Stereoscopy and Scanning Electron Microscopy of Brazil Nut ( Bertholletia excelsa H.B.K.) Shell, Brown Skin, and Edible Part: Part One- Healthy Nut

Stereoscopy and Scanning Electron Microscopy of Brazil Nut ( Bertholletia excelsa H.B.K.) Shell, Brown Skin, and Edible Part: Part One- Healthy Nut
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           H      :         H       e       a         l        t         h  ,         N       u        t       r        i        t        i       o       n        &         F       o       o         d Stereoscopy and Scanning Electron Microscopyof Brazil Nut ( Bertholletia excelsa   H.B.K.)Shell, Brown Skin, and Edible Part: PartOne— Healthy Nut  VildesM.Scussel,DanielManfio,GeovanaD.Savi,andElisaH.S.Moecke Abstract:  In this article, tissue layers and cells characteristics of the Brazil nut ( Bertholletia excelsa  H.B.K.) shell (tegument),brown skin ( testae  ), and edible part (cotyledons) were identified by stereoscopy (SM) and scanning electron microscopies(SEM). (a) The shell (a lignin rich, protective wall) varies in thickness throughout the nut structure and comprises differenttissue types (total 3)/texture (hard/mid-hard/soft), layers (2 to 5), colors (light to dark brown and white to cream), cellshape (amorphous/flattened on both surfaces; polygonal and cylindrical with thick, porous primary and secondary wallin cross-section), and vascular distribution (helically and polyedrical thickened vessels at soft tissue and locule/channelstructures). These variations are observed either in the shell faces, face corners, nut tips, or locule in  testae  . (b) Thebrown skin (shell nut part linked to both the shell and edible part) is made of flattened irregular-shaped parenchymalcells distributed in several layers with more flexible fibrous, thinner wall tissue than shell. It has both rough and smoothshiny texture on the upper and lower surfaces, respectively. However, the nut (c) edible part, that is the nut storage tissue,shows several different tissue/cell layers starting from  epidermis  (double/triple cells sequence of round and palisade shapes)layer–the  endosperm  tissue. The  parenchymal   tissues show cells of irregular shape with small and larger sizes distributedin regular and randomly layers, respectively, separated by a short  meristem  tissue layer. The  cortex  cells increase in size asthey approach the cotyledons junction. The Brazil nut part’s tissue layers and cells were identified by the SM and SEMmicroscopy methods applied, which provides knowledge for further understanding of nut alterations that may occur either in the forest or during the factory processing. Keywords:  Brazil nut, brown skin, cotyledon, edible part, electron microscopy, morphology, shell, stereoscopy, tegument Practical Application:  Knowledge about the characteristics and nature of the waste woody tissues from the Brazil nutfactories is of interest for potential applications in the industry. Understanding the nut tissues and cells structures helps in judging how much whole nut edible part gets spoiled/deteriorated (either raw or processed), for further development of procedures to prevent and/or control such spoiling/deterioration for achieving nut quality and safety (to be discussed inPart Two). Introduction Brazil nut trees (Angiospermae Division, Dicotyledon Class,Myrtiflorae Order) belong to the Lecythidaceae Family: genusand species  Bertholletia excelsa  H.B.K. They are mainly found inthe Amazon region of Brazil; and in small numbers in Bolivia,Peru, and the Guianas (Pacheco and Scussel 2006, 2011; Man-fio and others 2012a, 2012b). The Lecythidaceae family has 325types of trees in the American tropics and is divided into 20 gen-era, where  Bertholletia  is dominant with 75 species (O’Malley andothers 1988; Mori and others 2007). The flowering of an adultspecimen occurs between October and December, and the fruitsripen in the winter, corresponding to the Amazon rain season(Pacheco and Scussel 2006). MS 20140268 Submitted 2/18/2014, Accepted 4/20/2014. Authors Scussel,Manfio and Savi are with Laboratory of Mycotoxicology and Food Contaminants– LABMICO. Author Moecke is with Microscopy Laboratory, Food Science and Tech-nology Dept. Center of Agricultural Sciences, Federal Univ. of Santa Catarina–UFSC,Florianopolis, Santa Catarina, Brazil. Direct inquiries to author Scussel (E-mail: The pod (called as  ourico  by the Amazon natives) is the Brazil nutfruit, which is a large indehiscent (not opening at maturity), with10- to 15-cm-dia. capsule of very hard woody wall containing theseeds (10 to 25 units) known as the Brazil nuts. In botany, nut isone-celled grit, containing only one seed that rarely opens spon-taneously when ripe (Bonelli 2001; Serrano 2005). Brazil nuts areclassified as type I, II, and III (large, medium, and small) accordingto their sizes of 50/53.2, 40 to 50/43.9, and < 40/36.6 mm/g for length/weight ratio, respectively (de Mello and Scussel 2007). Thenuts are triangular (cross-sectioned) and elliptic (length-sectioned)in shape, with very hard and rough shell ( tegument  ; Muller andothers 1980, 1995; Clay and Clement 1993). Underneath the in-ner shell surface, the brown skin ( testae  ) is located, followed by theedible part ( two cotyledons ), corresponding to an average weight of 5.5, 0.101, and 4.9 g for each nut part, respectively (de Mello andScussel 2007; Manfio and others 2012a).Like other nuts (almond—   Amygdalus communis  L.; pistachio—  Pistacia vera  L.; hazelnut—  Corylus avellana  L., and walnut—   Juglans regia ), the Brazil nuts shell is rich in low digestibility(by the mammals gastrointestinal tract) and high combustibility C  2014InstituteofFoodTechnologists R  doi:10.1111/1750-3841.12502  Vol.00,Nr.0,2014    Journal of Food Science H1 Furtherreproductionwithoutpermissionisprohibited  H    :    H     e   a   l        t      h       ,N     u    t      r    i       t      i       o   n    &   F      o    o    d      SM and SEM of healthy Brazil nut... carbohydrates such as cellulose, hemicellulose, and lignin, whichgive the nut shell wall (sclerenchyma tissue) an extremely hard tex-ture and high resistance to damage characteristics and protectionand support to the nut edible part. Comparatively, Brazil nut hasthe highest lignin content (59.4%) when compared to hazelnutand almonds (33.5% and 27.7%, respectively; Bonelli and others2001). However, holocellulose (cellulose  +  hemicellulose) con-tent is rather low (48.5%) compared to other nuts, including olivestones (66.5%, 69.1%, and 63.5%). Despite all protections, infec-tious diseases can occur and nut spoilage (edible part) can developwith possible mycotoxin formation such as aflatoxins (Romeroand others 2003; Marklinder and others 2005; de Mello andScussel 2007, 2009; Olsen and others 2008; Pacheco and Scussel2009; FAO 2010).The very few studies conducted on the Brazil nut histologicalcharacteristics focused only on the edible nut part identified bylight microscopy (Spencer 1921; Camargo and others 2000).However, the shell and the brown skin, which are consideredwastes from the Amazonian dry-shelled Brazil nut factories,can be further better utilized either for fuel and dispersants (theshell—lignocellulosic rich), selenium/fiber food enrichment(brown skin—selenium and fiber rich), or other applications suchas arboform production (bio/fluid-plastic; Bonelli and others2001; Pacheco and Scussel 2011; Manfio and others 2012a,2012b; Europa 2014; Pfitzer and Nagele 2014; Tecnaro 2014).Considering that (a) there is no detailed information regardingBrazil nuts morphological and histological characteristics of all nutparts, neither by stereo (SM) or scanning electron microscopies(SEM), and the need for information either (b) on the wastes of Amazonian dry nuts factories for further application or (c) for abetter understanding on how deterioration can reach the ediblepart, this study was carried out to elucidated the shell, brown skin,and edible parts (from healthy in-shell nut) tissue layers and cellcharacteristics by both SM and SEM. Materials and Methods Materials Sample.  Whole in-shell Brazil nuts (1 kg) were separated intotheir parts—shell, brown skin, and edible part. The samples werekindly provided by the Renmero Brazil nut factory located atCameta, Para, in the Eastern Brazilian Amazon (Figure 1). A BC Figure 1–Brazil nut ( Bertholletia excelsa   H.B.K.) divided into its: (A) ediblepart ( cotyledons ), (B) shell ( tegument  ), and (C) brown skin ( testae ).  Equipment.  Stereo microscope (10 ×  and 0.71 to 11.5 ×  for ocular and objective lenses, respectively), model MZ16 (LeicaMicrosystems Ltd., Heerbrugg, Switzerland); stereo-light source(150 × ), model CLS (Leica Microsystems Ltd.); and color imagecapture camera with SM adaptor, model CoolSpnap-Pro cf   (MediaCybernetics, Silver Spring, Md., U.S.A.); light microscope, modelCH-Bl 45-2 (Olympus, Shinjuku, Tokyo, Japan); gold-coatingmachine, model EMScd500 (Leica, Leider, Ill., U.S.A.); and SEM(5000 × ), model JSM-6390LV Jeol (Peabody, Mass., U.S.A.) wereused in this study. Other materials.  Formaldehyde and ethanol, analar grade(Vetec, S˜ao Paulo, Brazil); nut cracker (Ciex, (Manaus, AM,Brazil); saw (Ticotico, S˜ao Paulo, Brazil); drying oven, model315 SE (Fanem, S˜ao Paulo, Brazil); tweezers (Mundial, S˜aoPaulo, Brazil), scissors (Tramontina, S˜ao Paulo, Brazil), scalpel(GSMtronix, S˜ao Paulo, Brazil), stubs (small metal blocks) with9.0 or 12 and 10 mm for diameter and height, respectively (Leica)and carbon double-sided tape (Electron Microscopy Sciences, Lei-der, Ill., U.S.A.). SM-image-capture software, model Image-ProExpress (Media Cybernetics, Silver Spring, Md., U.S.A.) are theother materials used in this study. Methods The in-shell Brazil nuts were (a) sectioned (whole) and (b)sliced (shell/brown skin/edible part) after cracking to separate thenut parts for (a)  SM   tissue layers and (b)  SEM   tissues and cellsidentification, respectively. Brazil nuts tissue layers distribution and characteristics —SM. Preparation of whole Brazil nut halves sections.  In-shell nuts weresectioned into halves across (Plan A) and lengthwise (Plan B) bymeansofafinesawasshowninFigure2(A.1andA.2).Inaddition,another 2 cross-sections (Plans C and D: closer to the 2 nut Tips1 and 2) were also obtained for Tips observation. The nut halves(Figure 2B) and Tips were then taken for layers identification. SM shell/brown skin/edible part distribution . Each nut half sectionwas observed through SM (Flint 1996) for layers distribution,color, and number per tissue in different magnifications. Themagnification used ranged from 71 ×  to 115 ×  and the softwareImage-Pro Express was applied for registering them. We obtaineda total of 30 photos and those with the characteristic structureswere selected. The advantage of using SM is that, it provides anupright and unreversed bidimensional image (with incidence lightillumination) when the subject cannot be fit in a slide. It shouldbe noted that prior to sectioning nut, the shell nut surface detailsshould be observed by SM for further elucidation by SEM. Brazil nuts tissues and cells characteristics—SEM. Preparation of nut parts slices.  The nut preparation was carried outby slicing and defating with organic solvents following the tech-niquesdiscussedbyDashek(2000),Dickson(2001),andGoodhewand others (2001) and stubs mounting with gold coating (Romeroand others 2003; Manfio and others 2012a) as follows: after thenutswere cracked utilizing acommercial nutcracker, each partwasseparated and reduced into both small cubes ( shell   and  brown skin  — cubes of around 3.0 × 3.0 × 2.0 mm for width/length/thickness,respectively) and slices ( edible part   —3.0- to 4.0-mm thick) with ascalpel. They were previously dehydrated (105  ° C for 4 hours— final moisture content: 1.9%) and fat extracted (only for the edibleparts) by adding acetone (30 min), stirred, and air dried. All sec-tions were transferred to glass jars covered lid and taken to theCentral Laboratory of Electron Microscopy (LCME) at UFSC. H2 Journal of Food Science   Vol. 00, Nr. 0, 2014           H      :         H       e       a         l        t         h  ,         N       u        t       r        i        t        i       o       n        &         F       o       o         d SM and SEM of healthy Brazil nut... Slices stubs mounting and gold coating  . (a) Stubs slices mounting— Brazil nut parts, previously discussed in “Preparation of nut partsslices,” were fixed on the stubs containing carbon double-sidedtape (5 to 6 mm length  ×  width; Figure 3A). Each stub canhold up to 4 histological cubes/slices. (b) Vacuum gold coating— the stubs were placed onto the machine holder (Figure 3B) andpositioned in the planetary gold coater, then closed, vacuum ap-plied (at 104 mbar), and gold coated. Metallizing nut slices witha thin gold layer (40 nm) converts nut slices into surface elec-tron conductors, which helps in visualizing the cells details bySEM. The stubs were removed and transferred to the microscopeequipment. SEM Brazil nut parts tissues and cells identification . For the SEMvisualization of the histological sections, stubs were fixed on theSEM planetary base and submitted to vacuum again. The tissueareas of interest of each sample type were scanned and their charac-teristic tissues and cells were observed by different magnification.The magnification used ranged from 30 × to 1700 × . We obtaineda total of 14257 photomicrographs and those with the character-istic structures properly displayed were selected, keeping 2 mainmagnifications: 300 ×  and 1700 ×  for better comparison amongthe different nuts (except for edible part and locule: 30 to 300 × and 100 to 1400 × µ m, respectively) and parts micrographs weretaken. (a.1) (a.2) Tip 1 Plan C  Tip 1Plan A Plan BloculeA B A BC CFaces corner link  Plan D  Tip 2Tip 2 Face Ashell wallbrownskinFace A FaceBedibleTip 1 edibleTip 2part part softtissueloculeFaceCfaces B&Clinkage (b.1) (b.2) Figure 2–Whole Brazil in-shell nut  sections  for layers and tissues SM and SEM observation: (A) cross [Plan A] and longitudinal [Plan B] (Plans C and Dcloser to Tips 1 and 2, basal and apical region, respectively) and (B) sectioned nuts obtained from Plans A and B. (Face A [curved shape] and B and C [straight shape], which touch the: inner pod surface and other nuts faces inside the pod, respectively—the linkage of B and C faces).   Brazil nut slicesstubsstubs AB Figure3–Brazilnutslices(shell,brownskinandediblepart)preparationforSEMevaluation:(A)stubswithnutpartsfixedondoublefacedtapeand(B)gold coater holder containing the slide fixed stubs positioned for vacuum gold coating and cells visualization through surface electron conductivity bySEM. Vol.00,Nr.0,2014    Journal of Food Science H3  H    :    H     e   a   l        t      h       ,N     u    t      r    i       t      i       o   n    &   F      o    o    d      SM and SEM of healthy Brazil nut... Results and Discussion From the halves of   whole in-shell   Brazil nut sectioned and theslices of   each nut part  , the tissue layers and their cells main char-acteristics were identified by SM and SEM. As expected, all nutparts (shell/brown skin/edible part) had different number of layers,were composed of different shapes and colors cells and the shellwall thicknesses varied throughout the nut structure. Figure 4and 5–9 show the  whole nut   sectioned with tissue layers distri-bution and  nut parts  cell layers differentiations by SM and SEM,respectively. Whole in-shell Brazil nut tissues layers distribution—SM The nut structure characteristics, layers distribution, and posi-tioning sequence, including those close to nut tips, were seen by Figure4–StereomicroscopyphotographsofBrazilnutpartscharacteristics:(A)surface—shell(A.1)centralfaceregion,(A.2/A.3)BandCfacelinkage;(B) cross-section—shell tips and nut body (outer; inner Tip 1 and 2 surfaces, main body and Tip 1 close views); (C) longitudinal section—shell/brownskin/edible part with (C.4 and C.6) locule and inner channel (tissues layers number, distribution, colors, and thickness ratios) (7 × to 115 × ). H4 Journal of Food Science   Vol. 00, Nr. 0, 2014           H      :         H       e       a         l        t         h  ,         N       u        t       r        i        t        i       o       n        &         F       o       o         d SM and SEM of healthy Brazil nut... SM either on  surface   (Figure 4[A.1–A.3]),  cross  and/or   longitudinal sections  (Figure 4B and C). Shell (tegument).  The highly lignified protective Brazil nutshell structure comprises (a) 3 faces (A, B, and C) linked to eachother, (which gives the nut a transversal triangular shape); the (b) 3faces corners (each 2 shell faces linkage); the (c) 2 nut tips (wherethe 3 shell faces connect at nut top); and the (d) locule in  testae   (thenut navel that links the nut to the pod and is located at the faces Band C corner linkage; Figure 2B and 4B.4 and C.4. It is also calledmicropyle). All of them have histological differences with a seriesof tissue layers number, color, and types, depending on which partof the nut shell structure they are located (face/faces corner/nuttips/locule). Surface.  Shell has a crinkled waxy  surface   throughout the nut, apartfrom a wavy imprint differentiation observed mainly along the B Figure 5–SEM micrographs of Brazil nut shell—shell  surface : (A) outer and (B) inner (linked to brown skin) and the (C) locule  in testae  (cavity) tissuesand cell characteristics (300 × and 1700/500 × ). Vol.00,Nr.0,2014    Journal of Food Science H5
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