foods

Article
Composition of Brazil Nut (Bertholletia excels HBK), Its
Beverage and By-Products: A Healthy Food and Potential
Source of Ingredients
Wilson V. Vasquez-Rojas 1,2 , Diana Martín 2 , Beatriz Miralles 3 , Isidra Recio 3 , Tiziana Fornari 2
and M. Pilar Cano 1, *

1 Department of Biotechnology and Microbiology of Foods, Institute of Food Science Research,

28049 Madrid, Spain; [email protected]
2 Department of Production and Characterization of Novel Foods, Institute of Food Science Research,
28049 Madrid, Spain; [email protected] (D.M.); [email protected] (T.F.)
3 Department of Bioactivity and Food Analysis, Institute of Food Science Research, 28049 Madrid, Spain;
[email protected] (B.M.); [email protected] (I.R.)
* Correspondence: [email protected]; Tel.: +34-910017937

Abstract: The consumption of plant-based beverages is a growing trend and, consequently, the
search for alternative plant sources, the improvement of beverage quality and the use of their by-
products, acquire great interest. Thus, the purpose of this work was to characterize the composition





 (nutrients, phytochemicals and antioxidant activity) of the Brazil nut (BN), its whole beverage
Citation: Vasquez-Rojas, W.V.; (WBM), water-soluble beverage (BM-S), and its by-products of the beverage production: cake,
Martín, D.; Miralles, B.; Recio, I.; sediment fraction (BM-D), and fat fraction (BM-F). In this study, advanced methodologies for the
Fornari, T.; Cano, M.P. Composition analysis of the components were employed to assess HPLC-ESI-QTOF (phenolic compounds), GC
of Brazil Nut (Bertholletia excels HBK), (fatty acids), and MALDI-TOF/TOF (proteins and peptides). The production of WBM was based on
Its Beverage and By-Products: A a hot water extraction process, and the production of BM-S includes an additional centrifugation
Healthy Food and Potential Source of step. The BN showed an interesting nutritional quality and outstanding content of unsaturated fatty
Ingredients. Foods 2021, 10, 3007. acids. The investigation found the following in the composition of the BN: phenolic compounds
https://doi.org/10.3390/
(mainly flavan-3-ols as Catechin (and glycosides or derivatives), Epicatechin (and glycosides or
foods10123007
derivatives), Quercetin and Myricetin-3-O-rhamnoside, hydroxybenzoic acids as Gallic acid (and
derivatives), 4-hydroxybenzoic acid, ellagic acid, Vanillic acid, p-Coumaric acid and Ferulic acid,
Academic Editors: Rosanna Di Paola,
bioactive minor lipid components (β-Sitosterol, γ-Tocopherol, α-Tocopherol and squalene), and a
Roberta Fusco, Rosalba Siracusa and
Ramona D’Amico
high level of selenium. In beverages, WBM had a higher lipid content than BM-S, a factor that
influenced the energy characteristics and the content of bioactive minor lipid components. The
Received: 8 November 2021 level of phenolic compounds and selenium were outstanding in both beverages. Hydrothermal
Accepted: 26 November 2021 processing can promote some lipolysis, with an increase in free fatty acids and monoglycerides
Published: 4 December 2021 content. In by-products, the BM-F stood out due to its bioactive minor lipid components, the BM-D
showed a highlight in protein and mineral contents, and the cake retained important nutrients
Publisher’s Note: MDPI stays neutral and phytochemicals from the BN. In general, the BN and its beverages are healthy foods, and its
with regard to jurisdictional claims in by-products could be used to obtain healthy ingredients with appreciable biological activities (such
published maps and institutional affil- as antioxidant activity).
iations.

Keywords: Brazil nut; cake (by-product); Brazil nut beverage; phenolic compounds; fatty acids;
proteins; tocopherols; squalene; antioxidant capacity; minerals; selenium

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and
The Brazil nut (Bertholletia excelsa HBK), also known as the Amazon nut, is a plant of the
conditions of the Creative Commons
family Lecythidaceae, native to the Amazon rainforest and concentrated along the Amazon
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
basin, marketed mainly by the countries of Bolivia, Brazil, and Peru. The socioeconomic
4.0/).
and environmental importance of the Brazil nut lies in its production system as a non-

Foods 2021, 10, 3007. https://doi.org/10.3390/foods10123007 https://www.mdpi.com/journal/foods

Foods 2021, 10, 3007 2 of 24

timber forest product coming mainly from natural areas (not crops) and the generation of
income in local populations [1].
In the nutritional and health field, in the last decade, there has been an increased inter-
est in nuts and their positive effects on human health, associating nut consumption with
the reduced risk of various degenerative diseases [2]. Studies on Brazil nut consumption,
and its effects on health, report interesting benefits, such as the reduction in risk factors for
cardiovascular disease, cancer prevention, and improved cognitive functions. These bene-
fits can be attributed to the biological properties, nutrients, and phytochemical constituents
that this nut possesses, such as its majority level of unsaturated fatty acids, selenoamino
acids, dietary fiber, minerals, phenolic compounds, tocopherols, and phytosterols [3].
Considering these potential benefits of the Brazil nut, its added value has been en-
couraged with a focus on the development of health-promoting foods, such a Brazil nut
beverage popularly known as “Brazil nut milk”, belonging to a sector of alternative bever-
ages to dairy milk. In recent years, this sector has shown an increasing in demand, driven
by several reasons such as the “negative” effects of cow’s milk in certain consumer groups,
e.g., protein allergy, lactose intolerance, the content of saturated fat, and the presence of
cholesterol; as well as the preference of people with a healthy diet and lifestyle, those
conscious of the environment, and concerns for animal welfare [4]. Although plant-based
milk usually has added sugar and is low in protein, its appreciable content of unsaturated
fatty acids, essential minerals, phenolic compounds, and other bioactive compounds makes
it a healthy food alternative. However, the market for plant-based milk is limited to a few
sources (mainly soy and almond), and further study is needed to focus on their bioactive
compounds, as well as their effect on processing and the remaining by-products [5]. The
scarce reports on Brazil nut beverages are mainly related to standardization (raw mate-
rial/water ratio, homogenization temperature, formulation, drying, among others) and
quality (sensory, nutritional, physicochemical, and microbiological) [6–9], with an absence
of studies related to its bioactive compounds and their biological properties, as well as the
potential use of its by-products generated in their processing.
Therefore, the present work seeks to characterize the nutritional attributes, the phyto-
chemicals, and the antioxidant properties of Brazil nuts, its beverage, and by-products, in
order to evaluate their potential as healthy food alternatives and healthy ingredients. In the
context of sustainable production, this study supports the investigation of the functional
beverages of the Brazil nut and the use of its generated by-products.

2. Materials and Methods

2.1. Plant Material
Brazil nuts, dry seed, and shelled (without woody tegument), were purchased from a
local market in Madrid (Spain) in April 2019. The vacuum packaged Brazil nuts were stored
in a refrigerated room with an average temperature of 4 ◦ C until analysis and processing.
All processing and analyses were carried out over a period of four months.

2.2. Solvents, Reagents, and Standards

Methanol (99.8% LC-MS) was purchased from VWR International (Barcelona, Spain).
Ultra-pure water (Mili-Q) was obtained from a Millipak® Express 40 system (Merck Milli-
pore, Darmstadt, Germany). The reagents acetone, sodium carbonate, n-hexane, formic
acid, and chloroform were purchased from VWR (Barcelona, Spain). Potassium perox-
ydisulfate (K2S2O8), potassium phosphate (KH2 PO4 ), sodium phosphate (NaH2 PO4 ),
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), fluorescein, N, O-bis
(trimethylsilyl) trifluoroacetamide (BSTFA), Folin–Ciocalteu reagent, 2,2-diphenyl-1-
picrylhydrazyl (DPPH), 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,20-
azobis (2-methylpropionamidine) dihydrochloride (AAPH), standards for GC analysis
(DL-α-tocopherol, squalene, oleic acid, CI-stearoyl-RAC Glycerol and β-sitosterol), and
for HPLC analysis (catechin, epicatechin, vanillic acid, protocatechuic acid, quercetin,
vanillin, gallic acid and hydroxybenzoic acid) were purchased from Sigma-Aldrich (Mis-
Foods 2021, 10, 3007 3 of 24

souri, MO, USA). For protein analysis: Tris-HCl and Trizma base (Sigma-Aldrich), SDS
(Merck, Darmstadt, Germany), glycerol (Panreac Química SAU, Castellar del Vallès, Spain),
β-mercaptoethanol (Sigma-Aldrich, Madrid, Spain), bromophenol blue (Merck, Darmstadt,
Germany), bis-trispolyacrylamide gels (Criterion XT, Bio-Rad, Hercules, CA, USA), XT
MES running buffer 20x (Bio-Rad Laboratories S.A, Madrid, Spain), and Coomassie Blue
(Instant blue, Expedeon, UK).

2.3. Production of Brazil Nut Beverage

The production of whole Brazil nut beverage (WBM) was obtained using the method
reported by Felberg et al. as a reference [6]. A centrifugation stage was added for partial
fractionation and as a strategy to reduce the level of fat and obtain the water-soluble
beverage (BM-S), as shown in Figure 1. Briefly, the Brazil nuts were ground to reduce
their size (particle size < 5 mm), then they were homogenized with water at 75 ◦ C using
the ratio 7:1 (water: raw material, v/w) for five minutes until the mixture reached a
homogeneous consistency via a high-speed homogenizer at 7500 rpm (OMNI Macro-ES
Programmable Homogenizer, OMNI International, Kennesaw, GA 30144, United States).
Then, this solution was immediately filtered with stainless mesh (≤2 mm) to obtain a hot
aqueous extract or WBM and cake by-product. For the fractionation of WBM, the liquid
was maintained at 5 ◦ C for 1 h and then it was centrifuged at 15,000× g at 5 ◦ C for 15 min,
producing its separation in three phases: the fat fraction (BM-F) from the upper phase,
the water-soluble fraction (BM-S) from the intermediate phase, and the sediment fraction
(BM-D) from lower phase. Each fraction was manually collected. Finally, all the fractions
were frozen with liquid nitrogen, freeze-dried, packed in vacuum bags, and kept at −20 ◦ C
until analysis.

Figure 1. Flow chart of the Brazil nut milk production and process by-products.
Foods 2021, 10, 3007 4 of 24

2.4. Methods of Analysis

2.4.1. Physicochemical Analysis
The centesimal composition of the samples was determined using the following
methods: ash, moisture, and dry matter were analyzed following AOAC standardized
methods [10]; the total protein content was determined by the elemental chemical analysis
of nitrogen (conversion factor: 6.25); the total lipid content was determined by the Folch
method [11]; the total carbohydrate was calculated by the difference. Regarding the physic-
ochemical properties, the pH was determined by direct reading on a digital potentiometer
(Metrohm 827 pH Meter, Metrohm, Herisau, Switzerland), the total acidity concentration in
the samples was calculated via potentiometric titration [10], and the ◦ Brix (soluble sugars)
was measured using a digital refractometer (PR-32α, ATAGO™, Tokyo, Japan).

2.4.2. Phenolic Compounds

Extraction
Phenolic compounds were extracted using the method reported by John and Shahidi,
with some modifications [12]. Each sample was previously defatted with hexane (1:5 w/v)
and homogenized for 5 min, in triplicate. The obtained solution was then filtered through
filter paper. The solid residue (defatted) was dried at room temperature for 12 h in the
dark and covered to avoid oxidation. It was then extracted with acetone, in a ratio of
1:15 (w/v), homogenized at 7500 rpm for 4 min in an ultra-speed homogenizer (OMNI
Macro-ES Programmable Homogenizer, OMNI International, Kennesaw, GA, USA), and
later submitted to reflux in a water bath at 60 ◦ C for 40 min with magnetic stirring. The
resulting slurry was centrifuged at 15,000× g for 10 min, and the supernatant was collected.
The sediment was re-extracted under the same conditions, then the supernatants were
combined, and the solvent was removed by vacuum at 40 ◦ C. The extract containing the
phenolic compounds was stored at −20 ◦ C until its use for the analysis of the total phenolics
and antioxidant activity. For the HPLC analysis of the individual phenolic compounds, the
extract was freeze-dried (Lyobeta-15, Azbil Telstar, S.L., Terrasa, Spain) for 72 h and the
samples were stored at −20 ◦ C until analysis.

Total phenolics Content (TPC)

The total phenolics in the samples (phenolic extracts) were analyzed spectrophotomet-
rically with Folin–Ciocalteu reagent according to the method of Cano et al. [13], with some
modifications. In a 96-microwell plate, the reactants were placed in the following order:
20 µL samples (extracts, standard, and blank) were reacted with 100 µL Folin–Ciocalteu
reagent (10%, v/v) and then alkalized with 80 µL of Na2 CO3 7.5% (w/v). The mixture was
stirred and kept in darkness for one hour at 20 ◦ C. The reading was taken spectropho-
tometrically at 756 nm (Varioskan Flash, Thermo Fisher Scientific, Waltham, MA, USA).
Gallic acid was used as the reference standard to elaborate the calibration curve with
concentrations in the range of 50–500 µg/mL. The results were expressed as mg of gallic
acid equivalents (GAE)/100 g dry weight (d.w.) or fresh weight (f.w.).

Individual Phenolic Compounds

Phenolic lyophilized extracts (BN, BM-S, and BM-D) were dissolved with methanol/water
(2:1, v/v) and filtered with 0.45 µm syringe filters before injection into HPLC UV/Vis.
Chromatographic analyses were performed according to the protocol reported by García-
Cayuela et al. [14], using a 1200 Series Agilent HPLC System (Agilent Technologies, Santa
Clara, CA, USA) with a reverse phase C18 column (Zorbax SB-C18, 250 × 4.6 mm, 5 µm;
Agilent) maintained at 20 ◦ C. Elution solvent A consisted of 1% formic acid (v/v) in water,
while solvent B was a mixture of methanol and formic acid (1%, v/v). Separation was
achieved using an initial solvent composition of 15% (B) during 15 min, increased to 25%
(B) within 10 min, ramped to 50% (B) within 10 min, increased to 75% (B) in 15 min, and
followed by a decreased period of 15% (B) in 5 min prior to isocratic re-equilibration at
15% (B) for 10 min. The flow rate was fixed at 0.8 mL/min, and the injection volume was
Foods 2021, 10, 3007 5 of 24

20 µL. The UV–vis photodiode array detector was set at 3 wavelengths (280, 320, 380 nm)
for monitoring different phenolic chemical families simultaneously.
The HPLC-DAD was coupled to a mass spectrometry detector (LCMS SQ 6120, Ag-
ilent, CA, USA) with an electrospray ionization (ESI) source operating in positive ion
mode. The drying gas was nitrogen at 3 L/min at 137.9 KPa. The nebulizer temperature
was 300 ◦ C, and the capillary had 3500 V potential. The coliseum gas was helium, and
the fragmentation amplitude was 70 V. Spectra, recorded m/z from 100–1000. Further
mass spectrometry analyses were performed using maXis II LC-QTOF equipment (Bruker
Daltonics, Bremen, Germany) with an ESI source and the same chromatographic condi-
tions. The ESI-QTOF detector worked in positive ion mode and recorded spectra m/z
from 50–3000. The operation conditions were 300 ◦ C, capillary voltage 3500 V, charging
voltage 2000 V, nebulizer 2.0 bar, and dry gas at 6 L/min. MS/MS analysis used the bbCID
(broadband Collision-induced dissociation) method at 30 eV.
Phenolic compounds were identified according to their retention times, UV/Vis, and
mass spectral data compared to those of commercial or purified standards. Quantitation
of most phenolic compounds was determined using the calibration curves of the corre-
sponding isolated standards. Quercetin glycosides were quantified by using the rutin
calibration curve.

2.4.3. Lipids and Other Minor Lipidic Compounds

Extraction
The lipids in the samples were extracted using the method of Folch, Lees, and Sloane
Stanley [11] as follows. Briefly, 1 g of sample was mixed with 20 mL of chloroform-methanol
(2:1, v/v) to be homogenized in a T-25 Ultraturrax (IKA-Werke GmbH & Co., KG, Staufen,
Germany) at 11,000 rpm for 3 min, followed by sonication by ultrasound bath for 3 min
and then centrifuged at 1050× g for 10 min. The supernatant was collected and stirred
with 4 mL of distilled water by vortex and then centrifuged again at 1050× g for 10 min.
The bottom organic phase was collected and filtered with paper and dried with anhydrous
sodium sulphate. The lipid solution obtained was dried on a rotary evaporator under a
vacuum at 40 ◦ C and kept at −20 ◦ C until analysis.

Fatty Acids
The fatty acids (FA) profile of lipid samples was determined after the derivatization
of fatty acid methyl esters (FAMEs). Briefly, 20 mg of sample was mixed with 0.5 mL
chloroform/methanol (2:1; v/v) and 1 mL of 0.1 M NaOH in methanol. This mixture was
heated at 60 ◦ C for 30 min, and then the reaction was stopped with the addition of 0.2 mL
of distilled water. The FAMEs formed were extracted with the addition of 1 mL of hexane.
After stirring, it was decanted, and the isolated upper phase was collected. Anhydrous
sodium sulfate was added to the solution of FAMEs to eliminate aqueous traces for 30 min.
Finally, the hexane was removed under a nitrogen atmosphere.
The obtained FAMEs were dissolved in hexane to a concentration of 1 mg/mL for
each sample and injected into the gas chromatograph Agilent 7890 A (Agilent Technologies,
California, PA, USA) equipped with a split-splitless injector and an autosampler, a flame
ionization detector, and a triple-axis mass spectrometer detector. FAMEs were separated
using an HP-5MS column (30 m length, 0.25 mm internal diameter, and 0.25 µm film
thickness). Helium was used as a carrier gas at 2 mL/min. The injection temperature
was 260 ◦ C, splitless mode, 1 µL, and the mass spectrometer ion source and interface
temperatures were 230 and 280 ◦ C, respectively. The chromatographic analysis started at
50 ◦ C and increased at a speed of 20 ◦ C/min until it reached 210 ◦ C, there it stayed for
18 min, then it went up to 230 ◦ C and remained there for 13 min. The running time was
40 min. The mass spectra were obtained by electronic impact at 70 eV. The scan rate was
1.6 scans/s at a mass range of 30–700 amu. The identification of fatty acids was performed
by the NIST MS Data library. The area under each FA peak in relation to the total area of all
FA peaks was used for quantification and expressed as a percentage of FA.
Foods 2021, 10, 3007 6 of 24

Minor Lipid Compounds: Free Fatty Acids, Monoglycerides, Tocopherols, Phytosterol,

and Squalene
For the analysis of these minor compounds, the lipid extracts were derivatized using
N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The procedure used was based on that
of Herrera et al. [15]. Briefly, 5 mg of the sample was mixed with 1 mL of the derivatizing
agent and then submitted to 75 ◦ C for 1 h, shaking every 15 min. Finally, it was injected
into the previously described chromatography equipment, and the separation procedure
was started at 50 ◦ C and was held for 3 min, increasing to 310 ◦ C at a rate of 15 ◦ C/min and
held for 25 min. For quantification, calibration curves were obtained from the standards
of α-tocopherol, squalene, oleic acid, stearoyl-RAC glycerol, and β-sitosterol, which were
also previously derivatized.

2.4.4. Proteins
SDS-PAGE Analysis
The electrophoresis assay was performed on Precast Criterion XT 4–12% Bis-Tris gels
using the Criterion cell (Bio-Rad, Hercules, CA, USA) as reported by Sanchón et al. [16].
The samples were dissolved at 0.8 mg of protein/mL in a buffer containing 0.05 M Tris-HCl,
pH 6.8, 8% (v/v) glycerol, 1.6% (w/v) SDS, 2% (v/v) β-mercaptoethanol, and 0.002% (w/v)
bromophenol blue. The concentration was calculated at 0.8 mg protein/mL buffer solution.
Then, they were heated at 95 ◦ C for 4 min, and 45 µL was loaded onto the 4–12% Bis-Tris
polyacrylamide gel as well as 15 µL of the molecular weight marker (Precision Plus Protein
unstained Standard (Bio-Rad, Bio-Rad, Hercules CA, USA). Electrophoretic separation was
carried out using XT-MES as a running buffer (BioRad). After 5 min at 100, 150 V were
applied. The gels were washed with Milli-Q water and were stained with Coomassie blue
G-250. Images were taken with a Molecular Imager® VersaDoc™ MP 5000 system (Bio-Rad,
Hercules, CA, USA) and processed with Quantity One® 1-D analysis software (Bio-Rad
Laboratories S.A, Madrid, Spain).

Mass Spectrometry Analysis of Peptides

To identify the proteins on the SDS-PAGE gels, bands were cut out from the gels
and transferred to a microcentrifuge tube. Subsequently, in-gel reduction, alkylation,
and tryptic digestion were carried out as previously reported by Miralles et al. [17]. An
Autoflex speed MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany) instrument was
used for mass spectra generation. The Mascot server (www.matrixscience.com, Matrix
Science, London, UK) was used to carry out protein identification searches against a
homemade database of Bertholletia excelsa and Pisum sativum proteins selected from the
UniProt database (https://www.uniprot.org/ (accessed on 22 May 2020)).

2.4.5. Minerals
Selenium
For the quantification of selenium (Se), the protocol proposed by López-Bellido et al. [18]
was followed, with some modifications. Briefly, the dry samples (approximately 0.5 g) were
pretreated by acid digestion, using HNO3 in a microwave (UltraWAVE Milestone, Shelton,
CT, USA). Then, the samples were diluted 1:2 (w/v) with 1% nitric acid and analyzed by
inductively coupled plasma mass spectrometry (ICP-MS model NexION 300 X, Perkin
Elmer® , Waltham, MA, USA) under the following operating conditions: nebulizer quartz
concentric, spray chamber PC3 Peltier Cooled Cyclonic, RF Power 1500 W, nebulizer gas
flow of 0.86 L/min, scanning mode in peak hopping, d.w.ell time of 50 ms, 20 sweeps,
1 reading, 3 replicates, analytes 78 Se and 80 Se, internal standard 72 Ge, and reaction mode
CH4 = 0.70 mL/min and RPq = 0.55. Calibration standards at 0.05, 0.1, 0.5, 1, 5, 10, and
50 µg/L were prepared from a 1000 mg/L stock solution of Se (SCP Science) in 1% nitric
acid. One internal standard (Ge at 30 µg/L) was used to correct for signal drift. The results
were expressed as µg/100 g d.w. or f.w.
Foods 2021, 10, 3007 7 of 24

Other Minerals
The analysis was performed by the high-resolution atomic absorption spectrometry
(HR-CS AAS) method according to the procedure suggested by Nielsen [19], with certain
modifications. First, the samples were digested: 1 g of the samples, with nitric acid and
hydrogen peroxide (both of high purity), were added and submitted to a programmed tem-
perature of 200 ◦ C using the microwave digestion system equipment (Ethos UP, Milestone
Srl, Sorisole, Italy). The digested samples were diluted with 50 mL of distilled water. In the
second stage, the HR- CS AAS was used (ContrAA 700, Analytik Jena AG, Jena, Germany).
Air-acetylene flame was applied for all minerals except calcium (Ca) and phosphorus (F).
The Ca required the more calorific acetylene nitrous oxide flame. F was determined using
a graphite furnace. Each element was determined by the previous calibration of specific
certified standards. The minerals obtained were expressed in mg/100 g d.w. or f.w.

2.4.6. Antioxidant Capacity

DPPH Assay
The antioxidant capacity was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl)
radical scavenging method according to the method of Abe et al. [20], with some modifica-
tions. In a 96-well microplate, the reaction was carried out as follows: a 50 µL aliquot of
the sample extract, previously diluted with methanolic solution (70%), was mixed with
250 µL of DPPH (0.5 mM) and after 25 min in the dark, the absorbance was measured
at 517 nm using spectrophotometric equipment (Varioskan Flash de Thermo Electron
Corporation® , Waltham, MA, USA) and analyzed with SkanIt Re for Varioskan 2.4.1®
software (Varioskan Flash de Thermo Electron Corporation® , Waltham, MA, USA). The
reference standard for obtaining the calibration curve consisted of a methanolic solution
of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) at different concentra-
tions (100–800 µM/mL). The antioxidant capacity was expressed as µmol Trolox equivalent
(TE)/g d.w. or f.w.

TEAC Assay
The radicals ABTS+ were generated according to Koroleva et al. [21], with some
modifications. ABTS and potassium peroxodisulfate (K2S2O8) were dissolved in water at
final concentrations of 7 and 140 mM, respectively. The ABTS stock solution was obtained
by mixing both reagents in a ratio of 17.6 µL K2S2O8/mL ABTS. To get a daily solution
of radical ABTS, the stock solution was diluted with phosphate-buffered saline (PBS,
75 mM, pH 7.4), constituted by potassium phosphate (KH2PO4) and sodium phosphate
(NaH2PO4), until an optical density of 0.70 ± 0.02 at 734 nm. TE solutions (10–100 µM)
were used for calibration. The reaction was initiated by mixing 30 µL of the sample
extract, or standard, with 200 µL of ABTS+, then left to stand 40 min and the absorbance
was measured spectrophotometrically at 734 nm. Values were calculated based on the
linear regression equation between the TE concentration and the decrease in absorbance.
Antioxidant capacity was expressed as µmol TE/g d.w. or f.w.

ORAC Assay
The oxygen radical antioxidant capacity (ORAC) was determined according to the
method proposed by Gómez-Maqueo et al. [22], with some modifications. The extracted
samples and TE were dissolved with phosphate-buffered saline (PBS) 75 mM at pH 7.4. A
TE curve was prepared using concentrations ranging from 10–45 mM. In a microwell plate,
20 mL of the sample extract, or standard, was added to each microwell, then 120 mL of
11.7 µM fluorescein solution was added. The microplate was incubated at 37 ◦ C for 10 min.
Afterwards, 60 mL of a 153 mM 2,20 -Azobis (2-methylpropionamidine) dihydrochloride
(AAPH) solution were added. In a 96-well microplate reader (Varioskan Flash de Thermo
Electron Corporation® , Waltham, MA, USA), plate lecture was registered every minute for
55 min at 37 ◦ C and with an excitation wave of 485 nm and emission wave of 530 nm. The
Foods 2021, 10, 3007 8 of 24

data analysis was performed by obtaining the area under the curve (AUC) and subtracting
the blank. The results were expressed as µmol TE/g d.w. or f.w.

2.4.7. Statistical Analysis

The values are expressed as mean and standard deviation. The obtained results were
evaluated with variance analysis (ANOVA), and differences between means were located
using Tukey’s test. The significant statistical differences were calculated at a p < 0.05 level.
The statistic software employed was IBM SPSS Statistic 20.

3. Results and Discussion

3.1. Production of Brazil Nut Beverage
Figure 1 show the production procedure reported in the Materials and Methods
section, using a ratio of BN/water 1: 7 (w/v). In this process, 100 g f.w. of BN with 700 mL
of water, 709 g f.w. of WBM and 611.6 g f.w. of BM-S, were obtained. That is, to obtain WBM
and BM-S beverage, 14.11 and 16.4% (w/v) of raw material were required, respectively.
In commercial plant-based milks, the raw material content varies in the range of 2–15%
(data from field survey, taken from the labels of the main brands of vegetal milk available
in Madrid, Spain.) (w/v), depending on the type of raw material, raw material/water
ratio, technological processes, and the prefixed characteristics of the final product. In the
literature regarding the production of Brazil nut beverages, the use of various BN/water
ratios have been reported, as 1:2 (w/v) [9] and 1:7 (w/v) [6]. The latter ratio was used in our
work due to the good yield of total solids at 77 ◦ C and the remarkable nutritional profile. A
remarkable aspect of BM-S was a better appearance of stability than WBM since fat and
sediment fraction was separated by centrifugation. It is true that this process would reduce
the formation of sedimentation (particles with higher density), and cream formation (oily
bodies of low density) during its storage, but does not affect the nutritional composition,
as verified in the following sections.
Regarding the by-products in the process of BN milk production, the cake is the first
residue from processing, with an amount of roughly 33 g d.w. from 100 g f.w. (~97.5 g d.w.)
produced from the initial raw material (BN). These results are similar to those reported
(36.14%, w/w, d.w.) in a previous study using partially defatted BN to obtain the BN
beverage [9], an important amount of food material for subsequent utilization. Furthermore,
on a fresh weigh basis, BM-D and BM-F residues represent less than 14% (w/w) of the
WBM, but on a dry basis they are very important and reach 31.4% (w/w) of BM-D and
49.2% (w/w) of BM-F, significative in large production volumes.

3.2. Physicochemical Properties

The centesimal composition of the Brazil nut, its beverages (BWM and BM-S), and
by-products (cake, BM-D and BM-F), are shown in Table 1. The macronutrients of BN,
in order of predominance, are the lipids (58.5/100 g f.w.), followed by carbohydrates
(19.6/100 g f.w.), proteins (16/100 g f.w.), and ash (3.3/100 g f.w.). The high caloric power
of 669.2 Kcal, mainly provided by lipid level, makes BN an energetic food with a valuable
nutritional contribution.
In BN beverages, the total solids content of the WBM beverage was about 9/100 g f.w.,
a higher level than BM-S with around 2/100 g f.w., when the vegetable milk commercials
can vary in a range of 3–23% (w/w) [5]. The nutrients that conform to the WBM were domi-
nated by total lipids with 5.24/100 g f.w., followed by carbohydrates with 1.94/100 g f.w.,
proteins with 1.43/100 g f.w., ash with 0.33/100 g f.w., and an energy content of 60.6
kcal/100 g f.w. These values are close to those obtained by Felberg et al. [6], who used a
similar process.
Foods 2021, 10, 3007 9 of 24

Table 1. Centesimal composition and physicochemical properties of the Brazil nut (Bertholletia excelsa
HBK), its beverages, and by-products.

Raw Material Beverages By-Products

Characteristic
BN 1 WBM *,1 BM-S 1 BM-D 2 BM-F 2 Cake 2
Protein 16.03 1.43 0.80 56.88 0.65 16.9
Lipid 58.52 5.24 0.25 14.6 81.3 44.2
Carbohydrate 19.61 1.94 0.83 16.65 16.29 35.45
Ashes 3.35 0.33 0.10 11.87 1.76 3.5
Energy (Kcal) 669.2 60.6 8.8 425.5 799.5 607.2
Total solids 1 97.51 8.93 1.98 31.41 49.21 36.17
Acidity 3 0.18 0.03 0.03 0.54 - 0.1
pH 6.4 6.5 6.5 6.7 - 6.6
Soluble solid (◦ Brix) 14.5 2.6 3.0 4.8 - 6.8
Abb.: Brazil nut (BN), Whole beverage (WBM), Water soluble fraction (BM-S), Sediment fraction (BM-D). * Values
of WBM were estimated from the fractions; 1 Expressed as g/100 fresh weight (f.w.); 2 Expressed as g/100 g fry
weight (d.w.); 3 Expressed as g citric acid/100 g.

In the case of the BM-S beverage, it had a higher predominance of proteins (0.8/100 g f.w.)
and carbohydrates (0.83/100 g f.w.), and a minimum composition of lipids (0.25/100 g f.w.),
ash (0.1/100 g f.w.), and a minimum energy content (8.8 kcal/100 g f.w.). In general, there
was a reduction of macronutrients in BM-S compared to WBM (mainly in lipid content,
which decreased up to roughly 20 times) due to the centrifugation process and separation
of the BM-D and BM-F fractions. Commercial vegetable milks have a wide compositional
variation: in protein from 0.1–3.3% (w/v), in lipids from 0.3–7.2% (w/v), in carbohydrates
from 0–13.4% (w/v), and in energy content from 21–121 kcal/100 mL [23]. Therefore, WBM
and BM-S are beverages comparable to commercial vegetable milk, but the WBM has a
high energy input, as opposed to the BM-S.
Regarding the by-products, the cake had a notable nutritional composition of lipids
(44.2/100 g d.w.), carbohydrates (35.45/100 g d.w.), proteins (16.85/100 g d.w.), and ash
(3.5/100 g d.w.). These values are consistent with those reported by Sartori et al. [9],
who produced the beverage from partially defatted BN. In addition, regarding the by-
products obtained after centrifugation, the BM-D fraction showed an important content
(about 57/100 g d.w.), probably due in part to the effect of the thermal denaturation of
some proteins and the increased hydrophobicity [24]. This fact was observed in soy and
almond milk when they were subjected to moderate heat treatments (around 60–80 ◦ C),
showing lower precipitation and protein denaturation than those produced when they
were treated at high temperatures (over 85 ◦ C) [25,26]. In the case of the BM-F fraction, it
had a predominance of lipid content (about 80/100 g d.w.), being related to the high-fat
content coming from the raw material.
In the physicochemical characteristics (Table 1), the pH ~6.5 of the Brazil nut and cake
were consistent with the previously published studies [27]. The pH of WBM and BM-S
were within the range (pH: 5–8) of most vegetable milks [5]. The high level of acidity (0.54 g
citric acid/100 g) of the sediment (BM-D) could be influenced by amino acids due to their
high protein content (~57%, w/w). Regarding the content of soluble solids, the BN has the
highest value with 14.5 ◦ Brix due to low moisture (2.5%, w/w), and the WBM and BM-S
have ~3 ◦ Brix, due to dilution in water of BN processing. The by-products (BM-D and
cake), in dry weight, have a low content of soluble solids that can be attributed to the effect
of the hydrothermal process for obtaining the beverage and the consequent extraction of
water-soluble substances for BN.

3.3. Phenolic Compounds

3.3.1. Total Phenolic Content (TPC)
The Folin-Ciocalteau method allowed the quantification of TPC present in the samples.
Table 2 show these data. The TPC of BN was 108 mg GAE/100 g d.w. (~100.1 mg
GAE/100 g f.w.), this value was similar to a previous report [20] but differed from that
Foods 2021, 10, 3007 10 of 24

obtained by John and Shahidi [12], who reported 519 mg GAE/100 g f.w. This difference
can be attributed to different factors inherent to the plant, industrial processes, and methods
of extraction and analysis.

Table 2. Total phenolic content of the Brazil nut (Bertholletia excelsa HBK), its beverages (WBM and
BM-S), and by-products (BM-D and cake), expressed in mg GAE/100 g.

TPC
Samples
Dry Weight Fresh Weight
Raw material BN 108.0 ±3.9 b 105.3 ±3.8 a
WBM * 79.5 ±1.5 b 7.1 ±0.1 c
Beverages BM-S 314.0 ±5.2 a 6.2 ±0.1 c
BM-D 83.4 ±3.1 b 28.7 ±1.1 b
By-products
Cake 49.2 ±2.7 c 17.8 ±1.0 b
Data are expressed as means ± SD (n = 3). Values with different superscript letters in the column are significantly
different (p < 0.05); * Value of WBM was estimated from their fractions.

Regarding the Brazil nut beverages, the WBM and BM-S showed similar total phenol
content, with 7.1 and 6.2 mg GAE/100 g f.w., respectively. These were higher values than
those reported for commercial vegetable milks, which ranged from 0.02–1.24 mg/100 mL [28].
However, some studies may show higher phenolic concentrations, such as almond milk
with 71 mg/100 g f.w. [29].
When analyzing the TPC level on a dry basis, according to Table 2, the BM-S (314 mg
GAE/100 g d.w.) was significantly higher (p ≤ 0.05) than the rest of the samples (<84 mg
GAE/100 g d.w.). According to the data of John and Shahidi [12], the phenolic extract of
BN contained roughly 97% (w/w, fresh weight) soluble phenolics and less than 5% (w/w,
fresh weight) bound phenolics, this would explain the higher TPC in the BM-S beverage.
Regarding the cake by-product, with 49.2 mg GAE/100 g d.w., it has a TPC nearly
to half of BN, a reduction consistent with the high concentration of soluble polyphenols
present in the BN transferred to the aqueous fraction (BM-S). There are no reports of TPC
in cake from the BN beverage. However, Gomes et al. [30], in their study of phenolic
extraction of cake (from defatting of the BN), obtained a TPC from 143–182 mg GAE/100 g.
In the cake (by-product) of soybean milk, known as “okara”, the total phenol content is
recorded to be from 620–2913 mg GAE/100 g d.w. [31,32]. Therefore, at the level of total
phenols, the cake from the BN beverage process is relatively low. As for the BM-D fraction,
precipitated by-product, with 83.4 mg GAE/100 g d.w., their total phenols would be to
a greater extent conjugated (esterified) with amino acids and peptides (due to their high
total protein content ~57% in dry weight), decanted by factors such as higher molecular
weight, lower aqueous solubility (as globulins and glutelins), and protein denaturation.
The fat fraction (BM-F) was not analyzed for total phenol content due to its high-fat content
(greater than 80%, w/w, dry weight) and the antecedents regarding its minimum TPC level
of BN oil, as 3.6 mg GAE/100 g oil [33].

3.3.2. Identification of Individual Soluble Phenolic Compounds

After the extractions, in an aqueous solvent (70% acetone), the soluble phenols of
each sample were characterized. The profile of phenolic compounds of the Brazil nut,
water-soluble fraction (BM-S), and sediment fraction (BM-D) was conducted by HPLC-
DAD (UV/Vis detection) and by HPLC- ESI-QTOF, analyzing retention times, UV spectra,
mass spectral data compared to those of commercial and purified standards, and to re-
ported data (Table 3). The UV-vis detection was simultaneously recorded at 280, 320, and
380 nm, achieving the identification of 24 compounds: 4 organic acids, 10 phenolic acids,
9 flavonoids, and 1 phenolic aldehyde. The HPLC chromatogram at 280 nm (Figure 2)
showed a better resolution of the peaks for the identification of the phenolic compounds
present in the samples. Table 3 show the HPLC retention times, UV/Vis spectra, and MS
Foods 2021, 10, 3007 11 of 24

spectral data of individual phenolics in the Brazil nut (Bertholletia excelsa), water-soluble
fractions (BM-S), and sediment fraction (BM-D).

Figure 2. HPLC C18 chromatogram of phenolic compounds from (a) BM-S, (b) Brazil nut (Bertholletia
excelsa HBK), (c) cake and (d) BM-D at 280 nm.

Peaks detected in the first five minutes of the run were identified as organic acids.
Those were citric acid (peak 1, Rt = 2.9 min), ascorbic acid (peak 3, Rt = 3.8 min), and
succinic acid (peak 4, Rt = 4.8 min), distinguished by their common pattern of fragment
ions and agreement with previous reports [12] and other fruits [34,35]. Regarding peak 2
(Rt = 3.59 min), its precursor ion m/z 87 was identified as pyruvic acid [36]. The phenolic
acids identified were mostly hydroxybenzoic acids (eight compounds in their free or deriva-
tives form of gallic acid, ellagic acid, 4-hydroxybenzoic acid, protocatechuic acid, and vanil-
lic acid) and, to a minor degree, by hydroxycinnamic acids (ferulic acid, and p-coumaric
acid) and phenolic aldehyde (vanillin). Peak 5 (Rt = 6.4 min) corresponded to gallic acid
with a precursor ion m/z 274 and a daughter ion at m/z 125, a match with previous studies
of the Brazil nut [12]. Peak 6 (Rt = 6.9 min), due to the fragment, formed m/z 169, the
compound was identified as a derivative of gallic acid [12]. Peak 7 (Rt = 21.7 min) was
identified as protocatechuic acid derivative, due to its pattern of fragmentation m/z 153,
159, and 124, and in agreement with the values reported in the Brazil nut by John and
Shahidi [12], who found a higher abundance of protocatechuic acid and its derivative
in the BN skin. Peak 10 (Rt = 28.4 min) had an important signal and was identified as
4-hydroxybenzoic acid, confirmed by the UV spectrum of its standard and similarity of
molecular ion [M-H]− m/z 137 observed in almond, as previously reported [38]. In peak
11, the UV detected spectrum (Rt = 30.2 min) corresponded to vanillic acid, with molecular
ion m/z 167, which at the same time is a product ion peak of 24 (Rt = 42.1 min), identified
as vanillic acid derivative, coinciding in a previous report [12,38]. Peak 13 (Rt = 32.4 min)
was tentatively identified as vanillin with a precursor ion m/z 151; although not commonly
reported in nuts, the precursor ion was also found in wheat [40]. Peak 17 (Rt = 36.28 min)
corresponded to the molecular ion [M-H]- m/z 164, indicative of p-coumaric acid, with
product ion m/z 119, with fragment denominates, such as decarboxylated coumaric acid
in walnut, according to Pycia et al. [39]. Peak 18 (Rt = 36.4 min), a compound with a high
signal, has a similr UV spectrum to ferulic acid (λmax 230 nm) and matched to the fragment
ion m/z 133 reported in walnuts [39]. Peak 20 (Rt = 37.9 min) showed a fragmentation
Foods 2021, 10, 3007 12 of 24

pattern which corresponded to an ellagic acid derivative, conformed to ion m/z 301 (cor-
responding to ellagic acid) found at peak 22 (Rt = 39.7 min), with values consistent to the
previous report on the Brazil nut [12].

Table 3. HPLC retention times, UV/Vis spectra and MS spectral data of individual phenolics in the Brazil nut (Bertholletia
excelsa HBK), BM-S, BM-D, and cake.

Peak 1 Rt (min) Assigned Identity UV λmax (nm) [M-H]− m/z MS/MS m/z References
1 2.9 Citric acid 2 209 191 111, 173 [12,34]
2 3.1 Pyruvic acid 2 227.8 (259.7) 87.06 59.01 [36]
3 3.8 Ascorbic acid 2 289 175.03 147.2, 87.00, 69.03 [34]
4 4.8 Succinic acid 2 228 117.02 72.91 [35]
125.02, 107.01, 97.03,
5 6.4 Gallic acid 274 169.01 79.02, 69.03, 51.02, [37]
41.04
6 6.9 Gallic acid derivative 272 187 125.02, 169.01 [12]
Protocatechuic acid
7 21.7 280 − 153.04, 109.03, 124.03 [12]
derivative
8 22.3 Catechin 230, 280 289.1 136.8, 150.7, 160.8 [12,38]
9 24.0 Catechin derivative 282 − 289.1 [12]
10 28.4 4-hydroxybenzoic acid 252 137.03 106.64, 93.03 [38]
11 30.2 Vanillic acid 259, 292 167.03 152.01, 108.02 [12,39]
109.01, 121.01, 123.03,
12 30.9 Epicatechin 279 289.1 [38]
125.01, 137.00
137.05, 123.05, 109.0,
13 32.4 Vanillin 274, 309 151.05 [40]
81.0
109.01, 125.00, 168.98,
14 33.4 Catechin gallate 231.8, 280.1, (324.8) 441.03 [41,42]
289.03
109.08, 125.08,137.08,
15 33.8 Epicatechin gallate 232.6, 280 (324.8) 441.19 151.10, 203.14, 245.16, [38]
289.16
Epigallocatechin 3-O
16 34.6 232; 280 (312) 457.3 305.6, 169.1, 125.02 [43]
gallate
17 35.4 p-Coumaric acid 227, 310 164.05 119.05, 91.05 [39]
18 36.4 Ferulic acid (292), 323 193.1 177.1, 161, 133.1 [39]
Taxifolin
19 37.0 231.9, (282.8) 309.2 303.05 285.05, 179.00, 125.03 [12]
(dihydroquercetin)
20 37.9 Ellagic acid derivative 252, 360 447 301, 257, 229 [12]
21 38.9 Quercetin 232, 323 301 179, 151 [12,44]
285, 283, 257, 229,
22 39.7 Ellagic acid 254, 368 301 [12]
184.92, 134.92
Myricetin-3-O-
23 40.0 253, 370 463 317 [12,45]
rhamnoside
24 42.1 Vanillic acid derivative 259, 294 329 167 [12]
1 2
Peak numbers correspond to those of Figure 2; Organic acids.

In the flavonoid group of the Brazil nut and derivatives samples, six types of flavan-
3-ols were detected (mainly catechin, epicatechin, and their gallate ester or derivatives),
followed by two flavonols (myricetin-3-O-rhamnoside and quercetin), and one flavanonols
(taxifolin). Peak 8 (Rt = 22.3 min), peak 9 (Rt = 24 min) and peak 14 (Rt = 33.4 min)
were identified as catechin, catechin derivative, and catechin-gallate, respectively. The
catechin had the highest signal in flavonoids and its identification was confirmed by the
UV spectrum of its standard and pattern ion m/z 289, a common value reported in the
Brazil nut and walnut [12,38].
Peak 12 (Rt = 30.9 min) and peak 15 (Rt = 33.8 min) showed a precursor ion m/z 289
and 441, that corresponds to epicatechin and epicatechin gallate, respectively, which was
also observed in almond [38]. Peak 16 (Rt = 34.6 min) with molecular ion [M-H]− m/z 457
was tentatively identified as epigallocatechin 3-O gallate, which exhibited the loss of a
typical fragment ion m/z 169 that corresponds to gallic acid. Peak 19 (Rt = 37 min) was
Foods 2021, 10, 3007 13 of 24

identified as taxifolin (dihydroquercetin), due to the common pattern ion m/z 303 and
fragmentation pattern m/z 285 and 125, values that were also observed in a previous report
on the Brazil nut [12]. Quercetin was clearly recognized in peak 21 (Rt = 38.9 min) by
its principal ion m/z 301 and fragmentation pattern m/z 179 and 151, consistent with
reports of walnut [44]. Myricetin-3-O-rhamnoside was identified in peak 23 (Rt = 40 min)
with [M-H ]− m/z 463, also observed in reports on the Brazil nut and hazelnut [12,45]. In
general, the phenolic characterization of BN is scarce in the literature. We found individual
phenols that were not detected or analyzed in previous reports, such as vanillin, catechin
gallate, epicatechin, epicatechin gallate, epigallocatechin 3-O gallate, and myricetin-3-O-
rhamnoside.

3.3.3. Individual Phenolic Compounds Content in BN and Derivates

The quantification of phenolic compounds in the Brazil nut, WBM, BM-S, and BM-D,
are shown in Table 4. The BN had a total phenolic content of 575 µg/g f.w., composed of
flavonoids (393.4 µg/g f.w.), phenolic acids (176.1 µg/g f.w.), and vanillin (5.6 µg/g f.w.),
which is a phenolic aldehyde. This phenolic composition is common in other nuts, showing
a predominance of flavonoids and phenolic acids [46].

Table 4. Individual phenolic content (µg/g) in Brazil nut (Bertholletia excelsa HBK), its beverages (WBM and BM-S) and
by-products (BM-D and cake) by HPLC-DAD-MS.

Rt (Min) Phenolic Compound BN 1 WBM *,1 BM-S 1 BM-D 2 Cake 2

Phenolic Acids
6.4 Gallic acid 6.3 ±0.2 0.7 ±0.0 0.5 ±0.0 17.7 ±0.8 6.4 ±0.3
6.9 Gallic acid derivative 6.9 ±0.3 0.6 ±0.0 0.4 ±0.0 17.7 ±0.2 6.5 ±0.3
21.7 Protocatechuic acid derivative 42.0 ±1.9 3.0 ±0.1 2.1 ±0.1 83.2 ±4.0 31.7 ±1.4
28.4 4-hydroxybenzoic acid 6.5 ±0.3 0.5 ±0.0 0.4 ±0.0 3.7 ±0.0 2.2 ±0.1
30.2 Vanillic acid 18.0 ±0.8 1.5 ±0.0 1.4 ±0.0 4.9 ±0.1 3.3 ±0.1
35.4 p-Coumaric acid 8.5 ±0.3 0.9 ±0.0 0.8 ±0.0 4.9 ±0.1 2.6 ±0.1
36.4 Ferulic acid 4.6 ±0.2 0.5 ±0.0 0.5 ±0.0 1.7 ±0.1 1.0 ±0.0
42.1 Vanillic acid derivative 1.6 ±0.1 0.2 ±0.0 0.2 ±0.0 0.6 ±0.0 0.5 ±0.0
37.9 Ellagic acid derivative 45.4 ±0.4 4.7 ±0.1 3.3 ±0.1 138.6 ±3.8 49.1 ±1.7
39.7 Ellagic acid 36.2 ±1.4 3.5 ±0.0 2.1 ±0.0 134.1 ±3.4 46.8 ±2.5
Phenolic aldehyde
32.4 Vanillin 5.6 ±0.2 0.5 ±0.0 0.5 ±0.0 1.3 ±0.1 1.2 ±0.0
Flavonoids
22.3 Catechin 175.4 ±7.9 21.6 ±0.5 20.2 ±0.5 127.6 ±3.0 79.7 ±3.3
24.0 Catechin derivative 86.1 ±2.6 9.2 ±0.5 8.9 ±0.5 25.7 ±1.2 18.3 ±0.9
30.9 Epicatechin 21.8 ±0.7 1.5 ±0.0 1.4 ±0.0 16.2 ±0.4 8.0 ±0.1
33.4 Catechin-gallate 71.0 ±1.7 8.5 ±0.1 8.2 ±0.1 24.8 ±0.6 16.7 ±0.3
33.8 Epicatechin-gallate 14.3 ±0.4 1.4 ±0.0 1.3 ±0.0 13.6 ±0.2 6.3 ±0.2
34.6 Epigallocatechin 3-O-gallate 7.4 ±0.3 0.3 ±0.0 0.2 ±0.0 11.0 ±0.3 4.3 ±0.1
37.0 Taxolin (dihydroquercetin) – – – – –
38.9 Quercetin 6.7 ±0.2 0.9 ±0.0 0.9 ±0.0 0.2 ±0.0 1.0 ±0.0
40.0 Myricetin-3-O-rhamnoside 10.6 ±0.5 0.2 ±0.0 0.2 ±0.0 6.8 ±0.4 3.6 ±0.0
Total phenolic acids 176.1 ±5.8 16.0 ±0.4 11.8 ±0.3 407.1 ±12.5 150.3 ±6.5
Phenolic aldehyde 5.6 ±0.2 0.5 ±0.0 0.5 ±0.0 1.3 ±0.1 1.2 ±0.0
Total flavonoids 393.4 ±14.4 43.7 ±1.2 41.1 ±1.1 225.9 ±6.1 138.0 ±5.0
Total phenolic compounds 575.0 ±20.4 60.2 ±1.6 53.3 ±1.4 634.3 ±18.6 289.4 ±11.5
* Value of WBM was estimated from their fractions; 1 Expressed on fresh weight basis.; 2 Expressed on dry weight basis.; – Detected but not quantified.

According to Table 4, the predominant flavonoids of BN, in free or derivative form,

were catechin and epicatechin, and at a lower level of concentration were quercetin, tax-
ifolin, and myricetin-3-O-rhamnoside. These compounds belong mainly to flavan-3-ols
and flavonols subgroups. This profile is consistent with the general characteristics of the
nuts, where the flavan-3-ols, flavonols, and anthocyanins are considered to be the main
flavonoids [47]. Moreover, previous studies also consider catechin and its derivatives as
one of the main flavonoids present in BN [30,48]. With respect to the phenolic acids of BN,
Foods 2021, 10, 3007 14 of 24

the most abundant phenolics were mainly hydroxybenzoic acids, free and its derivatives,
among them ellagic acid, protocatechuic acid, and vanillic acid. The results are consistent
with previous reports of BN, indicating the predominance of gallic acid and protocatechuic
acid [12,30,48]. In addition, the present study agreed with those reported for other nuts
comprised mainly of hydroxybenzoic acids. For example, in walnuts, pecans, and pista-
chios, gallic acid and its derivatives were the most abundant; in hazelnuts, the gallic acid
and the protocatechuic acid were most prevalent; and in almonds, protocatechuic, vanillic,
and p-hydroxybenzoic acid dominated [46]. Considering the above, the phenolic profile
obtained from the BN showed several common aspects with previous studies, but also
noted distinctions that contribute to the characterization.
Regarding the beverages obtained from Brazil nuts, according to Table 4, the WBM
and BM-S had a similar total phenol content of 60.2 and 53.3 µg/g f.w., respectively. In
both beverage samples, flavonoids, such as catechin, epicatechin, and their derivatives,
stand out, but an inverse fact was seen in BM-D and cake by-products, where phenolic
acids are more abundant than flavonoids (Table 4). These results suggested that most
of the flavonoids in BN are water-soluble due to the effect of beverage processing and
are transferred to the aqueous phase, but their presence is reduced in nonwater-soluble
matrices such as BM-D and cake. This fact agrees with the report of John and Shahidi [12],
who noted that phenolic acids, such as protocatechuic acid, gallic acid, and ellagic acid,
are mainly concentrated in the BN skin, which was the non-water-soluble matrix found
primarily in the BM-D. In the literature, reports on the phenolic profile of vegetable milks
are scarce, with the exception of soy milk, which showed a higher level of phenolic content
than that observed for Brazil nut beverages studied here (WBM and BM-S). In soy milk,
the level of flavonoids, such as isoflavonoids, are very important and may vary according
to the variety of soy legume [49].
The phenolic compounds identified as predominant in BN and its derivatives, such
as flavan-3-ols (catechin and epicatechin) and hydroxybenzoic acids (ellagic acid, vanillic
acid, and protocatechuic acid), are widely recognized in the literature with promising
effects associated with human health promotion and the prevention of disease risk by
acting as an antioxidant, anti-inflammatory, cardioprotective, and antimicrobial, among
others [50,51]. Therefore, Brazil nuts (BN), beverages of BN (WBM and BM-S), and its
by-products, represent a contribution to the diet through their phenolic phytochemicals.

3.4. Lipids and Other Lipophilic Components

3.4.1. Fatty Acid Profile
The identified fatty acids by gas chromatography are presented in Table 5. The fatty
acids of the Brazil nut (BN) mostly consisted of unsaturated fatty acids (UFA), more than
70% of total fatty acids, mainly linoleic acid (36.9%) and oleic acid (36.4%). The rest of
the fatty acids consisted of palmitic acid (15.9%), stearic acid (10.32%), and in minimal
amounts, palmitoleic acid (0.24%) and arachidic acid (0.1%). This fatty acid profile of the
BN was similar to those reported in the literature [48]. Moreover, it was observed that the
fatty acid profile of the Brazil nut (BN), WBM fractions, and cake, did not show a significant
difference (p < 0.05), demonstrating that the hydrothermal grinding (homogenization) to
obtain the beverage from BN does not affect the percentage composition of the fatty acid
between raw material and its beverages (WBM and BM-S) and by-products (BM-D, BM-D
and cake).
Foods 2021, 10, 3007 15 of 24

Table 5. Fatty acid content of the Brazin nut (Bertholletia excelsa HBK), WBM fractions, and cake.

WBM Fractions
Fatty Acids (FA) % BN Cake
BM-S BM-D BM-F
Palmitic (C16:0) 15.95 ± 1.06 a 15.35 ± 0.15 a 15.36 ± 0.05 a 15.30 ± 0.05 a 15.17 ± 0.11 a
Palmitoleic (C16:1) 0.24 ± 0.01 b 0.20 ± 0.02 b 0.21 ± 0.02 b 0.22 ± 0.02 b 0.22 ± 0.01 b
Stearic (C18:0) 10.32 ± 2.59 c 9.84 ± 0.24 c 10.09 ± 0.15 c 9.57 ± 0.03 c 9.52 ± 0.17 c
Oleic (C18:1) 36.40 ± 5.68 d 36.66 ± 1.23 d 36.59 ± 0.34 d 35.85 ± 0.18 d 35.33 ± 0.45 d
Linoleic (C18:2) 36.99 ± 7.22 e 37.86 ± 1.30 e 37.64 ± 0.47 e 38.97 ± 0.10 e 39.64 ± 0.52 e
Arachidic (C20:0) 0.10 ± 0.06 f 0.09 ± 0.01 f 0.11 ± 0.03 f 0.09 ± 0.01 f 0.11 ± 0.02 f
Total Saturated (SFA) 26.37 ± 3.71 g 25.28 ± 0.39 g 25.56 ± 0.23 g 24.97 ± 0.09 g 24.81 ± 0.30 g
Total Unsaturated (UFA) 73.63 ±12.90 h 74.72 ± 2.55 h 74.44 ± 0.82 h 75.03 ± 0.30 h 75.19 ± 0.98 h
Polyunsaturated (PUFA) 36.99 ± 7.22 i 37.86 ± 1.30 i 37.64 ± 0.47 i 38.97 ± 0.10 i 39.64 ± 0.52 i
Monounsaturated (MUFA) 36.64 ± 5.69 j 36.86 ± 1.25 j 36.80 ± 0.36 j 36.07 ± 0.19 j 35.55 ± 0.46 j
Data are expressed as means ± SD (n = 3). Values with different superscript letters in each row are significantly different (p < 0.05).

Regarding the beverages, the importance of fatty acids falls mainly on WBM beverage
due to the level of total lipids they contain (5.24/100 g f.w.), something that minimally
influences the BM-S beverage, which contains only 0.25/100 g f.w. in total lipids. The
predominant level of unsaturated fatty acids (MUFA and PUFA) compared to saturated
is common in vegetable milk alternatives, as is the case of soy, rice, almond, and cashew
milk alternatives, which usually contain more than 70% UFA of the total fatty acids, with
the exception of coconut milk, with a predominance of saturated fat [4]. This feature of
UFA is considered beneficial for potential health-promoting effects, such as reducing LDL
cholesterol, increasing HDL, and controlling cardiovascular events [52]. However, due to
the oxidation susceptibility and hydrolytic rancidity of the UFA in the beverage obtained
from BN, it is necessary to control the oxidative stabilization processes. Considering the
above and the composition of fatty acids (Table 5), the cake and BM-F by-products are food
ingredients with “healthy fat” characteristics.

3.4.2. Free Fatty Acids and Monoglycerides

Free fatty acids (FFAs) and mono- and diglycerides are associated with shelf life and
quality of edible oils and are normally removed by refining processes because they are
susceptible to autooxidation. In the case of vegetable milks the rancidification phenomenon
is attributed to lipolysis of triglycerides and increase of FFA, causing off flavor and the
deterioration of the beverage [53]. As expected, our work observed corresponding FFA
content (Table 6) and total lipid content (Table 1), in the samples.

Table 6. Free fatty acids, monoglycerides, phytosterols, β-Sitosterol, and squalene content of the Brazil nut (Bertholletia
excelsa HBK), its beverages (WBM and BM-S), and by-products (BM-D, BM-F, and cake).

Raw Material Beverages Byproducts

Compound
BN 1 WBM 1 BM-S 1 BM-D 2 BM-F 2 Cake 2
(mg/100 g) 346 ±61 a 51.3 ±12 ab 3.11 ±0.7 b 181.4 ±41 ab 794 ±172 c 80.5 ±4.1 ab
FFA
(g/100 g oil) 0.59 ±0.10 abc 1.0 ±0.2 abc 1.24 ±0.3 bc 1.24 ±0.28 b 0.98 ±0.2 abc 0.18 ±0.01 a
(mg/100 g) 110.8 ±32 a 113.5 ±34 a 5.66 ±0.8 a 304.3 ±65 a 1789 ±58 b 66.3 ±33 a
Monoglycerides
(g/100 g oil) 0.19 ±0.05 a 2.20 ±0.6 b 2.25 ±0.3 b 2.08 ±0.45 b 2.20 ±0.6 b 0.15 ±0.07 a
Total (mg/100 g) 18.9 ±6 a 3.1 ±0.3 c 0.22 ±0.04 bc 7.2 ±1.3 abc 47.8 ±5 d 25.5 ±0.8 ae
tocopherol (mg/100 g oil) 32.3 ±11 a 59.9 ±6.1 b 88.1 ±17 b 49.3 ±9 ab 58.77 ±6 ab 57.6 ±1.8 ab
(mg/100 g) 16.5 ±4.6 a 2.4 ±0.08 b 0.18 ±0.04 b 5.76 ±1.1 b 37.4 ±1 c 19.9 ±1.9 a
γ-Tocopherol
(mg/100 g oil) 28.3 ±7 ac 47.1 ±1.4 abc 73.5 ±14 b 39.5 ±7 c 45.98 ±1 abc 45.1 ±4.3 abc
(mg/100 g) 2.4 ±2 a 0.7 ±0.26 a 0.04 ±0.01 a 1.4 ±0.2 a 10.4 ±4 b 5.5 ±1.1 ab
α-Tocopherol
(mg/100 g oil) 4.02 ±1.3 a 12.8 ±4.8 a 14.61 ±2.9 a 9.78 ±1.5 a 12.79 ±2.5 a 12.5 ±2.5 a
(mg/100 g) 60.2 ±15 a 7.1 ±0.9 b 0.73 ±0.23 b 30.7 ±6 ab 105 ±11 c 62.1 ±3 a
β-Sitosterol
(mg/100 g oil) 102.8 ±26 b 138.4 ±17 ab 289.5 ±89 a 210.1 ±43 ab 129 ±13 ab 140.4 ±6 ab
(mg/100 g) 217.5 ±13 a 13.2 ±2.8 b 0.69 ±0.11 b 40.1 ±11 b 207.6 ±41 a 123.6 ±8 c
Squalene
(mg/100 g oil) 371.7 ±23 a 256.7 ±50 a 272.62 ±43 a 274.8 ±74 a 255.4 ±50 a 279.7 ±18 a
1mg/100 g is expressed on fresh weight basis; 2 mg/100 g is expressed on dry weight basis. Data are expressed as means ±SD (n = 2). The
different superscript letters within a row are significantly different (p < 0.05).
Foods 2021, 10, 3007 16 of 24

The FFA concentration of BN (raw material), expressed in oil basis, was around
0.6/100 g oil, consistent with one previously reported, in a range of 0.2–0.8/100 g oil [54],
a value within the quality standards established by the European community (≤2/100 g
oil, expressed in oleic acid) to be considered as virgin oil [55]. When the FFA of BN and
its beverages (WBM and BM-S, with 1 and 1.24/100 g oil, respectively) were compared,
there was no significant difference (p ≤ 0.05). However, the slight increase observed can be
attributed to triglyceride lipolysis that might occur during the homogenization process,
when subjected to high temperatures (75 ◦ C) in an aqueous medium.
Although FFA concentration is considered a promising quality indicator for vegetable
milks, reports are scarce. The FFA obtained for the WBM and BM-S beverage had a
concentration of 54.9 and 3.11 mg/100 g f.w., respectively, a lower value than almond milk
(around 380 mg/100 mL)[29] and soybean milk (99 mg/100 mL)[56], as previously reported.
As for the by-products (BM-D, BM-F fraction, and cake), there was also no significant
difference with the raw material in terms of FFA concentration (on oil basis), which ranged
from 0.18–1.24 mg/g oil, showing that they were not affected by the hydrothermal process
of beverage production.
In relation to monoglycerides, expressed in oil bases, the BN had 0.19/100 g oil,
comparable to most oilseeds in the range of 0.2–0.4/100 g oil. However, a significant
increase (p < 0.05) in monoglycerides was observed between the BN (raw material) and
beverages (WBM and BM-S, both with 2.2/100 g oil), which suggests the occurrence of
lipid hydrolysis during beverage processing. This can be attributed to a reaction with the
water molecules, oxygen, exposure to high temperature (75 ◦ C), and, to a lesser extent, by
enzymatic hydrolysis (a considerable part would be inactivated by heat treatment).
As for the by-products, a significant difference (p < 0.05) was found in the concen-
tration of monoglycerides. Despite the cake coming from the hot water grinding stage
(homogenization), its monoglyceride content was low (0.15/100 g oil) with around 2/100 g
oil. This can be explained by the protective and barrier effect of the matrix structure of the
cake, which contains within it the lipid molecules that were not released in the crushing,
thus avoiding contact and reaction with agents that promote lipolysis.

3.4.3. Phytosterols, Tocopherols, and Squalene

These compounds are found in minor quantities, mainly in edible oils of plant origin,
appreciated for their antioxidant capacity and biological properties. Therefore, it was
considered of interest to evaluate the composition of these minor compounds in the Brazil
nut, its derived beverage (WBM and BM-S), and by-products (BM-D, BM-F and cake).
According to the results obtained in Table 6, the BN had a total tocopherol content of
18.9 mg/100 g f.w., mainly comprised of γ-tocopherol (16.5 mg/100 g f.w.) and, to a lower
extent, by α-tocopherol (2.3 mg/100 g d.w.). It is interesting to remark that the γ-tocopherol
content obtained from BN (28.3 mg/100 g oil) may be higher than other edible oils, such
as sunflower oil (1.4–4.5 mg/100 g oil) and olive oil (0.9–1.3 mg/100 g oil), which would
be an interesting feature considering that γ-Tocopherol is recognized as a potent inhibitor
of free radicals against lipid oxidation [57]. Regarding the content of β-sitosterol, the BN
had 102.8 mg/100 g oil, which was consistent with a previous report but shows a relatively
low level if compared to other nuts (range of 97–468 mg/100 g oil) [58]. On the other hand,
the most abundant lipid bioactive minor component found in BN lipid was squalene with
371.68 mg/100 g oil, a great value compared to most other plant sources [59].
The hydrothermal process for the production of the beverage from BN, according to
the results of Table 6, in an oil base, had a positive effect on the increase of total tocopherol
and β-sitosterol in the beverages (WBM and BM-S) with respect to the raw material. This
increase could be linked to the extractability power of the hydrothermal process, hydrolysis
of conjugated (esterified) forms, their affinity to emulsion formation, and their hydrophobic
character as glycosylated sterols. In the literature, despite being scarce, the WBM beverage
(with 3.1 mg/100 g f.w.) is suggested have a total tocopherol comparable to the range
reported in vegetable milks, roughly 0.5–3.9 mg/100 mL [5]. Although there are no reports
Foods 2021, 10, 3007 17 of 24

of squalene content in vegetable milks, its high content in BN compared to other vegetable
sources (explained in the previous paragraph) could consequently estimate that the WBM
beverage (with 14.4 mg/100 g f.w.) contains a higher squalene concentration than other
vegetable milks.
In relation to by-products, according to Table 6 and expressed on a dry basis, BM-F
has the highest content of lipophilic bioactive minor components, followed by cake and
BM-D, which are consistent with the lipid content of each sample (Table 1). In oil bases, no
significant differences of these minor components were observed among the by-products,
nor within the raw material (BN), which reiterates their stability after beverage processing.
In a previous report, using cake (beverage by-product) from the Peruvian Brazil nut, a
similar composition was also found: total tocopherol (56.6 mg/100 g oil) and β-sitosterol
(183.9 mg/100 g oil) and squalene (531.7 mg/100 g oil) [60]. In general, it is possible
to say that the lipophilic bioactive components of the beverage by-products are stable
against the hydrothermal process (homogenization stage) and BM-F and cake contain a
substantial level.

3.5. Characterization of Proteins in Brazil Nut and Derivatives

The Brazil nut and its derivatives had a significant content of proteins, except for
the BM-F. In dry weight (w/w), BN had 16%, BM-S 40.5%, BM-D 57%, and cake 17% of
protein. This remarkable protein level encouraged us to characterize this fraction, which
was performed by SDS-PAGE electrophoresis and mass spectrometry of the protein bands
using MALDI TOF/TOF analysis. Figure 3 show the electrophoretic profile of the different
proteins characterized by the BN and its derivatives.

Figure 3. SDS-PAGE profile of Brazil nut products. Lane 1: Brazil nut (Bertholletia excelsa HBK);
Lane 2: BN protein isolate; Lane 3: BM-D; Lane 4: BM-S; Lane 5: Cake. A to F bands in lanes 1 and 2
were in-gel digested for identification by MALDI TOF/TOF.

The main bands appeared in all samples analyzed and corresponded to molecular
weights between 75–10 kDa. Mass spectrometry identification after in-gel digestion of
the proteins enabled the assignment of three different bands to 11S globulin (B, C, and
D band), which points to the occurrence of different electrophoretic forms of this protein
(see Table 7). In addition, E and F bands could be putatively assigned to the two reported
chains of 2S sulfur-rich storage protein and 2S albumin, although identification must be
contrasted by other methods. Similarly, band A, with the highest molecular weight but
with the lowest occurrence, matched with convicilin from Pisum sativum (Garden pea). The
proteins identified and the observed occurrence is comparable to previous reports where
11S protein was found to be the most abundant protein with around 60–70% of the total
Foods 2021, 10, 3007 18 of 24

protein content of the BN, followed by 2S (19–30%) and 7S (8–10%) [61]. Furthermore, in
Figure 3, all products kept a characteristic profile, but BM-S (Lane 4) showed a higher band
intensity above 20 kDa, suggesting a higher concentration of convicilin and 11S globulin
protein than BM-D (Lane 3) and cake (Lane 5). These proteins have been found to be
soluble at a neutral and alkaline pH, which explains this behavior [62]. Among Brazil nut
proteins, a 9 kDa methionine-rich 2S albumin has been identified as the major allergen in
this nut [63]. This protein could correspond with the electrophoretic band F present in all
products. Several experiments are in progress to evaluate the resistance of these proteins to
gastrointestinal digestion and to identify potential allergenic protein fragments.

Table 7. Identification of peptide fragments of Brazil nut (Bertholletia excelsa HBK) by in-gel digestion of elec-
trophoretic bands.

Band Tryptic Peptide (Ion) Protein Fragment Sequence Protein Name Entry Number
B 1145.44 194–202 RSQKQRGER 11S globulin Q84ND2 (Bertholletia excelsa)
B 1202.43 338–327 MMAPLWRLNA 11S globulin Q84ND2 (Bertholletia excelsa)
B 1145.49 434–442 RLSQEEARR 11S globulin Q84ND2 (Bertholletia excelsa)
QYRLEAEAGVSE
B 2256.85 42–60 11S globulin Q84ND2 (Bertholletia excelsa)
VWDYTDQ
C 1202.46 184–193 RHFFLAGNIQ 11S globulin Q84ND2 (Bertholletia excelsa)
C 999.37 90–97 LYYVTQGR 11S globulin Q84ND2 (Bertholletia excelsa)
C 882.39 131–137 QDQHQKV 11S globulin Q84ND2 (Bertholletia excelsa)
C 1202.55 185–194 HFFLAGNIQR 11S globulin Q84ND2 (Bertholletia excelsa)
D 1165.4 367–376 GETVFDDNLR 11S globulin Q84ND2 (Bertholletia excelsa)
D 1229.54 422–433 RGIPVGVLANAY 11S globulin Q84ND2 (Bertholletia excelsa)
GVSEVWDYTD
D 2315.75 49–70 11S globulin Q84ND2 (Bertholletia excelsa)
QQFRCAGVAAL

3.6. Selenium and Other Minerals

3.6.1. Selenium Content
The Brazil nut is recognized as a food with extraordinary concentrations of Selenium
(Se), an essential micromineral that performs various physiological functions and is at-
tributed with favorable health effects [3]. In the present study, the Se content of the Brazil
nut (BN), its beverages (B-S), and by-products (BM-D, BM-F, and cake) was determined by
ICP-MS (Table 8). Brazil nuts had a Se content of 36.28 µg/g f.w. This value was within the
range of 10–78 µg/g f.w. of the Se content previously reported [64].
Concerning the BN beverages, the WBM showed a higher Se content (15 µg/100 g f.w.)
than BM-S (8.3 µg/100g f.w.) This difference can be attributed mainly to the fact that the
WBM has a higher protein content, roughly double that of BM-S, a nutrient associated
with selenoamino acids. Another factor that contributed to the Se level of WBM was the
participation of BM-D, with 537 µg Se/100 g d.w. According to the Food and Nutrient
Database of USDA [65], commercial vegetable milks (soybean, almond, rice, and coconut
milk) register Se levels up to a maximum of 2.3 µg/100 g f.w., values much lower than
those registered in the WBM and BM-S beverage. However, some previous reports on BN
beverages showed higher Se levels than our study. Sartori et al. [9] obtained 106 µg Se
/100 mL, using a BN with Se content of 871.4 µg/100 g f.w. and defatting the BN before
the beverage is produced. The authors used a higher raw material to water ratio (1:2, w/v)
than in our work (1:7, w/v). In another study, when BN beverage powder (obtained by
spray-drying) was dissolved in water (10 g in 100 mL), the Se concentration was 120 µg/100
mL. This study had also used defatted BN and raw material to water ratio of 1:2 (w/v) [8].
Considering the above, the raw materials and technological processes employed are factors
that influenced the Se level of our beverages compared to others previously reported.
The presence of Selenium linked to proteins in Brazil nuts is well known in the
literature. Still, it is interesting to note the difference in Se level between BN (3721 µg/100 g
d.w.) and cake (229 µg/100 g d.w.) observed in the present study after beverage processing,
even though both have a similar protein content (about 16%, w/w, dry weight). This
Foods 2021, 10, 3007 19 of 24

variation could be explained by the solubility properties of proteins and its selenoamino
acids. According to protein identification (Table 7), albumin proteins (2S albumin, 2S SP1
and 2SP2) are known to be more soluble in aqueous media than 11S globulin and convicilin
(soluble in saline solution). In addition, the albumin protein of BN (about 30% of the
total protein) is considered to be the most abundant of the selenoamino acids (mainly
methionine and cysteine amino acids) [66].
Our work suggests that one BN (~5 g f.w.) could provide about 260% of the dietary
reference value (DRV) (70 µg Se/day); and a cup (~240 mL) of BM and BM-S beverages can
contribute around 50 and 30% of DRV, respectively. In by-products, the Se level of cake and
BM-D in 10 g d.w. are able to provide 32.7 and 76.7% of DVR. To this important contribution
of Se from BN and its beverages must be added its recognized good bioavailability, due
to its occurrence in organic form. According to Lima [64], by X-ray absorption near edge
structure (XANES) spectra study, the Se contained in BN an average of 91% was present in
its organic form, detected as C-Se-C compounds. This characteristic of Se and its recognized
biological value in health allow BN and the derivatives studied (beverages and by-product
with remarkable Se level) to be considered as an important source of this mineral and its
possible application in food as a healthy ingredient.

3.6.2. Other Minerals Content

Table 8 show the other minerals quantified in the Brazil nut and its derivatives,
omitting from Selenium. In the BN, the content of macrominerals showed the follow-
ing order of predominance: P (686.05 mg/100 g f.w.), K (576.21 mg/100 g f.w.), Mg
(468.85 mg/100 g f.w.), and Ca (186.83 mg/100 g f.w.). In microminerals, those that stood
out are: Zn (4.72 mg/100 g f.w.), Fe (3.12 mg/100 g f.w.), and Cu (1.83 mg/100 g f.w.). The
values obtained in the present study are similar to those reported by the USDA National
Nutrient Database [65] regarding Brazil nuts. Likewise, taking into account the report of
dietary reference values (DRV) for nutrients by EFSA [67], for adults over 18 years old, one
edible portion of BN (~6 units and 30 g) could provide about 40% of the daily requirement
in Mg, P, and Cu, followed by Zn (12.3%), Fe (8.5%), Mn (7.5%), and about 5% in Ca and K.

Table 8. Mineral content of Brazil nut (Bertholletia excelsa HBK), its beverages (WBM and BM-S), and by-products (BM-D,
BM-F, and cake).

Minerals Raw Material Beverages By-Products DRV 3

(mg/100 g) BN 1 WBM 1 BM-S 1 BM-D 2 BM-F 2 Cake 2 (mg/Day)

Mg 468.85 ±25 a 35.89 ±1.1 b 11.06 ±0.67 b 2301.5 ±62 c 19.27 ±1.06 b 513.03 ±8.8 a 325
K 576.21 ±40 a 77.70 ±0.7 b 55.13 ±1.25 b 1949.6 ±108 d 38.92 ±1.89 b 421.15 ±44 c 3500
Ca 186.83 ±2.6 a 13.96 ±2.3 a 2.36 ±0.27 a 1196 ±227 b 7.79 ±1.18 a 221.62 ±1.6 a 1000
±11.6
P 686.05 ±13 a 60.59 ±2.3 b 11.38 ±1.16 b 4605 ±151 c 23.17 ±4.38 b 725.11 a 550
±0.1 a b d ±0.27 e b ±0.1 c
Zn 4.72 0.43 ±0.01 0.02 ±0.00 36.4 0.59 ±0.08 5.71 11.5
Cu 1.83 ±0.1 a 0.21 ±0.0 b 0.16 ±0.00 b 3.86 ±0.03 d 0.16 ±0.02 b 1.48 ±0.0 c 1.5
Fe 3.12 ±0.1 a 0.22 ±0.0 bd 0.01 ±0.00 d 19.04 ±0.22 e 0.31 ±0.05 b 3.40 ±0.01 c 11.0
Mn 0.75 ±0.1 a 0.07 ±0.0 b 0.01 ±0.00 b 6.66 ±0.14 e <1.1 b 1.06 ±0.02 c 3
Se 70
3628 ±51 a 15 ±0.2 d 8.3 ±0.14 d 537 ±6 b 19 ±0.3 d 229 ±3 c
(µg/100g) (µg/day)
1Expressed on fresh weight basis; 2 Expressed on dry weight basis; 3 DRV, Dietary reference value based in Population reference intakes
and adequate intakes (PRI-AI) of minerals for adults (estimated for males and females ≥18 years old), EFSA [67]; Data are expressed as
means ±SD (n = 3). The different superscript letters within a row are significantly different (p < 0.05).

With respect to beverages, as shown in Table 8, in most minerals, WBM had slightly
higher concentrations than BM-S. This is understandable due to the participation of the
sediment in the WBM, which has a high level of minerals. When compared with other
commercial vegetable milks (from soybean, almond, rice, and coconut), reported by Vanga
and Raghavan [68], the WBM showed, in most minerals, a level within the range, i.e.,
at Mg (13–49 mg/100 g f.w.), K (47–364 mg/100 g f.w.), P (48–108 mg/100 g f.w.), Fe
(0.05–0.84 mg/100 g f.w), and Z (0.38–0.75 mg/100 g f.w.). In BM-S, a lower mineral
Foods 2021, 10, 3007 20 of 24

content than commercial vegetable milks was noted. Regarding calcium, both WBM
(13.9 mg/100 g f.w.) and BM-S (2.4 mg/100 g f.w.) had lower concentrations than other
commercial plant milks, which typically have values greater to 100 mg/100 mL as they
are often fortified to mimic the calcium content of cow’s milk [68]. In the aspect of daily
nutritional intake (DRV of Table 8), a cup (~240 mL) of WBM beverage may contribute
mainly Mg with 26.5%, P with 26.4%, and Cu with 34%. As for the BM-S beverage, only Cu
with 26%. The rest of the minerals had a contribution of less than 10% in both WBM and
BM-S beverages.
Regarding by-products, it was observed (Table 8) that the BM-D had higher mineral
concentrations (p < 0.05) than cake and BM-F. Regarding its daily nutritional contribution
(according to DRV), an intake of 10 g d.w. of BM-D may to provide P with 84%, Mg with
71%, Zn with 32%, Cu with 27%, Mn with 22%, Fe with 17%, and Ca with 12%. The cake,
with an intake of 100 g d.w., may provide Mg with 160%, P with 132%, Cu with 102%, Zn
with 50%, Mn with 36%, Fe with 30%, and Ca with 22%. According to the above, BM-D
and cake by-products represent an important source of Mg, P, Zn, and Cu and Mn, Fe, and
Ca to a lesser degree.

3.7. Antioxidant Capacity

In this work, three in vitro methods were used to evaluate the antioxidant capacity
of the BN samples, as shown in Table 9. The antioxidant level of the Brazil nut showed
consistency to the previous report, i.e., 2.6 µmol TE/g f.w. by DPPH assay [20], ~11 µmol
TE/g f.w. by TEAC, and ~26 µmol TE/g f.w. by ORAC [12].

Table 9. Antioxidant capacity of the Brazil nut (Bertholletia excelsa HBK), its beverages (WBM and
BM-S), and by-products (BM-D and cake), expressed as µmol TE/g.

Raw Material Beverages Byproducts

Assay
BN 1 WBM 1 BM-S 1 BM-D 2 Cake 2
DPPH 1.72 ±0.11 a 0.08 ±0.00 b 0.07 ±0.00 b 1.52 ±0.04 c 0.27 ±0.03 d
TEAC 14.39 ±0.46 a 0.82 ±0.04 b 0.71 ±0.03 b 11.03 ±0.89 c 3.34 ±0.15 d
ORAC 10.98 ±0.45 a 0.61 ±0.01 b 0.47 ±0.01 b 14.18 ±0.77 c 6.73 ±0.40 d
1Expressed on fresh weight basis; 2 Expressed on dry weight basis. Data are expressed as means ± SD (n = 3).
The different superscript letters within a row are significantly different (p < 0.05).

Regarding the BN beverages, WBM and BM-S showed a similar antioxidant capacity,
with no significant difference (p < 0.05). In the DPPH assay, WBM and BM-S had 8
and 7 µmol TE/100 g f.w., respectively, values that are in the range of other commercial
vegetable milks that range from 4.7–30.5 µmol TE/100 mL by DPPH [28].
With respect to the antioxidant capacity of the BN by-products (Table 9), the BM-D
showed greater antioxidant power than the cake. These results had a relationship with the
TPC content shown in Table 2. In the literature, there are no reports about the antioxidant
capacity of the cake obtained as a by-product of beverage processing of BNs, but our result
is similar to the cake obtained from defatted BNs by pressing (4.07 µmol TE/g d.w. by
TEAC assay), reported by Gomes [30]. If compared to the antioxidant power (by TEAC
assay) of the cake produced by soymilk (called “okara”), with 19–29 µmol TE/g d.w. [69],
the antioxidant capacity of the BN cake was minimal.

4. Conclusions
The present study reports a comprehensive compositional characterization of the
Brazil nut (BN), its derived beverages, and processing by-products. The BN showed an
interesting nutritional quality related to its bioactive constituents (such as unsaturated fatty
acids, phytosterols, tocopherols, squalene, phenolic compounds, proteins, and selenium).
Twenty-four phenolic compounds were identified in Brazil nuts and products, flavan-3-
ols and the hydroxybenzoic acids, the phenolics more abundant. Brazil nut beverages
showed an interesting content in phenolic compounds, and their nutritional value was
Foods 2021, 10, 3007 21 of 24

comparable to other commercialized vegetable milks, WBM being the beverage with the
higher content of nutritional components as the minor bioactive lipids (total tocopherols,
β-sitosterol, and squalene). The selenium content of the Brazil nut beverage was higher
than those reported for other commercial vegetable milks. For this reason, the BN beverage
represented an important alternative source to the daily requirement of Se in the human diet.
Additionally, the hydrothermal processing for beverage production has a significant effect
on the content of some minor lipid components (FFA, MG, total tocopherol, β-sitosterol, and
squalene), by the hydrolysis of triglycerides and certain conjugated compounds. Regarding
the BN by-products, the fat fraction (BM-F) is mainly composed of oleic and linoleic
acids and other minor lipid components; the sediment fraction, a by-product produced in
minimal quantities, showed high content of protein, selenium, other minerals, and phenolic
compounds; and the cake retained many of the nutritional components of the BNs. The
present study provides some support for future studies related to the improvement of the
technological processes used to produce BN beverages, to influence the stability of these
processes (physical and oxidative) regarding bioactive compounds present in the beverage,
as well as the development of a smart formulation of BN beverages according to the target
consumer (older people, children, others).

Author Contributions: Conceptualization, M.P.C. and T.F.; methodology, W.V.V.-R., M.P.C., D.M.
and B.M.; software, W.V.V.-R.; validation, W.V.V.-R., M.P.C., D.M. and B.M.; formal analysis, W.V.V.-R.,
D.M. and B.M.; investigation, W.V.V.-R., M.P.C., T.F. and I.R.; resources, M.P.C.; data curation, M.P.C.,
T.F., I.R. and W.V.V.-R.; writing—original draft preparation, W.V.V.-R., D.M. and B.M.; writing—
review and editing, M.P.C., T.F. and I.R.; visualization, M.P.C. and T.F.; supervision, M.P.C., T.F. and
I.R.; project administration, M.P.C.; funding acquisition, M.P.C. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Spanish Ministry of Science and Innovation, project
number RTA2015-00044-C02-02 and PID2020-118300RB-C21.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: Author Wilson V. Vasquez thanks the scholarship “Presidente de la Repub-
lica” postgrade program (RJ 4285-2018/MINEDU/VMGI-PRONABEC-OBE) from the Ministry of
Education of the Republic of Peru.
Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication
of this paper.

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