Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

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Biocatalysis and Agricultural Biotechnology

journal homepage: www.elsevier.com/locate/bab

Synchronization between photosynthetic responses to seasonality

during fruit development and fatty acid profile of mesocarp oil in
macauba (Acrocomia aculeata)
Cassia Duarte Oliveira a, 1, Bianca Marise Pereira e Silveira a, 2,
Natalia Fernanda de Assis b, Gustavo Resende Rios b, Advanio Inácio Siqueira-Silva c,
José Carlos Baffa Júnior a, Pollyanna Amaral Viana a, Eduardo Gusmão Pereira b, *
a Instituto de Ciências Exatas e Tecnológicas, Universidade Federal de Viçosa (UFV)- Campus Florestal, Florestal, MG, Brazil
b Instituto de Ciências Biológicas e da Saúde, UFV - Campus Florestal, Florestal, MG, Brazil
c Universidade Federal do Oeste do Pará (UFOPA), Santarém, Pará, Brazil

ARTICLE INFO ABSTRACT

Keywords: Macauba palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart. Arecaceae) is a novel oil crop with
Fatty acid composition wide industrial applicability. However, little is known about the photosynthetic responses to sea-
FT-IR spectroscopy sonality during fruit development in macauba palm and its effects on the fatty acid profile of
Novel oil crop mesocarp oil and its physicochemical and spectroscopic characteristics. The fatty acid profile
Photosynthesis
changed in six fruit developmental stages (ranging from 180 days after anthesis to full ripening
Physicochemical analysis
and dispersion). This occurred during the transition from dry to rainy season concomitant with
adjustments in photosynthetic responses. The increase in mesocarp oil yield in the fruit was syn-
chronized with the higher photosynthetic capacity at the beginning of the rainy season, being
characterized by a raise in oleic acid. The dynamics of soluble solids, acidity, pH, total sugars and
starch were proportional to the spectroscopic changes and allowed to determine the ideal harvest
point: 360 days after anthesis, with fruits still in the bunch. The macauba palm fruits in this stage
showed suitable physical and chemical characteristics for industrial use, as well as higher oil
yield. The monitoring of chemical changes with Fournier transformed infrared spectroscopy tech-
niques would aid fast and precise detection of the fruit development and its ideal harvest point.

1. Introduction
Macauba palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) is a novel oil crop, native to South America, with abundant distribution
in the central-west and southeast regions of Brazil. In this country, the oil from the fruits of the macauba palm has the potential to be-
come the main source of raw material for biodiesel production (César et al., 2015; Evaristo et al., 2016a). The oils extracted from the
fruits of the macauba have high industrial value, with potential use in different sectors, such as food, cosmetics and energy (Resende
et al., 2020). Macauba fruits are characterized by the high content of oil produced, around 6.2 kg of oil per hectare (Motoike and
Kuki, 2009).

* Corresponding author.
E-mail address: [email protected] (E.G. Pereira).
1 Programa de Pós-graduação em Ciência dos Alimentos, Universidade Federal de Lavras, UFLA, Lavras, MG, Brazil.
2 Programa de Pós-graduação em Ciência e Tecnologia de Alimentos, Universidade Federal dos Vales do Jequitinhonha e Mucuri, UFVJM, Diamantina, MG, Brazil.

https://doi.org/10.1016/j.bcab.2022.102423
Received 8 February 2022; Received in revised form 30 June 2022; Accepted 8 July 2022
Available online 13 July 2022
1878-8181/© 2022 Elsevier Ltd. All rights reserved.
C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

Macauba palm has high rusticity and drought tolerance, also occurs in acidic soils and can be grown in open areas and pastures
(Castro et al., 2017; Plath et al., 2016; Rosa et al., 2019). However, currently in Brazil, the commercial exploitation and plantations of
macauba palm are still incipient and the fruit extraction from natural populations predominates. Usually, during the extraction
process, the fruits are collected several days after falling to the ground. This can cause great damage, such as contamination with mi-
croorganisms from the soil, which can accelerate the degradation of this fruit as well as losses in oil quality (Evaristo et al., 2016b).
Although few studies indicate the viability for the collection of fruits directly from the bunch, before abscission, the harvest period
can influence the fatty acid composition of the oil and the pulp physicochemical traits, compromising its use in the production of
biodiesel and other applications (Coimbra and Jorge, 2012; Evaristo et al., 2016b; Resende et al., 2020).
The oil extracted from the mesocarp of ripe macauba fruits is characterized by its orange color due to the presence of carotenoids.
This oil is predominantly composed of unsaturated fatty acids, with oleic acid being the main constituent (Ciconini et al., 2013;
Moreira et al., 2020). The developmental period of macauba palm fruits occurs during one year and is divided into three stages
(Mazzottini-dos-Santos et al., 2015; Montoya et al., 2016). The first phase characterizes the histo-differentiation of the pericarp last-
ing until 90 days after anthesis (DAA). At this stage, it is not possible to identify the different internal parts of the fruit, since the tis-
sues of the exocarp and endocarp are not fully formed and have high water content. This initial phase occurs in the rainy summer in
the tropical region of Brazil and coincides with the final ripening stage of the fruits developed in the last year, in which the same indi-
vidual presents bunches in both ripening stages of the fruits.
The beginning of lipids accumulation takes place in the second stage, which lasts up to 300 DAA and is characterized by seed dif-
ferentiation and accelerated fruit growth. In this phase, the fruit reaches its maximum size, mainly due to an increase in the mesocarp
and endosperm size. There is also a reduction in the water content of the fruits, due to their substitution for reserve substances. The
initial phase of this stage occurs during the dry season, with the accumulation of lipids concentrated at the beginning of the rainy sea-
son. In the third stage, it is possible to completely differentiate the internal parts of the fruits. This period is characterized by the
ripening of the mesocarp until abscission, which lasts up to 360 DAA (Mazzottini-dos-Santos et al., 2015; Montoya et al., 2016). Be-
sides lipids, the main substances that are stored at the end of fruit ripening are the carbohydrates, and together with the proteins pre-
sent mainly in the seed, have a low mineral content (Mazzottini-dos-Santos et al., 2015). Tracking the specific changes in oil concen-
tration and fatty acid profile in macauba palm fruit could be tricky, due to the difficulties and complexity of laboratory procedures.
Some efforts have been made to predict the oil concentration in the mesocarp of macauba palm fruit (Anderson et al., 2017; Matsimbe
et al., 2015). However, during fruit development, the changes in oil concentration are related to the changes in physicochemical
traits, which can be assessed also with Fournier transformed infrared (FT-IR) spectroscopy techniques and would allow estimating the
fruit collection time.
Seasonal changes during the annual period of fruit development in macauba palm would affect the photosynthetic process respon-
sible for assimilates used in the synthesis of carbohydrates and oil in the final ripening stage. A reduction in water availability in the
soil during the dry season would affect the development of fruits through its effects on photosynthetic metabolism, concerning stom-
atal and non-stomatal responses (Ali et al., 2021). The transition from the dry season to the rainy season in palms requires adjust-
ments in stomatal control and the maintenance of efficient use of light energy, especially in the presence of strong photosynthate sinks
during flowering and fruit development (Legros et al., 2009; Mialet-Serra et al., 2008). In palm trees the photosynthetic rate influ-
ences the fruiting time especially under drought (Vogado et al., 2020), however until now, there was no information about how the
photosynthetic responses to seasonality are related to fruit development and reserve metabolism in a tropical palm.
Therefore, this study aimed to assess the photosynthetic adjustments in response to seasonality during fruit development in
macauba palm and its effects on the fatty acid profile and mesocarp oil concentration. In addition, we sought to characterize the
changes in physicochemical properties of the oil-rich mesocarp during fruit development and final ripening stage, in comparison with
its spectroscopic characteristics based on the FT-IR technique.

2. Material and methods

2.1. Study area
The experiment was carried out at the Federal University of Viçosa (UFV) Campus Florestal, Minas Gerais, Brazil. The climate in
Florestal is classified as tropical. The average annual rainfall is 1434 mm. The average annual temperature is 21.5 °C, with a mini-
mum temperature of 10.4 °C and a maximum of 29.6 °C. During the experiment, from July 2014 to January 2015, the total precipita-
tion was 967 mm and the average temperature and relative humidity of 20.7 °C and 68.7% respectively (Fig. 1A).
Acrocomia aculeata ((Jacq.) Lodd. ex Mart.), Arecaceae, is an arborescent and perennial palm tree that is wide-spread in tropical
America (César et al., 2015). During the experiment, macauba palm plants about 5 m tall and in full production were selected in a
natural population occurring in a pasture area (870 mamsl, 19° 52′ 32.16″ S and 44° 24′ 47.52″ W). The inflorescences in each palm
were marked before anthesis and the fruits from 24 adult macauba palm plants were collected at the following stages of develop-
ment: 180, 240, 270, 300 and 360 DAA. In the final stage (360 DAA), were collected fruits in the bunch and fruits recently fallen on
the ground (less than three days). Initially, the fruits were weighted and the exocarps were broken manually. The fruits were pulped
with a stainless-steel knife. The mesocarp from fruits at all developing stages, including the fruits collected in the ground, was
weighted and a portion was stored at freezer temperature (−20 °C) until the time of physicochemical analysis and the other portion
was used for oil extraction.
At each stage of fruit development, the determination of water content in soil and macauba leaves was performed, together with
photosynthetic analysis.

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C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

Fig. 1. Climatic variables (precipitation and temperature; A), soil water content (SWC; B) and relative water content (RWC; C) in leaves of macauba palm over the days
after anthesis (DAA). Different letters represent a statistically significant difference between the fruit developmental stages by the Tukey test (α = 5%). Values are the
means ± standard error (n = 7).

2.2. Soil water content and leaf relative water content measurements
The soil water content (SWC) and the leaf relative water content (RWC) were measured at noon on the corresponding sampling
dates. The SWC was determined on a gravimetric basis from samples collected at 0–10 cm depth, calculated as the difference between
wet weight (W) and dry weight (DW) using the following equation: SWC (%) = 100 × [(W-DW)/DW].
The RWC was determined in samples from middle leaflets from a fully expanded leaf. Three leaflet segments (with approx. 3 cm2)
were removed from each leaf sample, and the fresh weight (FW) was immediately measured. The turgid weight (TW) was determined
after rehydration of the leaf segments immersed in distilled water and kept at 4 °C for 24 h. The DW was determined by placing the

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C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

leaf discs in an oven at 65 °C to reach a constant weight. The RWC was calculated as the difference between the different leaf weights,
using the following equation: RWC (%) = 100 × [(FW – DW)/(TW – DW)] according to Barrs and Weatherley (1962).

2.3. Gas exchange, chlorophyll a fluorescence and relative chlorophyll content

Gas exchange measurements were performed between 07:00 a.m. and 11:00 a.m., on the corresponding sampling dates; using a LI-
6400xt infrared gas analyzer (Li-Cor Inc., Lincoln, NE, USA). Light (1500 μmol m−2 s−1) was provided by the LED light source in the
leaf chamber fluorometer (6400–40, Li-Cor Inc.), which had an area of 2 cm2. The measurements were performed in detached leaflets
from the middle portion of the second or third leaf below the spear-leaf (Pires et al., 2013), within 120 s of detachment. Field tests
with detached leaflets were made previously to the measurements to confirm the lack of loss in the photosynthetic capacity during the
time of measurement (see complete description in Sup. Material Fig. S1). During all measurements, the CO2 levels were kept at
400 μmol mol−1 using the control system (6400–01, Li-Cor Inc.), with a mean leaf temperature and air humidity of 27.5 °C and 46%,
respectively. The following gas exchange variables were evaluated: net photosynthetic rate (A, μmol m˗2 s˗1), stomatal conductance
(gs, mol m˗2 s˗1), transpiration (E, mmol m˗2 s˗1), the ratio of internal to external CO2 concentration (Ci/Ca) and the instantaneous wa-
ter use efficiency (Wt = A/E, μmolCO2 mmolH2O m˗2 s˗1).
The chlorophyll content index was measured using the portable ClorofiLOG meter (CFL1030, Falker, Porto Alegre, RS, Brazil). The
measurements were performed three times in the middle part of the same leaflet used for gas exchange analysis, and the mean of the
three measurements was calculated as a replicate.
Chlorophyll a fluorescence was measured on the same leaves used for the gas exchange measurements, using a Mini-PAM fluorom-
eter (Heinz Walz, Effeltrich, Germany). The initial fluorescence (F0) and the maximum quantum efficiency of photosystem II (PSII)
(Fv/Fm) were determined in dark acclimated leaflets for at least 30 min (Genty et al., 1989). Then, the leaves were exposed to photo-
synthetic photon flux density (PPFD) of 1500 μmol m−2 s−1 for 40 s, followed by a saturating light pulse, to determine the effective
quantum yield of PSII (ϕPSII) (Genty et al., 1989) and the non-photochemical quenching (NPQ) (Bilger and Bjorkman, 1990).

2.4. Oil extraction and fatty acid profile

The cut pulp sections from fruits at the same developmental period were dried at 60 °C for 15 h, homogenized, weighted and then
subjected to extraction. The extraction of the oil from the macauba fruit's pulp was carried out in a Soxhlet extractor, with 300 mL of
petroleum ether P.A at 70 °C for 8 h (IAL, 2008). The oil concentration in the mesocarp of the six fruit developing stages was calcu-
lated by the difference between the initial and final mass of extracted oil (IAL, 2008). The yield of macauba pulp oil (g kg−1fruit) was
also determined by considering the oil concentration in pulp and the respective fruit mass.
For chromatographic analysis, a 100 μL sample of macauba pulp oil was added to a test tube with 1 mL of the methanol P.A./wa-
ter solution 50%/50% (v/v). Then, the test tube was heated in a water bath at a temperature of 70 °C for 1 h. It was cooled with tap
water and 2 ml of distilled water was added. The extraction was done three times with 1 mL of hexane. The test tube was shaken by
hand, opening the cap at small intervals because of the pressure. Subsequently, the solution supernatant (organic part) was collected.
Chromatographic analysis was performed using a gas chromatograph model (CG Solution, Shimadzu, Japan), equipped with a flame
ionization detector (FID). The compounds were separated and identified in a Carbowax capillary column (30 m x 0.25 mm). For chro-
matographic separation, 1 μL of the sample was injected in a Split = 10 system. The nitrogen gas was used as a carrier with a linear
velocity programmed to 43.2 cm s−1 and the gases hydrogen and synthetic argon formed the flame in the detector. The injector and
detector temperatures were controlled isothermal at 200 °C and 220 °C. The initial temperature of the column was 150 °C, maintained
for 5 min, increasing by 4 °C per min. until reaching 220 °C (maintained for 20 min). The flow of carrier gas in the column was
1 mL min−1. The chromatographic conditions were set using a mix of standards purchased from Sigma-Aldrich® (USA) which con-
tained known mass and proportion of the evaluated fatty acids: palmitic acid, stearic acid, oleic acid, linoleic and linolenic acids. For
quantification, the peak area was normalized following a comparison of the retention time of the samples with the commercial chro-
matographic standards.

2.5. Physicochemical characterization of the pulp of macauba fruits

The physicochemical analyses performed were total titratable acidity (TTA), pH, total soluble solids (TSS), reducing sugar, non-
reducing sugar, total sugar and starch.
The TTA was determined using 5 g of pulp homogenized in 50 ml of distilled water, with the addition of 2 drops of phenolph-
thalein solution and the suspension titrated with 0,1 M NaOH (IAL, 2008).
The pH was measured using 5 g of pulp diluted in 50 mL of distilled water (IAL, 2008). The concentration of TSS was determined
using a portable digital refractometer (Quimis -Q-107D162) at 20°C. Three drops from each sample of homogenized macauba pulp
were applied directly in the refractometer and the levels of TSS were expressed in °Brix (IAL, 2008).
Samples of the dry mesocarp of the fruits were used for the extraction and quantification of total sugars, reducing sugars, non-
reducing sugars and starch. The total sugar concentration was determined by spectrophotometry, using the phenol-sulfuric method
(Dubois et al., 1956). The concentration of reducing sugars was determined by spectrophotometry, using the dinitrosalicylic acid
method. Non-reducing sugars were quantified by the difference between total and reducing sugars (Miller, 1959).
For the extraction of starch, the portion of mesocarp remaining after the extraction of total sugars was used. The starch concentra-
tion was determined in 500 mg of mesocarp, extracted with hot ethyl alcohol (Hodge, 1962).

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2.6. FT-IR spectroscopy analysis of the pulp

Changes in the chemical composition of the fruits were also characterized by the FT-IR spectroscopy technique. Spectra were ob-
tained, in the region from 4000 to 600 cm−1, using a Frontier spectrometer (PerkinElmer, model “FT-IR Spectrum 100S″, USA) in
transmission mode, using 0.2 g of the dry mesocarp of macauba palm fruits.

2.7. Experimental design and statistical analysis

The experiment was carried out in a completely randomized design (DIC), with six sampling times (days after anthesis), except for
SWC, RWC and photosynthetic variables which had five sampling times. For the oil extraction, physico-chemical and FT-IR character-
izations five replications were used, and seven replications for photosynthetic analysis, SWC and RWC. The results obtained were ana-
lyzed statistically using the software R (R Core Team, 2018). Analysis of variance (ANOVA) was performed, followed by Tukey's test
(5% probability) and principal component analysis (PCA).

3. Results and discussion

3.1. Photosynthetic adjustments in macauba palm during fruit development and climate seasonality in the field
During the evaluated period, the SWC ranged from 7% in the dry season (240 DAA) to 25% in the rainy season (360 DAA) (Fig.
1B). The leaf RWC showed its minimum values (40%) in 180 DAA (July 2014) and increased until the beginning of the rainy season
(in October 2014), with values between 60 and 75% in 360 DAA (Fig. 1C).
The reductions of 73% and 40% in SWC and RWC, respectively, in the driest period, did not result in significant reductions in net
photosynthesis in macauba palm (Fig. 2A). The higher values found in 300 DAA were the result of a higher stomatal conductance (gs)

Fig. 2. Net photosynthetic rate (A; A), stomatal conductance (gs; B), water use efficiency (Wt, C), chlorophyll index (D), effective quantum yield of photosystem II
(ϕPSII; E) and non-photochemical quenching (NPQ; F) of macauba palm over the days after anthesis (DAA). Different letters represent a statistically significant differ-
ence between the fruit developmental stages by the Tukey test (α = 5%). Values are the means ± standard error (n = 7).

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in the same period (Fig. 2B). With steady water availability in the soil during the rainy season, a higher gs would allow maximum pho-
tosynthetic capacity during the period of higher sink demand due to fruit filling (Mazzottini-dos-Santos et al., 2015; Montoya et al.,
2016). The lower gs in the dry season have ensued the rise observed in RWC at 240 DAA, and in the same way, the higher gs is related
to a reduction in RWC at 300 DAA (Figs. 1C and 2B). The effective stomatal control of photosynthetic capacity in macauba palm has
been reported in other works, regarding its exposition to drought (Rosa et al., 2019) and light stress (Dias et al., 2018). The higher
stomatal closure during the dry season is commonly documented for palm species (Legros et al., 2009; Mialet-Serra et al., 2008;
Vogado et al., 2020). The highest values of water use efficiency (Wt) were reached during the transition from the end of the dry to the
rainy season (between 240 and 300 DAA), without significant differences between 180 and 360 DAA (Fig. 2C). The maintenance of a
higher Wt during all the evaluated periods is related to the stomatal control of macauba's photosynthesis. The partial closure of stom-
ata in the dry season led also to a significative lower transpiration rate (E) and the ratio of internal to external CO2 concentration (Ci/
Ca) (Sup. Material Fig. S2). The photosynthetic adjustments concerning effective stomatal control and thus the recovery of RWC con-
tributed to the plant strategy to overcome the seasonality during fruit development (Montoya et al., 2016). The high sink demand
during fruit development contributed to the maintenance of high carbon assimilation even in the dry season. The demand for carbo-
hydrates in sink organs, such as developing fruits of macauba, is a major factor that contributes to higher photosynthetic capacity
(Demmig-Adams et al., 2017)
The chlorophyll index did not change due to seasonality regardless of the DAA in macauba palm plants (Fig. 2D). In the same way
as net photosynthesis, the highest values of effective quantum yield of PSII (ϕPSII) were found at 300 DAA (Fig. 2E). The macauba
palm plants showed higher non-photochemical quenching (NPQ) during the dry season and at the end of the rainy season (Fig. 2F).
Besides stomatal control, macauba palm plants were able to adjust the light energy use to keep the photosynthetic efficiency during
the dry season, as showed by the high values of maximum quantum efficiency of PSII (Fv/Fm) (Fig. S2C). The dissipation of excess en-
ergy through NPQ at minimum SWC due to the dry season was an effective mechanism that prevented damage to the PSII reaction
center. The increase of NPQ at 360 DAA (Fig. 2F) might be an important photoprotection mechanism to avoid damage in PSII mainly
during source-sink balance (Demmig-Adams et al., 2017) at the end of fruit ripeness which overlaps with the initial fruit develop-
ment.

3.2. Changes in lipid concentration of macauba mesocarp during fruit development

The mesocarp weight in the fruit increased significantly (p<0.05) over the DAA (Fig. 3A). Maximum productivity was achieved
for fruits harvested directly from plants in full maturity (360 DAA), differing from fruits harvested from the soil and other evaluated
points (Fig. 3A). The lower mesocarp weight of the fruits collected from the soil could be due to the loss of water during the remaining
time in the soil or due to the loss of dry mass due to respiratory metabolism to provide energy for the continuous metabolism of the
fruit, as also observed by Tilahun et al. (2020). Natural phenomena in the environment, such as changes in temperature, relative hu-
midity and oxygen, also contribute to the decrease in the water content of the fruits (Tilahun et al., 2020).
The oil concentration in the mesocarp and the yield of macauba pulp oil in the fruit increased after 270 DAA, reaching the maxi-
mum values with the final ripening stage of the fruits (Fig. 3B and C). However, only for the mesocarp oil yield in fruits, higher values
(p<0.05) were found for the fruits harvested in the plant at 360 DAA, in comparison with the fruits harvested in the ground (Fig. 3C).
The lower oil yield in the fruits harvested on the ground, when compared with those harvested in the bunch in the same period
(360 DAA) indicates the possible deterioration through microorganisms and chemical and physical reactions, highlighted by the
higher humidity in the ground during fruit dispersion. The longer permanence of fruits in the soil increase the free fatty acid content,
due to the action of lipase enzymes present in the fruit and in the microorganisms that colonize the interior and on the surface of the
fruit (Evaristo et al., 2016b). To ensure better yields and avoid continuous deterioration, it is recommended to dry the fruits before
storage for future oil extraction (Ciconini et al., 2013). Moreover, when the fruits are collected from the soil, they are more suscepti-
ble to excessive irradiation, desiccation and oviposition of insects (Evaristo et al., 2016b; Montoya et al., 2016), which can generate
lipid oxidation and degradation of biological components, such as cell membranes and nucleic acids, resulting in loss of quality of the
oil extracted from the pulp. The rate of oil oxidation is also related to the content of alkyl esters and the position of the carbon double
bond (Evaristo et al., 2016b)
The increase in mesocarp lipid concentration is probably the result of the accumulation of storage compounds in the fruit over its
developmental stages. The fibrous mesocarp of macauba palm fruits is formed by oil-rich parenchymatic cells, lignified sclerenchyma-
tous fiber bundles, idioblasts (containing phenolic compounds) and large mucilaginous ducts (Montoya et al., 2016; Reis et al., 2012).
The lipid concentration in mesocarp from the final ripening stages in the present study (Fig. 3B) was higher than those obtained by
Oliveira et al. (2013) in macauba fruits and lower than those observed by Del Río et al. (2016). However, Ciconini et al. (2013) ob-
served no variation in mesocarp oil contents in fruits harvested from macauba palms from the Brazilian Cerrado and Pantanal. The ge-
netic variability mighty, therefore, plays a significant role in determining the oil yield in different A. aculeata accessions (Costa et al.,
2018; Sá et al., 2022). However, the wide range of values found for mesocarp oil concentration in the macauba fruits also depicted the
influence of environmental factors during fruit development, considering the tropical occurrence of macauba population. Environ-
mental factors such as lower or moderate temperatures can negatively affect the dry weight gain of the pulp (Resende et al., 2020).
Besides temperature, the water availability in the soil during fruit development can also affect the fresh weight of fruits and conse-
quently its oil concentration. The water deficit can impair the synthesis of carbohydrates and prevent the expansion of the cells, tis-
sues and organs of these fruits (Resende et al., 2020).

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Fig. 3. Mesocarp weight in fruits (A), oil concentration in mesocarp (B), and yield of mesocarp oil in fruits (C) of macauba palm collected over the days after anthesis
(DAA) (● fruits collected from the plant) (○ fruits harvested from the ground). Different letters represent a statistically significant difference between the fruit develop-
mental stages by the Tukey test (α = 5%). Values are the means ± standard error (n = 5).

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3.3. The fatty acids profile of the pulp oil from developing fruits of macauba
The characterization of the oils extracted from the fruits of the macauba palm at the different DAA revealed a dynamic change in
fatty acids profile through fruit developmental stages (Fig. 4). Palmitic and stearic acids reached the highest values at the 240 DAA,
declining through the fruit developmental phase until the lowest values were found at 360 DAA in fruits harvested on the ground, but
without significant differences with fruits collected at 300 DAA (Fig. 4A and B). Oleic acid showed increased levels over the fruit de-
velopmental time from 240 DAA to 360 DAA in the fruits harvested on the ground (Fig. 4C). Both linoleic and linolenic acids showed
higher levels at the developmental stage of 180 DAA, reaching lower values from 270 DAA to 360 DAA, irrespective of fruits collected
in the tree or in the ground (Fig. 4D and E). The fruit development and its final ripening process with an increase in oleic acid are syn-
chronized with the photosynthetic performance during the rainy season to sustain carbohydrate dynamics and lipid accumulation,
mainly oleic acid. The use of macauba pulp oil for industrial application would be suitable by using fruits with high values of oleic
acid, reaching greater lipid concentration at the final ripening stages (360 DAA collected in the bunch or in the ground), since for this
purpose the oils must be rich in unsaturated fatty acids (Navarro-Díaz et al., 2014). Similar fatty acid composition at the end of the
ripening stage, with oleic acid being the most abundant, has been reported by other authors (Coimbra and Jorge, 2012; Del Río et al.,
2016).
In the 180 and 240 DAA period, linoleic and palmitic acid predominated in the pulp of macauba fruits, respectively (Fig. 4). The
linoleic fatty acid is an essential fatty acid, and a precursor in the omega 3 and 6 synthesis routes (Sun et al., 2016). It is difficult to
compare the fatty acid profile with reported literature, since many papers do not describe in which developmental stage (e.g. DAA)

Fig. 4. Fatty acids profile in macauba palm fruits over the days after anthesis (DAA): palmitic acid (A), stearic acid (B), oleic acid (C), linoleic acid (D) and linolenic
acid (E). (● fruits collected from the plant) (○ fruits harvested from the ground). Different letters represent a statistically significant difference between the fruit devel-
opmental stages by the Tukey test (α = 5%). Values are the means ± standard error (n = 5).

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the fruits were collected or even do not inform the harvest period (dos Santos Ribeiro et al., 2017; Favaro et al., 2017; Rodrigues et al.,
2018). Even not informing the fruit developmental stage, Hiane et al. (2005) observed a predominance of oleic acid (66%), palmitic
acid (16%), stearic acid (6%) and linoleic acid (5%) in macauba palm fruits, probably collected at or after 270 DAA, based on the fatty
acid profile of the present study. Considering only ripe fruits collected directly from the soil between November to January
(2008–2009), Coimbra and Jorge (2012) found similar fatty acid composition in the pulp oil of the macauba fruit: oleic (53%),
palmitic (25%), linoleic (14%), palmitoleic (4%) and linolenic (2%).
An increase in oleic acid and a reduction in palmitic and stearic acids were observed when the fruits were compared in 360 DAA
on the ground and in the bunch (Fig. 4A–C). This occurred due to the metabolic activities of lipid synthesis, these saturated fatty acids
being converted to oleic acid through the action of enzymes and lipid chain elongation reactions. A reduction in the levels of linoleic
and linolenic unsaturated fatty acids over time may have probably occurred due to the oxidation of double bonds. A similar response
was also observed by Lieb et al. (2019), who evaluated the accumulation of palmitic, palmitoleic and oleic acids and the reduction of
linolenic and linolenic acids in macauba fruits collected in three different developmental stages (green, ripe and fully ripe, classified
according to the fixation to the bunch and color of the exocarp). The reduction in polyunsaturated fatty acids and an increase in
mono-unsaturated ones are well reported also for E. guineensis (Kok et al., 2013). The macauba oil appears as a promising source of
oils for human consumption in a near future, as an alternative to oil palm, which is responsible for 33% of vegetable oil and 45% of
edible oil consumed worldwide (Resende et al., 2020). However, with the advantage that the cultivation of macauba is possible in ar-
eas of low vapor pressure deficit in the air, such as the Cerrado, or even in pastures (Resende et al., 2020)

3.4. The physicochemical analysis provides a signature of the macauba fruit ripening
Similar to the fatty acid profile, carbohydrates concentration changed over the developmental stages in macauba palm fruits (Fig.
5). The dynamics of carbohydrate accumulation and consumption are related to the synthesis of oil in macauba fruits which also oc-
curs in the final ripening stage (Montoya et al., 2016). For starch concentration, higher values were observed on days 240 and 300
DAA when compared with fruits harvested on the bunch at 360 DAA (p<0.05) (Fig. 5A). The reduction of the starch levels and in-
crease of the oil concentration in the mesocarp at the end of fruit development (Figs. 3B and 5A) were also observed by Montoya et al.
(2016). The starch concentration has a negative correlation with the oil concentration, since this carbohydrate is used during the syn-
thesis of mesocarp oil by the macauba (Montoya et al., 2016). This negative correlation was observed in our study during all the
stages of fruit development, which might be related to the genetic variability and the influence of environmental conditions. In
macauba fruits, as observed in this study (Fig. 5A) and supported by others (Mazzottini-dos-Santos et al., 2015; Montoya et al., 2016),
the starch values decreased particularly at the final developmental stage of the fruits. Similar to macauba, a reduction in starch con-
centration and an increase in oil at the end of ripening were also observed in other types of palm fruit, such as E. guineenses (Gao et al.,

Fig. 5. Concentration of starch (A), total sugars (B), reducing sugars (C) and non-reducing sugars (D) in the mesocarp of macauba palm fruits over the days after an-
thesis (DAA). (● fruits collected from the plant) (○ fruits harvested from the ground). Different letters represent a statistically significant difference between the fruit
developmental stages by the Tukey test (α = 5%). Values are the means ± standard error (n = 5).

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C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

2019; Loei et al., 2013). Starch is characterized as a transitory storage carbohydrate present in fruits and can be converted into solu-
ble sugars or intermediates, such as trioses, for the formation of fatty acids (Montoya et al., 2016). This fraction of lipids and carbohy-
drates, which are the main substances stored by fruits, characterizes the increase in the mass of the mesocarp in the final period of
ripening. This is probably because the increase in pulp mass is related to an increase in dry mass (Mazzottini-dos-Santos et al., 2015).
Starch is synthesized in plastids and stored in different organs of the plant. It is considered one of the main polysaccharides of
plant reserves. Because it is a dense molecule and poorly soluble in water, it has to be broken down into smaller, mobile molecules to
be used in the fruit ripening process. The starch reduction is related to the increase in reducing and non-reducing sugars during the
ripening process (Buckeridge et al., 2000; Montoya et al., 2016).
The concentration of total sugars, including reducing and non-reducing sugars, also showed a reduction at the end of the ripening
stage, mainly at 300 DAA (p<0.05), without significant differences between fruits at 360 DAA collected in the ground or in the bunch
(Fig. 5B–D). The reduction in the concentration of reducing sugars observed over the DAA in macauba palm fruits was reported by
Montoya et al. (2016) and Sá et al. (2022). Carbohydrates are essential components and include reducing sugars such as glucose and
fructose, non-reducing sugar such as sucrose, starch and cellulose, which play a fundamental role in the flavor and structure of the
fruit (Andrade et al., 2020).
Non-reducing sugars presented lower values at 300 DAA, which differ (p<0.05) from 270 days (Fig. 5D). Non-reducing sugars can
be easily transported through plant tissues without undergoing chemical oxidation (Wang et al., 1993). Among non-reducing sugars,
sucrose is the main one transported by plants and used for fruit growing, and it can be found in macauba fruit and other palm trees
such as coconut and oil palm (Mialet-Serra et al., 2005; Montoya et al., 2016; Sá et al., 2022; Tomlinson, 2006; Wang et al., 1993).
The decrease in total sugar concentration in macauba palm fruits at 300 DAA was not related to the TSS, in which the opposite re-
sponse was observed (Figs. 5B and 6A). The TSS in macauba palm fruits showed an increase at 300 DAA, differing significantly (p
<0.05) from the fruits collected at 180 DAA (Fig. 6A). Andrade et al. (2020) found a slightly lower value of TSS (3° Brix) in ripe
macauba fruits collected at 360 DAA after fall. Probably other constituents of TSS, such as organic acids, proteins, lipids and minerals,
are representing this increase until 300 DAA. However, the fruit ripening stage did not affect the concentration of soluble solids in
other palm fruits, such as Mauritia flexuosa (Milanez et al., 2018).
The TTA was highest in the final ripening stage, at 360 DAA, both for the fruits harvested in the bunch and in the soil, differing sig-
nificantly (p<0.05) from the other harvest periods (Fig. 6B). This increase in acidity is related to the increase in oil acidification. The
autocatalytic hydrolysis releases the fatty acids from the triacylglycerol, increasing the acidity of the oil, and consequently of the
pulp, as observed by Tilahun et al. (2020). Another factor that may be related to the increase in acidification of the oil and, conse-
quently, of the pulp, is the presence of the endogenous and microbial enzymes that increase the hydrolysis of the triacylglycerol, re-
leasing free fatty acids and other oil-related compounds during the ripening (Cavalcanti-Oliveira et al., 2015; Tilahun et al., 2020).
Milanez et al. (2018) also observed an increase in the pH and acidity of M. flexuosa fruits at different periods of ripening. This
variation in acidity might be due to the production of organic acids during ripening, that are consumed during metabolic processes
of the fruits, such as respiration. Thus, when the production of organic acids is greater than necessary for the respiration process,
they are accumulated, resulting in increased acidity of these fruits.
The TTA is an important parameter assessing the conservation status of a product, which can be altered by hydrolysis, oxidation,
or fermentation (Alves et al., 2000). The contact of the fruits with the soil at 360 DAA also resulted in a significant reduction in pH
values when compared to fruits collected at 300 DAA (Fig. 6C).

3.5. The principal component analysis (PCA) revealed the synchronization of macauba fruit development and oil accumulation with
seasonality
The PCA showed two main groups, with the best correlation on axis 1 (Fig. 7). The first one is characterized by the presence of
linoleic, stearic, palmitic, linolenic acids and reducing sugars, which in general accumulated mainly at the beginning of fruit develop-
ment (until 240 DAA). At the intermediate fruit development stage (from 270 to 300 DAA) a scattered pattern was observed in the
PCA analysis. The oil concentration was influenced by pH and also by starch, reducing, and non-reducing sugars, however, those vari-
ables showed a better correlation with the initial stages of fruit development, mainly at 270 DAA. The increase in SST that took place
at 300 DAA was highlighted in the PCA and marked the beginning of the transition from immature fruits to the beginning of the ripen-
ing process. This response is also associated with the increase in the photosynthetic capacity of macauba palm (Fig. 2) coordinated
with the rainy season and the increased demand for carbohydrates in sink organs (fruits) (Demmig-Adams et al., 2017; Legros et al.,
2009). The second group is related to all significant changes that occurred at the end of the developmental stage at 360 DAA, regard-
ing fruit collection in the bunch or in the soil. The PCA analysis clarified the correlation between oleic acid and TTA, together with the
increase in the mesocarp weight and oil concentration at the end of the developmental period of the fruits. The increase in TTA can be
useful for estimations of oleic acid concentration mainly in fruits collected in the ground, and represents a better proxy for fruit ripen-
ing than the TSS, as highlighted by the PCA. The PCA showed that the increase in the oleic acid proportion in the oil at the end of the
ripening stage is negatively related to the levels of other fatty acids present in the fruit over the analyzed time (Figs. 4 and 7). This
clustering pattern considering the changes in carbohydrates and lipids accumulated in mesocarp from macauba fruits over the devel-
opmental period was also observed by Sá et al. (2022) in macauba plants from different accessions. Sá et al. (2022) also found higher
lipid content and accumulation of sugars in macauba accessions from Minas Gerais State, highlighting the influence of genetic vari-
ability on oil yield (Costa et al., 2018). The extension of variability is related to the interactions between the plant's genetic potential
with its physiological responses to the climatic seasonality during fruit development, as observed in this study.

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C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

Fig. 6. Concentration of total soluble solids (A), total titratable acidity (B) and pH index (C) present in the mesocarp of macauba fruits over the days after anthesis
(DAA). (● fruits collected from the plant) (○ fruits harvested from the ground). Different letters represent a statistically significant difference between the fruit devel-
opmental stages by the Tukey test (α = 5%). Values are the means ± standard error (n = 5).

11
C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

Fig. 7. Principal component analysis of the main components among fatty acids profile, physicochemical characteristics and oil lipid concentration over the time (days
after anthesis - DAA).

3.6. The spectroscopic (FT-IR) traits highlighted the changes in chemical functional groups over the ripening stages of macauba palm fruit
Despite the differences in intensity for absorption bands of the FT-IR spectra, the same compounds were present in all fruit devel-
opmental stages. However, the signature and the intensity of the FT-IR (Fig. S3, Table S1) were able to provide information about the
ripening processes of macauba fruits. The absorption band in the region of 3314 cm−1, in which the stage at 240 DAA presented the
most intense band, probably referring to a stretch of the functional group OH, may be related to the accumulation of carbohydrates
(Fig. 5 A and B) in the developing fruits (Montoya et al., 2016).
In the region of 2926 and 2872 cm−1, two thin bands of medium intensity were observed, which supposedly correspond to
stretches of asymmetric and symmetrical C–H bonds, respectively. The fruits collected in the ground showed a significant reduction in
the transmittance values in those bands, while a higher intensity was observed in fruits from the stage at 270 DAA. The thin band, ob-
served in the 1734 cm−1 region, indicates a possible stretch of C = O of the ester, indicating the presence of oil. The esters have two
absorption regions, ranging from 1750–1730 and 1300-1000 cm−1 (Guillén and Cabo, 1997; Siudem et al., 2019). The band of
medium width observed in the region of 1602 cm−1, possibly represents a C = O connection stretch of the aldehyde, present in the
sugar composition. The higher values observed in this band at DAA 240 and 270 are correlated to the higher amount of total sugars
observed (Fig. 5). Peaks observed between 1237 and 1368 cm−1 might inversely be related to the abundance of acyl groups and oleic
acid (Jaswir et al., 2003). The acute band of greater intensity, observed in the region of 1070 cm−1, points to a probable elongation of
the C = O ester bond and the highest values of oleic acid in 360 DAA (Table S1). The bands close to 700 cm−1 correspond to the CH2
and CH vibrations (Siudem et al., 2019). The relatively small variation between the ranges is related to the sample nature, since the
macauba pulp used in this study presented some interferents, such as structural and non-structural carbohydrates. Del Río et al.
(2016) evaluated the spectroscopic properties of the oil extracted from macauba pulp and kernel and they also found a predominance
of unsaturated and saturated triglycerides, respectively. The analysis proposed here, by using the dried mesocarp of macauba fruits
for the FT-IR characterization, provides a simple method for monitoring the ripening and thus the oil concentration for harvesting, in
addition to the acyl profile of the lipids present in these oils (Rodríguez et al., 2019).

4. Conclusion
The photosynthetic adjustments in macauba palm during fruit development and seasonality include effective stomatal control
with consequent maintenance of RWC and Wt, coupled with adjustments in light use efficiency and dissipation of excess energy
through NPQ when soil water availability reached limiting values, without compromising chlorophyll content. The increase in meso-
carp oil concentration in fruits was synchronized with the higher photosynthetic capacity at the beginning of the rainy season, being
characterized by a raise in oleic acid. Those responses might also be associated with the influence of environmental conditions and
the genetic variability that determines fruit development. Considering the yield and the quality of oil for industrial purposes, the har-
vest of the fruits of the macauba palm can be carried out at 360 DAA, with the fruits still in the bunch. After dispersion, the acidity
tends to increase, thus reducing the quality of the pulp, but with an increase in the proportion of oleic acid. The changes in titratable
acidity of the pulp accompanied by reductions in carbohydrate concentration can be a proxy for fruit harvesting point at the end of
the ripening period, with higher oil yield. Alternatively, the monitoring of chemical changes with Fournier transformed infrared spec-
troscopy techniques would aid a fast and precise detection of chemical changes during macauba palm fruit development.

Author contribution statement

EGP: Funding acquisition and resources, Project administration, Supervision, Conceptualization, Formal analysis, writing-
reviewing and editing. CDO, BMPS, GRR and NFA: Investigation, Formal analysis, Writing – original draft. AISS, JCBJ and PAV: Con-
ceptualization, Investigation, Formal analysis,writing-reviewing and editing. All authors approved the manuscript.

12
C.D. Oliveira et al. Biocatalysis and Agricultural Biotechnology 43 (2022) 102423

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgments
The authors thank Bruno Luan Rosa for technical assistance in the field during fruit collection and a special acknowledge to
Alexandre C. Vicente Campos and Prof. Cláudio dos Santos Ferreira for their help on FT-IR spectroscopy analysis. This work was sup-
ported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (grant numbers APQ-01244-13 and APQ-00733-19
to EGP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 470116/2013–7 to EGP). E. G.
Pereira also thank CNPq for the research productivity grant.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.bcab.2022.102423.

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