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Mesocarp of Brazil nut (Bertholletia excelsa) as inspiration for new impact

resistant materials

Article  in  Bioinspiration & Biomimetics · May 2019

DOI: 10.1088/1748-3190/ab2298

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 Mesocarp of Brazil Nut (Bertholletia excelsa) as Inspiration for New Impact
Resistant Materials
M. Sonego1, C. Fleck2, L.A. Pessan1
1
Federal University of São Carlos (UFSCar), Graduate Program in Materials
Science and Engineering (PPG-CEM), São Carlos, SP, Brazil;
2
Materials Engineering, Technische Universität Berlin, Berlin, Germany

Abstract— Aiming to produce bioinspired impact and puncture resistant

materials, the mesocarp of Brazil nut (Bertholletia excelsa) was characterized. The
mesocarp composition was investigated by chemical extraction and its
microstructure was analyzed by optical microscopy and microtomography
(microCT). Compression test evaluated the force need to open the mesocarp shell.
Shore D hardness test and nanoindentation measured the local mechanical
properties at different length scales. Brazil nut mesocarp has a higher content of
lignin (56 %) than other nutshells and is mainly composed of sclereids and fibers
cells arranged together and not in separated layers as usually found in nature.
Mesocarp has an internal and external layer with fibers oriented from peduncle to
opercular opening and a middle layer where entangled fibers are latitudinally
oriented. To open a Brazil nut mesocarp, compression forces of 10079 ± 1460 N
(parallel to latitudinal section) and 14785 ± 4050 N (perpendicular to latitudinal
section) are needed. Such forces are higher than the forces need to open most
nutshells, if fracture force is normalized by shell thickness. Shore D hardness test
showed that hardness is uniform in the mesocarp, although it is higher in the center
of the thickness than close to the inner or outer surface. The cell wall of fibers have
a higher reduced modulus then the cell wall of sclereids although they have a
similar hardness. These microstructural and mechanical results indicate that Brazil
nutshell has great potential as a source for bioinspiration and motivates further
studies.

Keywords— Bioinspiration, Brazil nutshell, mechanical properties, microstructure

1
 I. INTRODUCTION

For thousands of years, nature has accumulated countless possibilities for

optimized solutions to engineering problems using limited raw material, room
temperature, aqueous solutions, solar energy and renewable resources. [1]
Recently, this natural accumulated knowledge has increasingly being studied and
applied to solve technical problems in studies of biomimicry and bioinspiration.
The fruit walls of nuts and drupes are of special interest for the development
of impact and puncture resistant bioinspired structures. [2] They protect the seed
from falls after fruit maturation and from predators with sharp teeth and high biting
strength. The high impact and puncture resistance of many fruit walls come from a
combination of hierarchical structure and the existence of different vegetal tissues,
which can inspire the development of new lightweight materials. [3] These
bioinspired materials can be applied as containers for transportation of dangerous
goods, as helmets and other protective clothing and as protective shields in
vehicles against impact. [3]
Macadamia (Macadamia sp.) nutshell is one example of an especially
strong and tough seed shell. It shows a sandwich structure formed by four layers of
sclerenchymatous tissue and two inner layers of non-sclerenchymatous tissue that
form an efficient and damage tolerant shell. [4], [5] According to Jennings and
Macmillan [5], Vickers hardness of macadamia nutshell (density of 1.3 g/cm 2)
reaches average values of up to 180 (± 30 MPa). Fracture strength spans 25 to 80
MPa, Young’s modulus 2 to 6 GPa and work of fracture 100 to 1000 J/m2. This
mechanical performance is comparable to the performance of engineering
materials. Macadamia nutshell has the same Vickers hardness as commercially
pure aluminum (1100-0) and it fractures at stresses that are twice the yield strength
and half the ultimate tensile strength of this engineering alloy although this metal
has twice the density of macadamia nutshell.
Citrus maxima and coconut (Cocos nucifera) are further examples of impact
resistant vegetal structures. The mesocarp of Citrus maxima is an open-pore foam
with a gradual increase in pore size from the inside to the outside. [6] It has low a
Young’s modulus, 0.14-0.45 MPa in compression and 0.39-0.98 MPa in tension,

2
and a fracture strain of 60% under tension. In cyclic quasi-static compression tests,
Citrus maxima pericarp dissipates 75% of energy during the first cycle, and in free
fall experiments it has a deceleration of 3100 m/s2. [6] The coconut pericarp is a
combination of a mesocarp with flexible and poorly interconnected fibers, which
promotes high energy absorption, and a stiff and tough endocarp formed by highly
lignified and densely arranged sclereid cells. [6], [7] It can withstand falls from more
than ten meters without any damage to the fruit. [6] In a recent study [8], its high
toughness (rising R-curve with a maximum at 3.2 MPa.m 1/2) has been explained by
the tortuous crack path running through boundaries of sclereid cells and crack
trapping and deflection by open channels which are part of the endocarp structure.
The natural structure of macadamia nutshell and the pericarp of Citrus maxima and
coconut can inspire the production of materials with good energy dissipation,
shock-protection, and puncture resistance through the combination of solid layers
with spongy fiber-reinforced structures. [6]
The endocarp of the Cocoyol palm tree (Acrocomia mexicana) is a hard and
tough natural material composed by functionally graded sclereids cells.[9] The
geometry of its sclereids varies from polyhedral shape to bundles of elongated
cells. The external layer is harder due to a denser packing of polyhedral sclereids
while the entangled elongated sclereids near the inner edge leads to tortuous crack
propagation in uniaxial compression. Such complex microstructure results in
strength versus toughness performance superior to wood and comparable to
synthetic composites performance. [9]
Babassu (Orbignya speciosa) nutshell also inspired the design of new
materials. It has a thin exocarp, a starch mesocarp and a lignified endocarp. [10]
Staufenberg et al. [10] produced a composite of cellulose fibers (L), polylactide
(PLA) and polypropylene (PP) bioinspired by the babassu nutshell layers that has
impact strength 1.6 times higher than the reference PLA/PP/L regular composite.
Another biological example of outstanding mechanical properties and
astounding fracture resistance is the Brazil nutshell (Fig. 1 (a)). It is the globular dry
and woody fruit of Brazil nut tree (Bertholletia excelsa) which is relatively big, with a
diameter of 10 to 12 cm.[11] Figure 1 shows the tree and gives a survey of the

3
fruits.
The single fruits can weigh up to 1 kg, and they demonstrate an impressive
impact resistance as they survive a free fall from trees of up to 50 meters height
(Figure 1(b)), which is equivalent to a (small) skyscraper with 16 (!) floors. [12] The
Brazil nut fruit has three layers, shown in Figure 1(c): exocarp composed of dead
cells which rot and are released after maturation, a mesocarp formed by a
glabrous, fibrous layer with a thickness of approximately 1 cm and an endocarp,
which is a thin, glabrous, cartaceo, slightly fibrous and septate layer. [13], [14] The
mesocarp, the thickest and the most robust layer, is responsible for the mechanical
resistance of the Brazil nut fruit: the exocarp is very often already rotten when the
fruits fall to the ground, and the endocarp is so thin that it may safely be assumed
to play hardly any role for the mechanical impact properties. Therefore, the focus in
this study was on the mesocarp. The natural function of the shell is to protect the
seeds from predators during ripening, and from impact when they fall from the
trees, ensuring maximum dispersal of seeds. In the Amazon forest, the natural
habitat of Bertholletia excelsa, agoutis are the only animals with sharp enough
teeth and/or sufficient biting strength to break the nutshell and consume the seeds.
Agoutis usually cache some seeds for future consumption, and if they do not find
them again, plants germinate thus helping the propagation of the species. [11]
Interestingly, this dispersal mechanism is more efficient than releasing the seeds in
the surroundings, as Bertholletia excelsa did thousands of years ago.
The Brazil nut fruit is classified as dehiscent, as in prehistoric times, it
released its seeds through an opercular opening. [15] With time, the fruit evolved to
store the seeds by decreasing the opercular opening diameter and adapted its
seed dispersal strategy to depend on scatter-hoarding rodents, such as agouti. A
fruit with a woody and thick shell that is hard to open may motivate the animal to
cache seeds for future consumption, helping the dispersal of the species.
Bertholletia excelsa fruit is a capsule which stands out among nuts, drupes
and other dry fruits by its comparatively large dimensions and robustness, capacity
to survive to high falls and by its challenging opening, even for human
technologies. Brazil nut fruits are bigger than most dry fruits. Its dimensions are

4
rather comparable with coconut fruit. The latter is even bigger, but their pericarp is
thicker and is made of three very different layers to protect its seed. In contrast,
Brazil nut fruits are mainly protected by one mesocarp layer, which is, furthermore,
thinner than the coconut pericarp. Definitely, nature must have a good reason to
spend so much energy to create this robust structure and rely on only one animal
as the species disperser. A mechanical and microstructural comparison between
several dry fruits may bring new ideas to understand nature strategies to create a
protective capsule or nutshell, what is also very important to human technological
development.
This work characterized the microstructure of the mesocarp, the mechanical
performance of the whole mesocarp under compression and its hardness
properties at different length scales. The mesocarp microstructure was evaluated
by chemical analysis, optical microscopy and microtomography (microCT).
Additionally, the mechanical and microstructural characteristics of Brazil nut
mesocarp will be compared with literature information of others typical nutshells
and dry fruits, as macadamia and coconut. This is important to understand the
principles bringing about the outstanding mechanical performance of Brazil nutshell
and to enable us to produce bioinspired materials with improved impact energy
absorption.

II. MATERIALS AND METHODS

A. Materials

Brazil nut fruits, shown in Figure 1, were obtained from Juina – MT, Breves -
PA, and Jaboticabal – SP, in Brazil.

5
Figure 1: Bertholletia excelsa: (a) Edible seed, tegument and dry fruit; (b) tree in
Amazon forest; (c) longitudinal section showing endocarp (green), mesocarp (red)
and exocarp (blue). Peduncle and opercular opening are highlighted.

The fruits were cleaned with water, dried at room temperature and finally cut
with a band saw in two different orientations. The exocarp, the endocarp and the
seeds were discarded, so that just the mesocarp remained for analysis. Figure 2
shows the nomenclature adopted in this study for denominating sections, cuts, and
cell orientation.

6
Figure 2: (a) Brazil nut fruit showing (b) latitudinal and (c) longitudinal sections and
the orientation nomenclature adopted in this study.

B. Chemical analysis

To measure its composition, the mesocarp was milled with a knife mill
(Wittman Battenfeld, MAS1), and subsequently with a hammer mill (Servitech, CT-
058I), and finally sifted. The steps of the chemical analysis are given in Table 1.

7
Table 1: Steps of the chemical analysis of Brazil nut mesocarp.

Analysis of Method Particle Based on

size [mm]

1 Moisture content Weight loss 0.5-0.35 ASTM D4442-15

2 Extractives removal Soxhlet extraction 0.18-0.35 ASTM D1105-96

3 Lignin content Klason method 0.18-0.35 ASTM D1106-96

4 Holocellulose content Chloration 0.18-0.35 [16]

5 Separation of α-cellulose NaOH maceration 0.18-0.35 [16]

6 Ash content Thermogravimetry 0.5-0.35 [17]

The moisture content was calculated using the original weight (wet basis)
and the weight after drying in an oven with air circulation (Tecnai, TE-394/1). A
scale (Shimad Witzu, AY220h) with a precision of 0.1 mg was used for the weight
measurements on eight samples that were measured every three hours until
weight change was below 0.1 %.
Before any measurements of the lignin or cellulose contents, the extractives
of mesocarp, such as tannin, were removed. A solution of ethanol-acetone (1:2)
was chosen, followed, after four hours, by pure ethanol for six hours because it is
very efficient to extract plant constituents, and furthermore, it is less toxic than the
ethanol-toluene (1:2) solution usually applied. [18], [19] In the final extraction stage,
boiling water was used as a solvent. The system was heated in a water bath and
the boiling water in contact with the sample was changed every hour. After
completion of the third extraction with water, the sample was filtered, dried and
weighed.
After Klason method (lignin), chloration (holocellulose) and NaOH
maceration (α-cellulose) the same weighting procedure was adopted. It consists in
filtering the respective final solution under vacuum with filter paper (porosity 14 µm)
and with a fritted-glass crucible (porosity number 3), both previously weighed. The

8
content of filter paper and crucible was then dried and weighed.
The lignin content of the mesocarp was measured by the Klason method.
Dried and extractive-free mesocarp was firstly cold mixed with aqueous solution of
72% (w/w) H2SO4 for two hours. Then it was boiled in aqueous solution of 3%
(w/w) H2SO4 for four hours, keeping a constant solution volume by adding more
water. The solution was filtered as mentioned before.
An aliquot of the filtered solution was used to estimate the soluble lignin
content by ultraviolet-visible light spectrophotometry with a spectrophotometer from
Varian, model 50 UV/Vis, adopting scan range from 200 nm to 800 nm, scan rate
of 24000 nm/min and data interval of 5 nm. The white solution used in the analysis
consisted of 1 % (w/w) H2SO4 aqueous solution.
The soluble lignin content was calculated by equation 1, where A 215 is the
absorbance at 215 nm and A280 is the absorbance at 280 nm. [12]

4.53 215 280

( ⁄ ) (1
300

The holocellulose (hemicellulose and α-cellulose) extraction by chloration

was performed according to the method described in [16]. Dried and extractive-free
mesocarp, ethanoic acid and sodium chloride were heated in a water bath at
70 °C. The sodium chloride reacts with ethanoic acid forming hydrochloric acid,
which oxidizes lignin, thus allowing holocellulose extraction. [20] Additional
reagents were added every hour until the sample was white and then filtered and
weighed as mentioned.
The procedure used to separate α-cellulose from holocellulose started by
macerating holocellulose with a glass rod in a sodium hydroxide 17.5 % (w/w)
aqueous solution for ten minutes. The system was left resting for 30 minutes
before more water was added to decrease the concentration of sodium hydroxide
to 0.05 % (w/w). This solution was stirred for one hour. Finally, the final solution
was filtered and weighed.
Thermogravimetry analysis was used to measure the ash content of
mesocarp using TGA model G50 from TA Instruments. The procedure is described
9
in Table 2.

Table 2: Thermogravimetry procedure to estimate the ash content of

mesocarp. Based on [17].

Heating
Temperature
Step Action Atmosphere Time [min] rate
[°C]
[°C.min-1]

1 Heating 23 to 35 - 5

2 Isotherm 35 20 -

3 Heating N2 - 10 ml.min-1 35 to 110 - 30

4 Isotherm 110 30 -

5 Heating 110 to 500 - 5

6 Heating 500 to 700 - 20

-1
O2 - 90 ml.min
7 Isotherm 700 5 -

Thermogravimetry analysis also evaluated if chemical analysis successfully

isolated each component of mesocarp. Lignin, cellulose, and α-cellulose obtained
from previous chemical analysis and mesocarp (without extractives) were
decomposed and its differential thermogravimetric spectra (DTG) were compared.
In an inert atmosphere (nitrogen - 10 ml.min-1), each sample was heated to 700 °C
using heating rates of 5 °C.min-1 and kept at this temperature for 30 minutes. The
decomposition procedure was described in [21].

10
 C. Microscopy

Latitudinal sections of the mesocarp were cut with a band saw (see Figure
2) and subsequently ground with sandpaper of decreasing roughness (grit
numbers 800, 1200, 2400 and 4000) to evaluate the mesocarp thickness by light
microscopy (VHX 100: Keyence Deutschland GmbH). Additionally, pieces of
mesocarp were embedded in polyester resin, also ground with sandpaper (grit
number 120, 320, 600 and 1200) and polished with alumina suspension with a
particle size of 1 m. The identification of cells was made using these mesocarp
pieces in an Olympus BX41M-LED optical microscope.

D. Microtomography (microCT)

MicroCT scans of an whole mesocarp and pieces of it were made with

phoenix nanotom m 180 kV/20 W X-ray microCT and nanoCT Computed
Tomography System from GE Measurement & Control. Different resolutions were
adopted depending on the size of the scanned piece. The images of whole
mesocarp, intermediate pieces (20 x 15 x 10 mm) and small pieces ( 1 x 1 x 1 mm)
have voxel size of 0.06 mm, 0.009 mm and 0.001 mm, respectively. Image J - FIJI
[22] and Thermo Scientific Avizo Software were used for reconstruction and
visualization.

E. Compression test of mesocarp

After removal of exocarp, Brazil nut fruit was tested under compression
forces and the effect of endocarp and the stored seeds was ignored. Compression
tests were carried out using an Instron machine model 5569 with a maximum force
of 50 kN. Force was measured by a load cell with a maximum force of 50 kN.

11
Crosshead displacement rate was set to 5 mm/min. To hold the spherical
specimen in place in the correct orientation, the specimens were loaded to a
maximum force of 0.05 N with a force rate of 0.05 N/min before the actual
compression test. All tests were performed in ambient air at room temperature.
Compression forces were applied parallel and perpendicular to the latitudinal
specimen section (Figure 2).

F. Hardness

The hardness of the mesocarp was measured on different length-scales:

i) Nanoindentation was used to measure the reduced modulus (Er) and the
hardness (H) of the cell walls. Sections were embedded in polyester resin, ground
with sand paper (grit numbers 120, 320, 600 and 1200) and polished with alumina
suspension. The measurement was done in a scanning nanoindenter (Ubi 1,
Hysitron IInc.) using a tetrahedral Berkovich diamond indenter tip. For accurate
positioning of the indents, an imaging scan of the cell wall was performed on the
indenter stage by means of in situ scanning probe microscopy (SPM) with the
indenter tip and a scan size of 30 µm x 30 µm. The intended indent positions were
marked on the SPM image and indentation was executed from this mode.
Afterward, the position of all indents was verified by SPM. We used a loading and
unloading rate of 8.3 µN/s. Maximum load was 250 µN, and it was held constant
for a dwell time of 10 s. The data were analyzed with the TriboScan software
(Hysitron Inc.) according to the method described by Oliver and Pharr. [23] Five
indentations were made in the cell walls of five different fibers and sclereid cells
each, in different regions of the embedded sample.
n NOV analysis and a Turkey’s post hoc pairwise comparison was
made to evaluate if the average values of Er and H for fibers and sclereids are
statistically different at a confidence level of 95% (α 0.05 .
ii) Shore D hardness map of mesocarp sections (Figure 3) evaluated
possible differences in local mechanical properties. Shore D test, based on ASTM
D2240, measure the penetration of an indentor (tip ray of 0.100 ± 0.012 mm), into
12
the specimen under a force produced by a 5 kg mass during a penetration time of
1 second. The Shore D hardness is inversely related to the penetration and is
dependent on the elastic modulus and viscoelastic behavior of the specimen. It
was adopted once it can estimate the hardness without evaluation of an indentor
mark, which is not an easy task for a natural material.

Figure 3: Schematic drawings showing the Shore D indentation map on mesocarp

sections: (a) longitudinal section, (b) latitudinal section and (c) three regions (A, B
and C) in a longitudinal section to evaluate differences over the thickness of the
mesocarp layer.

Shore D hardness was also used to compare the local mechanical

properties of different nutshells. For that, walnut shell, macadamia shell, coconut
endocarp, Brazil nut tegument and mesocarp were embedded in polyester resin
and a slice with parallel and flat surfaces of approximately 6 mm of thickness was
cut with a diamond saw. The resin was useful to provide stability to the slice of thin
specimens, as walnut shell and Brazil nut tegument. Five indentations were made
at different positions in the thickness of each shell.

13
 III. RESULTS AND DISCUSSION

A. Chemical composition

Mesocarp of Brazil nut has a moisture content of approximately 9.7 % ±

0.3%. The lignin, holocellulose and α-cellulose contents obtained by chemical
extraction, and the ash content determined by thermogravimetry are shown in
Table 3.

Table 3: Composition of Brazil nut mesocarp estimated by chemical extraction

Component Content [% (w/w)]

Moisture 9.7
Ash 6.2
Extractives 2.5
Insoluble lignin 56
Soluble lignin 2.2
α-cellulose 15.9
Hemicellulose 15.7
Holocellulose (hemicellulose and α-cellulose) 31.6

Thermogravimetric analysis was used to evaluate the products of chemical

extraction and the extraction efficiency. Figure 4 shows the thermogram (TGA) and
the differential thermogravimetric spectra (DTG) of mesocarp (without extractives),
lignin, holocellulose, and α-cellulose, all results from chemical extraction.
The DTG curve (Figure 4(b)) of mesocarp has the first peak between 200 -
300 °C. This peak is due to decomposition of hemicellulose that is also present in
the DTG curve of holocellulose, but it is absent in the DTG curve of α-cellulose,
showing that its isolation was successful. When isolated, the α-cellulose has just a
high and narrow decomposition peak at 330 °C. However, this decomposition peak
is found around 315 ° when α-cellulose is part of mesocarp or of holocellulose.
Lignin slowly decomposes over a large temperature range, with the highest
14
decomposition rates at 300 °C and 470 °C. The DTG curves in Figure 4(b) show
that chemical extraction was efficient. There is no cellulose peak in the DTG curve
of lignin, proving the successful removal of cellulose by the Klason method.
Moreover, DTG curves of holocellulose and α-cellulose have no peaks at
temperatures higher than 400 °C, which would be lignin peaks. The separation of
holocellulose in α-cellulose and hemicellulose also shown to be satisfactory as
DTG of α-cellulose has no peak between 200 - 300 °C, which is accounted to
hemicellulose residues.

Figure 4: Thermogravimetric analysis of mesocarp (without extractives), lignin,

holocellulose and α-cellulose obtained in the chemical extraction: (a) thermogram
(TGA) and (b) differential thermogravimetric spectra (DTG)

B. Microstructure

Figure 5 shows a section through a Bertholletia excelsa fruit, exposing the

endocarp, mesocarp and exocarp. The endocarp is very thin (approximately 200
µm) as compared to the other two layers. The exocarp of most fruits decays and
falls off after maturation such that it is usually not observed. The fruit shown in
Figure 5 (a) still contains the exocarp and endocarp layer and its interfaces with
mesocarp are shown in Figure 5 (b) and (c), respectively. The interface between

15
mesocarp and exocarp has defects and large pores, what facilitates its removal. In
contrast, mesocarp and endocarp are well adhered, as can be seen in Figure 5 (b).

Figure 5: Bertholletia excelsa fruit: (a) exocarp (blue), mesocarp (red), endocarp
(green), tegument (yellow) and seed (pink) are highlighted; (b) thin endocarp layer
and (c) exocarp layer with rotten tissue

Figure 6 shows the two types of vegetal cells identified in the mesocarp.

16
Figure 6: Vegetal cells found in the mesocarp of Brazil nuts: light micrographs of
(a) sclereids, (b) transversely cut fibers and (c) longitudinally cut fibers (d) 2D slice
through microCT reconstruction showing the distribution of fibers and sclereid cells:
arrows indicate natural defects, (e) 3D volume reconstructions of microCT data in
two different resolutions: the different cell types and the defects are painted in
different colors.

17
 Sclereids (Figure 6(a)), also known as stone cells, are short, isometric, often
dead cells which are part of the sclerenchymatous tissue. The secondary walls of
sclereids are highly lignified resulting in a hard and inflexible tissue. [24] The
mesocarp further contains fibers, another sclerenchyma tissue. The cross-section
of fibers is shown in Figure 6(b) while the longitudinal section is shown in Figure
6(c). Fibers are elongated cells with thick and lignified cell wall, which are usually
organized as a bundle. They are elastic and provide strength and elasticity to the
vegetal tissue.[24] Although the cell wall of both cells has the same thickness,
sclereids are larger (area of 1014 ± 424 µm2) than fibers (area of 238 ± 127 µm2),
thus, when cross-sections are compared, sclereids (Figure 6(a)) seem to be hollow
cells while the inside of fibers (Figure 6(b)) is mostly filled up with cell wall material.
As shown in Figure 6 (d) and (e), fibers and sclereid cells appear to form an
intricate mixture, together building the mesocarp. The fibers are organized as
bundles, and they form a net while the spherical sclereids cells fill the gaps
between fiber bundles. It was further observed numerous natural defects, like open
channels and cracks, in the mesocarp structure, as shown in Figure 6 (d) and (c).
These defects are traces of the previous vascular bundles that distributed water
and nutrients through the mesocarp when the cells were still alive.
Figure 7 shows the thickness of mesocarp in a latitudinal section.

18
Figure 7: (a) Thickness of mesocarp in a latitudinal section where the region under
the circle has a higher magnification in (b). White arrows highlight the longitudinal
section of a fiber bundle, lighter regions are sclereid cells and dark regions are
cross sections of fibers in a bundle.

The elongated, brighter, regions are longitudinal sections of fiber bundles

while the darker spherical regions are cross sections of fiber bundles. Sclereids
cells fill the space between the fibers bundles and are better seen at higher
magnifications (Figure 7(b)). The fiber orientation in the mesocarp is complex
(Figure 7 (a)). Three layers can be distinguished: an internal layer (layer I), from
the inner side to the dotted line, which has dark and circular regions - cross-
sections of fiber bundles. Therefore, in layer I, the fiber bundles are oriented from
the peduncle to the opercular opening. In the second, intermediate layer (layer II),
between the dotted lines, the fiber bundles seem to be entangled. However, the
bundles nevertheless show a preferential orientation with their long axis parallel to
the latitudinal direction. The third, outer layer (layer III), close to the surface of the
nutshell, hardly shows any preferential orientation of the fibers, but circular dark
regions are also seen, indicating that fiber bundles are again oriented from
peduncle to opercular opening. Furthermore, in the center of the mesocarp, the
fiber bundles are bigger and the number of sclereid cells is smaller. Considering
these three layers, mesocarp is a sandwich structure with fiber bundles in one
direction in layer I and III and in the opposite direction in layer II.

19
 C. Global mechanical properties

Figure 8 shows the typical behavior of mesocarp when compressed in two

different directions, parallel (Figure 8(a)) and perpendicular (Figure 8(b)) to the
latitudinal section. As stress and strain distributions in a non-uniform, hollow
sphere with a thick shell are not easily calculated, Figure 8 shows the force and
displacement data.
The deformation behavior in both directions is similar. However, when the
force is applied parallel to the latitudinal section (Figure 8 (a)), the shells support
an average maximum force of 10079 N (± 1460 N). For the perpendicular loading
direction (Fig. 9 (b)), the average value is 14785 N (± 4050 N), which although
higher, also has a greater standard deviation. For a better comparison, a boxplot of
the fracture force of samples is also shown in Figure 8 (a) and (b). The greater
standard deviation of specimens in Figure 8(b) is caused by one specimen, which
does not have any visible defect, although it has a small shell diameter and
thickness. Figure 8(c) shows a schematic drawing of the fracture mechanism and
images from the specimen which force versus displacement curve is shown in
Figure 8(a).

20
Figure 8: Typical behavior of the whole mesocarp in a compression test: (a) force
applied parallel to the latitudinal section, (b) force perpendicular to the latitudinal
section, (c) schematic drawing of the fracture mechanism (I – III) and images (1-3)
of specimen during the test (a).

As to be expected, at the beginning of loading the regions in contact with the

compression plates are deformed and small cracks nucleate there. This first phase
is shown schematically by the grey regions in (Figure 8 (c), left-hand sketch; I). The
main crack, (II) - Figure 8 (c), causes a sudden decrease in force. This main crack
and the related force drop were observed in all specimens of both loading
directions. The main crack (II) nearly always went through the peduncle, as shown
in Figure 8 (c), suggesting that the peduncle is a weak region in the structure. After
the sudden force drop, the force increases again even though the main crack

21
grows further (Figure 8 (c) – image 2). Interestingly, the force increases to almost
half the maximum force even for substantial crack lengths. Finally, in phase III, the
force slowly decreases with the formation and propagation of additional cracks
(Figure 8 (c) – image 3) until the total collapse of the mesocarp.

D. Local mechanical properties

The hardness of the mesocarp was measured at different length scales,

ranging from the cell wall to mapping of the whole shell.
Figure 9 shows nanoindents made in the cell walls of fibers and sclereids.
The reduced modulus (Er) and the hardness (H) values are summarized in Table
4. The nanoindentations were made in the cross section of fibers (Figure 9 (a)) and
sclereids (Figure 9(b)) but also in the longitudinal section of fibers (Figure 9 (c)),
which report is uncommon in literature due to the great difficult to measure it.
Lucas et al. [25] has made nanoindentation in both sections of fibers of M.
parviflora seed and also obtained a low value of Er for the longitudinal section of
fibers, as shown in Table 4.

Figure 9: In situ scanning probe microscopy (SPM) images showing indents in (a)
cross-section of fiber; (b) sclereids and (c) longitudinal section of fibers. Indents are
highlighted by white circles while arrows indicate residues from polishing.

22
Table 4: Reduced modulus (Er) and hardness (H) measured by nanoindentation

Nutshells Er [GPa] H [GPa]

Brazil nut mesocarp - Fiber cross section 13.02 ± 2.78 0.30 ± 0.04
Brazil nut mesocarp - Fiber longitudinal section 7.30 ± 2.92 0.25 ± 0.08
Brazil nut mesocarp - Sclereid 8.29 ± 2.54 0.31 ± 0.05
Macadamia* 10,2 0,65
Brazil nut tegument* 5,4 0,33
Hazelnut * 7.3 0.33
Walnut * 7.0 0.45
M. parviflora shell – Fiber cross section ** 15.7 ± 0.9 0.40 ± 0.11
M. parviflora shell – Fiber longitudinal section ** 7.2 ± 0.7 0.40 ± 0.11
M. parviflora shell – pseudospherical cells** 10.7 ± 1.1 0.49 ± 0.11
Cocoyol (Acrocomia mexicana)*** 9.1±1.28 0.31 ±0.06
* Values from [26]
** Value from [25]
*** Value from [9]

The ANOVA analysis and a Turkey test evaluate if the mean values of Er
and H of fibers and sclereids are significantly different, as summarized in Table 5.

Table 5: Comparison of reduced modulus (Er) and hardness (H) of sclereids and
fibers cell walls with ANOVA analysis combined with Turkey test.

Er H

Fiber cross section x Fiber longitudinal section ≠ =

Fiber cross section x Sclereid ≠ =

Fiber longitudinal section x Sclereid = ≠

23
 A Shore D hardness map (Figure 3) in the longitudinal and latitudinal
sections and in the thickness of mesocarp evaluated its local mechanical
properties. Latitudinal and longitudinal sections have a hardness of 74.8 ± 2.2
(Figure 10(a)) and 74.9 ± 4.0 (Figure 10 (b)), respectively. It is possible to see that,
disregarding a low numb "indenation number" of outliers, hardness does not vary
significantly through latitudinal orientation (Figure 10(a)) and through longitudinal
orientation (Figure 10(b)), showing certain uniformity in mesocarp structure.
Shore D hardness through mesocarp thickness (from inside to outside) was
measured in three different regions (A, B and C in Figure 3(c)). Region A is close
to peduncle, region B is in the middle section and region C is close to opercular
opening. The three regions have close Shore D hardness with the same behavior
(Figure 10(c)). Shore D hardness is lower close to the inner surface, it increases in
the center, with an average value close from that measured in the longitudinal and
latitudinal sections and it decreases again when indents are made close to the
outer surface.

24
Figure 10: Shore D hardness maps as shown in Fig. 3: (a) latitudinal section, (b)
longitudinal section and (c) thickness layers I, II and III near peduncle (Region A),
in the center (Region B) and near the opercular opening (Region C). (Continue line
represents the mean hardness and dotted lines are standard deviations)

25
 IV. DISCUSSION

The pericarp structure of Brazil nut has differences as compared to other dry fruits.
Coconut and babassu also have a pericarp with three layers; in these fruits,
however, each layer has a mechanical purpose. Coconut, for example, has a
smooth skin-like exocarp that may avoid stress concentrations on the surface, a
flexible mesocarp made of interconnected fibers, which provides an improved
cushioning effect during impact and a hard endocarp to protect the seed. [6], [8],
[27] Babassu has a softer mesocarp between two harder layers of exocarp and
endocarp. [10] Citrus maxima achieves excellent damping properties through a
spongy gradient structure where the pore size increases from the outside of the
fruit wall towards the inside.[6], [28] In Bertholletia excelsa fruit, the endocarp is like
a membrane and exocarp is a rotten tissue: The Brazil nut, therefore, relies mostly
on the thick mesocarp and on its tegument for protection. Cocoyol fruit also
depends mostly on one layer, its hard and tough endocarp, to protect its seed. [9]
Thus, Bertholletia excelsa as well as cocoyol nutshell, must have developed
strategies to combine impact resistance and hardness in just one layer to protect
its seeds. These strategies may consider cell wall composition, the cell wall
thickness, cell-cell adhesion, cell geometry, tissue density and tissue
organization.[29] Some of these factors are discussed here.
Figure 11 shows a comparison between Brazil mesocarp results and other
nutshells. Figure 11(a shows the lignin and holocellulose (hemicellulose and α-
cellulose) content of several nutshells [30] in comparison with mesocarp. Moreover
in Figure 11 (b), Shore D hardness of several nutshells were measured and
compared to hardness of Brazil nut mesocarp. The mechanical performance under
compression forces of several nutshells is also shown, in Figure 11(c). Comparison
of fracture forces of different nut and seed shells is difficult due to the different
sizes and shell thickness of the different species. However, comparison is possible
by dividing the maximum fracture force by the mesocarp mean thickness. All shells
were tested with forces applied parallel to their latitudinal section. [4]

26
Figure 11: Comparison between different natural materials (nuts, seeds, drupes
shells): (a) Lignin and holocellulose content [30], (b) Shore D hardness test and (c)
fracture forces in compression tests normalized by mean shell thickness [4].

Although the amount of holocellulose present in most nutshells is similar

(Figure 11(a)), the mesocarp of Brazil nut is more highly lignified than other
nutshells. Lignin is a reticulum of short linear chains crosslinked in a random way.
In vegetal systems, the main functions of lignin are to increase cell wall rigidity,
joining of different cells, improving the mechanical behavior under compressive
loads and decreasing the cell water permeability. [31] The effect of lignin in the
bending strength of stem may be greater than the cellulose effect, once mutants

27
with 82% less cellulose than wild type of Arabodopsis are stronger than mutants
with 50% reduction of lignin content.[29], [32], [33] The performance under tensile
forces are also affected by lignin content, as showed by Gludovatz et al. [8]. In this
study, old and more lignified coconut endocarp has a higher density and a less
porous cell wall as well as higher tensile strength, Young’s modulus and KIC values.
[8]. The higher content of lignin in Brazil nut mesocarp when compared to others
nutshells may be one of the reasons for the better performance in the compression
test, with higher values of fracture force relative to the thickness of the shell wall
(Figure 11(c)). On a smaller length scale, the higher lignin content increases the
reduced modulus and nanohardness of the cell wall [34]. Gindl et al. [34],
concluded that properties of cell walls with higher lignin content, measured by
nanoindentation, are caused by filling of the free spaces with lignin and an increase
in cell wall packing density. For Brazil mesocarp, however, the higher content did
not lead to a higher reduced modulus or hardness (compare Table 4), where these
properties are very similar for different nutshells. Shore D hardness measurements
(compare in Figure 11(b)) also indicate that the high lignin content in mesocarp
does not improves its local mechanical properties, as the hardness of mesocarp is
similar or even smaller than the hardness of other nutshells.
Regarding cell hierarchical level, sclereids and fibers are the main
constituents of many nutshells, such as coconut, macadamia, and babassu.
However, in most nutshells, these cells are in different layers. For instance, in
coconut, shell fibers are preferentially present in the exocarp and the mesocarp
while sclereids are predominant in the endocarp. [35] Macadamia shells have a
sandwich structure where a compact arrangement of mainly fibers is arranged
between adjacent layers of sclereid cells. [4] In babassu fruit, the fiber bundles are
also surrounded by spherical cells, as in Brazil nut, but these cells are parenchyma
cells with starch granules, which are very different from the lignified sclereid cells
with their thick cell walls. [10] In Brazil nut mesocarp, the sclereids cells fill the
spaces between fiber bundles, which are arranged as a complex and entangled
net. Cocoyol nutshell also has a combination of spherical and elongated cells,
although the elongated sclereids cells of cocoyol are not as long as fibers.[9]

28
 Additionally to fibers and sclereids, there are numerous natural defects from
previous vascular bundles (compare Figure 6(e)) in Brazil nut mesocarp. Similar
vascular bundles are also present in other nutshells, as macadamia and coconut
endocarp, where they work as toughening elements by deflecting and even
trapping cracks. [8], [36]
Depending on the predominant orientation of fiber bundles, a sandwich
structure with three layers can be distinguished in mesocarp (compare Figure
7(a)): in layers I and III many bundles run in the direction from the peduncle to the
opercular opening while, in layer II a larger number of bundles are latitudinally
oriented. Moreover, Shore D hardness showed that layer II is harder than the other
two layers (compare Figure 10(c)). Stiffness gradients inside plant structures are a
common natural strategy to enhance adhesion among different tissues and
decrease stress concentrations. [29] This is achieved, for example, by degree of
lignification (stem of Washingtonia robusta [37]) or differential cell wall thickening
(Phyllostachys pubescens [38]). A hardness gradient is also present in cocoyol
nutshell, where the outer layer is harder than the inner layer due to different
sclereid geometries and tissue densities.[9] In Citrus maxima, the density gradient
due to different pore size affects the fruit mechanical behavior, once the largest
pore size deforms the most in compression tests. [28] In Brazil nut mesocarp, layer
II seem to have a greater number of fibers (see Figure 7 (a)) what explain the
greater hardness. Additionally, differences on the geometries and packing of
sclereids, although not observed, may also explain the hardness differences
among mesocarp layers.
The sandwich structure of mesocarp according to fiber bundle orientation
explains the higher average maximum force reached when compression forces
were applied perpendicular to latitudinal section (Figure 8(b)). In this configuration,
the main crack has to cross layer II, the hardest and the most fibrous layer,
breaking each individual fiber. Differently, when loading is parallel to the latitudinal
section, the main crack propagates in the interface between fibers of layer II, a
much easier path. The fact that load increases considerably with main crack
propagation indicates the presence of toughening mechanism and high dissipation

29
of energy during propagation which should be investigated in greater detail.
Cocoyol nutshell, can carry loads of 70 MPa after failure strain mostly due to the
entanglement of elongated sclereids that leads to a more globalized plastic
deformation, arrests the crack propagation resulting in a larger energy dissipation
during fracture under compression forces. [9] In coconut, the high energy
dissipation is caused by the aligned fiber cells of its mesocarp layer, which must be
ruptured one by one. [6], [28] The strategy of Citrus maxima to dissipate energy is
the large partly plastic deformation of its spongy structure. [28] Once Brazil nut
mesocarp has elongated cells like fibers and sclereids similar to a hard closed-cell
foam, some of these strategies may be taking place.
Nanoindentation measurements (compare Table 4 and 5) were used to
investigate the properties of sclereids and fibers separately. In nanoindentation of
plant cell walls, Er strongly depends on the microfibril angle (MFA) while H is more
affected by the cell wall matrix. [39], [40]. Therefore, fibers are expected to have
the same H but different Er values in the longitudinal and cross section, as we, in
fact, observed. As sclereid cells are spherical or elliptical, they do not have a
helical orientation of microfibrils with a defined MFA. This explains why the reduced
modulus (Er) of sclereids is smaller than the one measured on the cross sections
of fibers. However, the Er of sclereids and the Er of longitudinal sections of fibers
are statistically equal what may indicate a similar orientation of microfibrils.
Curiously, the hardness of sclereids is statistically equal to the hardness of fibers in
the cross section but different when longitudinal fiber sections are measured. By
correlating the Er values with results from chemical analysis it is possible to
estimate the microfibril angle of the fibers, based on a property chart, calculated
by. Jager et al. [41] Using this property chart and the measured moisture and
cellulose contents (10% and 30%, respectively) of mesocarp, the measured Er for
fibers (13.02 GPa) indicates a MFA of 13°. This is a relatively low value suggesting
the fibers have a high elastic modulus. [42]
Finally, the Shore D hardness maps showed that mesocarp is relatively
uniform with similar hardness values all over, even close to peduncle or opercular
opening. The mean Shore D hardness values of mesocarp are close to the values

30
measured for polypropylene (Shore D 74) and bone (Shore D 86). Thus, it is
relatively hard for a biological tissue, supporting its excellent use as bioinspiration
source. [43]

V. CONCLUSION

The mesocarp of Brazil nut has great potential for developing bioinspired
impact and puncture resistant structures. This work reports on the investigation of
its composition, microstructure, and mechanical properties on different length-
scales. The mesocarp is a sandwich structure with three layers, formed by highly
lignified sclerenchyma tissue comprising mostly of sclereid and fiber cells. The
fibers are organized as bundles and the sclereid cells fill the gaps between these
bundles. In mesocarp sandwich structure, layer I and III have fiber bundles from
peduncle to opercular opening, while in layer II the bundles are latitudinally
oriented. The shells sustain impressive compression forces of up to 10 kN before a
crack is formed, usually at the peduncle. Interestingly, maximum forces for loading
in the longitudinal direction are higher than in the latitudinal direction: sections
oriented in the longitudinal direction have fiber bundles oriented perpendicular to
loading in middle layer (layer II), the thickest and the hardest layer. While, on the
length-scale of the cell walls, mechanical properties, as measured by
nanoindentation, vary depending on the direction of loading and the cell type, the
mesocarp exhibits a uniform hardness in longitudinal and latitudinal sections, but a
hardness gradient from the inside to the outside, if measured by Shore D. Many
differences exist between Brazil nut fruit and other nutshells. Even then, its
mechanical performance is better or comparable to other notorious natural
structures what foment further research of Brazil nut mesocarp.

31
 VI. ACKNOWLEDGMENT

VII.

The authors gratefully acknowledge the financial support provided by FAPESP

(Brazil) through the Ph.D. scholarship of M. Sônego (grant number 2015/25523-1).
They further thank Prof. Dr. Astrid Haibel, Beuth Hochschule für Technik Berlin,
Germany, for performing the microCT analysis.

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