ± 0.2a 4.4 ± 0.7a 1.0 ± 0.1a 3.6 ± 0.5a 1.4 ± 0.2a 1.4 ± 0.7a 4.6 ± 0.6ab 26.4 ± 2.1a 24.0 ± 0.2a 30.6 ± 1.1a 19.0 ± 1.1a

4.9 ± 0.4a 2.4 ± 0.2a 1.8 ± 0.1a 4.0 ± 0.0a 1.8 ± 0.1a 2.5 ± 0.1a 6.8 ± 0.2a 5.1 ± 0.0b 1.4 ± 0.1a 1.3 ± 0.0a 1.4 ± 0.1a 4.3 ± 0.3a 1.0 ± 0.1a 3.5 ± 0.1a 1.3 ± 0.0a 0.9 ± 0.2a 4.5 ± 0.2a 26.9 ± 1.3a 24.0 ± 0.3a 30.8 ± 0.7a 18.3 ± 1.2a

5.0 ± 0.4a 3.4 ± 1.5a 2.1 ± 0.3a 4.0 ± 0.4a 1.7 ± 0.3a 2.5 ± 0.6a 8.0 ± 0.9a 5.9 ± 0.4a 1.6 ± 0.1a 1.4 ± 0.0a 1.4 ± 0.3a 6.6 ± 2.9a 1.1 ± 0.1a 5.0 ± 1.5a 1.5 ± 0.1a 1.1 ± 0.1a 4.0 ± 0.2b 23.1 ± 3.1a 22.2 ± 2.8a 33.0 ± 2.6a 21.7 ± 3.2a

Values represent means ± standard deviations (n = 3). Significant differences of means (p ≤ 0.05) within a row are indicated by different letters. For abbreviations, see Section 2.1 and Table 1. Positional distribution of fatty acids was neglected. a Co-eluting TAGs in trace amounts. b Including the minor TAGs CyCyLa/CyCCa, CyLaL/CCLa/CyCOa, CyLL, CLaL/CyML, CLL, COL, MLL, COO, LaPP/MMP/LaMS/CySSa/CPSa, MPP/MMS/ LaPS/CSSa, PPP/LaSS/MPS, PSO, MSS/PPS, and SSO.

molecule by two methylene units [37]. However, eminently high deviations between experimental and calculated LRIs as well as reduced linearity of data were determined (MSSE = 5246; R2 = 0.9946; n = 78). Multiple regression analysis of unsaturated TAGs using capacity factors according to Barron and Santa-María [38] enhanced the accuracy of data (MSSE = 1357), albeit an exact prediction of unsaturated TAGs still remained unsatisfactorily compared to saturated TAGs (R2 = 0.9969). In contrast, multiple regression analysis and LRI calculation considering the number of unsaturated fatty acids within the TAG molecule (a1 = −2.1084; a2 = −0.1116) revealed the best approximation of calculated and experimental data (Table S1). The high accuracy and linearity of calculated LRIs (MSSE = 736; R2 = 0.9980) rendered the reliable prediction of retention times of unknown unsaturated TAGs possible.

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Fig. 3. HPLC-DAD chromatograms at 210 nm of TAGs from mesocarp (a) and kernel oils (b) of fully ripe fruits (maturity stage C) with their respective ECNs. The insert (a') displays an enlarged section of TAGs with ECN 48 compared to an unripe mesocarp sample (maturity stage A, gray line). Co-eluting TAGs are not labelled. For abbreviations, see Section 2.1 and Table 1. Positional distribution of fatty acids was neglected.

3.4.1. Mesocarp oils The mesocarp oils had high proportions of unsaturated TAGs (35.6–38.8% di- and 54.2–58.4% triunsaturated TAGs), being consistent with data reported for Brazilian A. aculeata samples [14]. OOO was the major TAG in all maturity stages assessed (20.8–23.8%), followed by POO (13.0–17.3%), OOLn (10.3–12.7%), and OOL (9.3–10.4%). In contrast to the 37 TAGs reported herein, merely PSO (37.5%), OOO (35.2%), and PPO (5.8%) have been previously found in the mesocarp oil of ripe Brazilian fruits applying GC-MS analysis [14]. As presented in Fig. 4, TAGs with ECN 44–48 mainly contributed to the characteristic profile in mesocarp lipids of fully ripe fruits, representing high 91.3 ± 2.4%. During ripening, only slight changes in the TAG composition were observed. TAG synthesis in the mesocarp of the related E. guineensis was found to be completed at early 18 weeks after anthesis, reaching a steady state afterwards [31]. In the oils analyzed herein, PoOO and PPoO significantly increased during fruit ripening, whereas the proportions of OLnLn/PoPoLn and OOLn decreased. In general, TAGs composed of C16:0 as well as the monounsaturated C16:1n7 and C18:1n9 tended to accumulate, whereas those containing the polyunsaturated C18:2n6 and C18:3n3 decreased during maturation. These findings were in accordance with the results of the fatty acid analysis (see Table 1) and those already described for TAG accumulation in ripening E. guineensis fruits [31]. TAGs containing C18:1n9 (O) and C18:1n7 (V) slightly differed in their retention times; however, were not sufficiently resolved by HPLC. With progressing maturation, faint peak shoulders eluting prior to TAGs containing high proportions of oleic acid, e.g., OOO and POO (see Fig. 3a') emerged, possibly representing corresponding vaccenic acid isomers. This assumption was supported by the accumulation of C18:1n7 during ripening (Table 1). Chromatographic separation of corresponding TAGs carrying oleic and vaccenic acid from sea

Fig. 4. Relative proportions of mesocarp and kernel TAGs of fully ripe fruits (maturity stage C) classified according to their ECNs.

3.4. Distribution of triacylglycerols The composition of TAGs in the mesocarp and kernel oils at different maturity stages are compiled in Tables 2 and 3. Representative HPLC-DAD chromatograms of mesocarp and kernel TAGs with respective peak identification according to Table S1 are shown in Fig. 3. In some instances, TAGs remained unresolved in the HPLC-DAD chromatogram. However, elevated intensities of precursor and product ions in the MS experiments permitted the tentative assignment of the prevalent TAGs within such unresolved peaks. For the purpose of simplification, such co-eluting, less abundant TAGs (see Tables 2 and 3) are not considered in the following sections. 11

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buckthorn (Hippophae rhamnoides L.) pulp oil has been previously achieved [39]. However, the latter contains elevated proportions of vaccenic acid (6.2–10.7%) compared to the Acrocomia oils assessed herein (0.3–4.4%) [40]. Owing to their identical mass fragmentations and the insufficient chromatographic separation, the differentiation between abundant TAGs with C18:1n9 and those with C18:1n7 representing minor constituents was neglected. Whereas fatty acids and TAGs remained almost constant during the development of A. aculeata mesocarp, significant changes in the concentrations of its lipophilic micronutrients, such as carotenoids and αtocopherol, have been previously reported [21].

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.nfs.2019.02.002. References [1] A. Henderson, G. Galeano, R. Bernal, Field Guide to the Palms of the Americas, Princeton University Press, Princeton, New Jersey, USA, 1995. [2] FAO, Food and Fruit-Bearing Forest Species. 3: Examples from Latin America, FAO Forestry Paper 44/3, Rome, Italy, (1986). [3] N.E. de Lima, A.A. Carvalho, A.W. Meerow, M.H. Manfrin, A review of the palm genus Acrocomia: neotropical green gold, Org. Divers. Evol. 18 (2018) 151–161. [4] A. de Santana Lopes, T. Gomes Pacheco, T. Nimz, L. do Nascimento Vieira, M.P. Guerra, R.O. Nodari, E.M. de Souza, F. de Oliveira Pedrosa, M. Rogalski, The complete plastome of macaw palm [Acrocomia aculeata (Jacq.) Lodd. ex Mart.] and extensive molecular analyses of the evolution of plastid genes in Arecaceae, Planta 247 (2018) 1011–1030. [5] G. Ciconini, S.P. Favaro, R. Roscoe, C.H.B. Miranda, C.F. Tapeti, M.A.M. Miyahira, L. Bearari, F. Galvani, A.V. Borsato, L.A. Colnago, M.H. Naka, Biometry and oil contents of Acrocomia aculeata fruits from the Cerrados and Pantanal biomes in Mato Grosso do Sul, Brazil, Ind. Crop. Prod. 45 (2013) 208–214. [6] USDA, Oilseeds: World Markets and Trade, United States Department of Agriculture, Foreign Agricultural Service, Washington, D.C., 2018. [7] S. Falasca, A. Ulberich, S. Pitta-Alvarez, Development of agroclimatic zoning model to delimit the potential growing areas for macaw palm (Acrocomia aculeata), Theor. Appl. Climatol. 129 (2017) 1321–1333. [8] S.L.S. Moreira, C.V. Pires, G.E. Marcatti, R.H.S. Santos, H.M.A. Imbuzeiro, R.B.A. Fernandes, Intercropping of coffee with the palm tree, macauba, can mitigate climate change effects, Agric. For. Meteorol. 256–257 (2018) 379–390. [9] M. Plath, C. Moser, R. Bailis, P. Brandt, H. Hirsch, A.-M. Klein, D. Walmsley, H. von Wehrden, A novel bioenergy feedstock in Latin America? Cultivation potential of Acrocomia aculeata under current and future climate conditions, Biomass Bioenergy 91 (2016) 186–195. [10] A.D.S. César, F.D.A. Almeida, R.P. de Souza, G.C. Silva, A.E. Atabani, The prospects of using Acrocomia aculeata (macaúba) a non-edible biodiesel feedstock in Brazil, Renew. Sust. Energ. Rev. 49 (2015) 1213–1220. [11] M.C. Coimbra, N. Jorge, Proximate composition of guariroba (Syagrus oleracea), jerivá (Syagrus romanzoffiana) and macaúba (Acrocomia aculeata) palm fruits, Food Res. Int. 44 (2011) 2139–2142. [12] M.C. Coimbra, N. Jorge, Fatty acids and bioactive compounds of the pulps and kernels of Brazilian palm species, guariroba (Syagrus oleraces), jerivá (Syagrus romanzoffiana) and macaúba (Acrocomia aculeata), J. Sci. Food Agric. 92 (2012) 679–684. [13] L.D.H.C.S. da Conceição, R. Antoniassi, N.T.V. Junqueira, M.F. Braga, A.F. de FariaMachado, J.B. Rogério, I.D. Duarte, H.R. Bizzo, Genetic diversity of macauba from natural populations of Brazil, BMC Res. Notes 8 (2015) 406. [14] J.C. del Río, A.B. Evaristo, G. Marques, P. Martín-Ramos, J. Martín-Gil, A. Gutiérrez, Chemical composition and thermal behavior of the pulp and kernel oils from macauba palm (Acrocomia aculeata) fruit, Ind. Crop. Prod. 84 (2016) 294–304. [15] A.B. Evaristo, J.A.S. Grossi, L.D. Pimentel, S. de Melo Goulart, A.D. Martins, V.L. dos Santos, S. Motoike, Harvest and post-harvest conditions influencing macauba (Acrocomia aculeata) oil quality attributes, Ind. Crop. Prod. 85 (2016) 63–73. [16] S.P. Favaro, C.F. Tapeti, C.H.B. Miranda, G. Ciaconini, M.M.M. Amélia, R. Roscoe, Macauba (Acrocomia aculeata) pulp oil quality is negatively affected by drying fruits at 60 °C, Braz. Arch. Biol. Technol. 60 (2017) e17160373. [17] C.H. Lescano, I.P. Oliveira, L.R. Silva, D.S. Baldivia, E.J. Sanjinez-Argandoña, E.J. Arruda, I.C.F. Moraes, F.F. Lima, Nutrients content, characterization and oil extraction from Acrocomia aculeata (Jacq.) Lodd. fruits, Afr. J. Food Sci. 9 (2015) 113–119. [18] S.G. Montoya, S.Y. Motoike, K.N. Kuki, A.D. Couto, Fruit development, growth, and stored reserves in macauba palm (Acrocomia aculeata), an alternative bioenergy crop, Planta 244 (2016) 927–938. [19] S.B. Reis, M.O. Mercadante-Simões, L.M. Ribeiro, Pericarp development in the macaw palm Acrocomia aculeata (Arecaceae), Rodriguesia 63 (2012) 541–549. [20] C.P. Trentini, K.A. Santos, E.A. da Silva, V.A.D.S. Garcia, L. Cardozo-Filho, C. da Silva, Oil extraction from macauba pulp using compressed propane, J. Supercrit. Fluids 126 (2017) 72–78. [21] R. Schex, V.M. Lieb, V.M. Jiménez, P. Esquivel, R.M. Schweiggert, R. Carle, C.B. Steingass, HPLC-DAD-APCI/ESI-MSn analysis of carotenoids and α-tocopherol in Costa Rican Acrocomia aculeata fruits of varying maturity stages, Food Res. Int. 105 (2018) 645–653. [22] C.A. Harvey, C. Villanueva, H. Esquivel, R. Gómez, M. Ibrahim, M. Lopez, J. Martinez, D. Muñoz, C. Restrepo, J.C. Saénz, J. Villacís, F.L. Sinclair, Conservation value of dispersed tree cover threatened by pasture management, For. Ecol. Manag. 261 (2011) 1664–1674. [23] V.M. Lieb, L.K. Schuster, A. Kronmüller, H.-G. Schmarr, R. Carle, C.B. Steingass, Fatty acids, triacylglycerols, and thermal behaviour of various mango (Mangifera indica L.) kernel fats, Food Res. Int. 116 (2019) 527–537. [24] Q. Gu, F. David, F. Lynen, P. Vanormelingen, W. Vyverman, K. Rumpel, G. Xu, P. Sandra, Evaluation of ionic liquid stationary phases for one dimensional gas chromatography–mass spectrometry and comprehensive two dimensional gas chromatographic analyses of fatty acids in marine biota, J. Chromatogr. A 1218 (2011) 3056–3063.

3.4.2. Kernel oils LaLaLa/CyLaP (7.1–7.5%) and OOL (6.8–7.0%) represented the most abundant TAGs in kernel oils of fruits harvested from the bunch (maturity stages A and B). LaLaLa has been previously identified to be the major TAG in kernels of ripe Brazilian Acrocomia fruits prior to abscission [14]. Regrettably, merely the distribution of TAGs according to their total atom carbon numbers in the molecule has been determined in the aforementioned study. OOL (8.0 ± 0.9%), OOO (6.6 ± 2.9%), and LaLaLa/CyPL (6.1 ± 0.7%) prevailed in the oils of abscised fruits (C). Unlike the mesocarp oil, kernel TAGs were welldistributed over a wide range of ECNs (Fig. 4). As already seen for fatty acids (Table 1), TAG composition of the kernel oils remained almost constant during ripening. Highly saturated TAGs decreased by trend, whereas unsaturated TAGs accumulated without showing statistically significant differences. TAGs composed of medium-chain saturated (C8:0–C14:0) as well as long-chain polyunsaturated fatty acids (C18:2n6), representing particularly TAGs with ECN 30–42, tended to decrease. Similar to the A. aculeata mesocarp oil observed herein and E. guineensis [31], TAGs with C16:0 and C18:1n9, i.e., in particular those with ECN 44–50, accumulated in the kernels during ripening. 4. Conclusions The composition of mesocarp and kernel lipids extracted from Costa Rican A. aculeata fruits differed. Di- and triunsaturated TAGs composed of long-chain fatty acids (C16:0, C18:1n9) prevailed in the mesocarp oil. In contrast, the kernel showed a more complex lipid profile, containing almost equal proportions of saturated and unsaturated TAGs. The predominant fatty acid of the kernel fat was C12:0 (41.6–42.9%), followed by the monounsaturated C18:1n9 and the saturated mediumchain fatty acids C14:0 and C16:0. Moreover, this study enlarges the knowledge on A. aculeata oil composition and the possible impact of fruit maturation. Detailed GC-FID, GC-MS, and HPLC-DAD-APCI-MS and ESI-MSn analyses revealed almost consistent fatty acid and TAG proportions in both mesocarp and kernel oils among all maturity stages assessed. Whereas total lipid contents of the kernel remained unaffected, those of the mesocarp increased, particularly at the end of maturation. Consequently, diminished oil yields are to be expected when unripe mesocarp is processed. To avoid manual fruit collection and microbial contaminations, harvesting of the entire bunch at progressed maturity is recommended to optimize the production of edible oil. Further lipids in mesocarp and kernel oils such as free fatty acids, partial glycerides, wax esters, phospholipids, or phytosterols may be subject matter of future research. Acknowledgments This work was supported by the Adalbert-Raps-Foundation (Kulmbach, Germany). R.S. is grateful for a travel grant provided by the Hanns-Seidel-Stiftung (München, Germany). The authors are grateful to Klaus Mix for his technical assistance in moisture determination. The authors thank Green Integrated Energies S.A. (San José, Costa Rica) for the help in the identification and harvest of samples. 12

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