Research ArticleReceived: 31 March 2012 Revised: 29 May 2012 Accepted: 3 June 2012 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5793

Critical points of Brazil nuts: a beginningfor food safety, quality control and AmazonsustainabilityAndriele M Lima,a Evonnildo C Goncalves,b Soraya S Andrade,b

Maria SR Barbosa,b Karla FP Barroso,a Mayara B de Sousa,a Larissa Borges,c

Joze LF Vieiraa and Francisco M Teixeiraa∗

Abstract

BACKGROUND: One difficulty of self-sustainability is the quality assurance of native products. This research was designed tostudy the risks and critical control points in the collection, handling and marketing of Brazil nuts from native forests and urbanfairs in the Brazilian Amazon by characterisation of morphological aspects of fungi and posterior identification by molecularbiology and determination of aflatoxins by high-performance liquid chromatography.

RESULTS: Several corrective actions to improve product quality were found to be necessary in both sites. Growth of fungi wasobserved in 95% of fragments of Brazil nuts from both sites during the between-harvest period. Aflatoxin levels indicated that,although fungal growth was observed in both sites, only Brazil nuts from the native forest showed a high risk to human health(total aflatoxin level of 471.69 µg kg−1).

CONCLUSION: This study has shown the main issues related to the process design of Brazil nuts, supporting the necessity forresearch on new strategies to improve the quality of nuts. Also, the habit of eating Brazil nuts stored throughout the year mayrepresent a risk to farmers.c© 2012 Society of Chemical Industry

Keywords: Brazil nuts; sustainability; quality control; aflatoxin; Good Manufacturing Practices; HACCP

INTRODUCTIONThe Amazon biome is one of the greatest biological treasures onEarth, but it has faced the problem of deforestation as well asthe stress of climate change during the last century.1 There isconsiderable interest in the promotion of self-sustainable forestproducts as a strategy for its conservation.2 Brazil nut (Bertholletiaexcelsa) fits this role quite well, because it is an internationallyregistered product and its harvesting is carried out exclusively innative forests.3 Solely in the Brazilian Amazon, about 45 000 tonsof Brazil nuts are collected annually, with a sales value amountingto US$33 million.2

The importance of Brazil nut is also reflected in its appli-cation in the cosmetics industry and by its nutritional value.Besides being the richest source of selenium, an importantnutrient for the immune system, it also has a higher bioavail-ability of this element when compared with the consumptionof selenomethionine.4 The possibility of generating sustainabilitycoupled with the contribution to food quality and healthcareof native communities characterises the importance of self-sustainable forest products.5 However, the lack of scientificknowledge and appropriate technologies generates a signifi-cant demand for training and capacity building to enable thegeneration of wealth and employment without destroying theforest.6 From the academic point of view, the bioprospect-

ing of these products requires standardised procedures withcontrol at all stages of production, providing an excellent op-portunity for staff training and technology transfer, leadingto the detection of faults during the production process andpromoting the implementation of corrective and preventiveprocedures.

Although a recent study has shown the absence of a risk topublic health related to the presence of potential pathogensin nuts produced in Brazil and consumed in Australia,7 it is

∗ Correspondence to: Francisco M Teixeira, Faculdade de Farmacia, Institutode Ciencias da Saude, Avenida Augusto Correa, N◦ 01, Campus Universitariodo Guama, CEP 67150-110 Universidade Federal do Para, Belem, Para, Brazil.E-mail: ft martins@yahoo.com

a Faculdade de Farmacia, Instituto de Ciencias da Saude, Avenida AugustoCorrea, N◦ 01, Campus Universitario do Guama, CEP 67150-110 UniversidadeFederal do Para, Belem, Para, Brazil

b Laboratorio de Polimorfismos de DNA (LPDNA), Instituto de Ciencias Biologicas,Avenida Augusto Correa, N◦ 01, Campus Universitario do Guama, CEP 67150-110 Universidade Federal do Para, Belem, Para, Brazil

c Laboratorio Nacional Agropecuario (LANAGRO-PA), Avenida Almirante Bar-roso, N◦ 1234, Marco, CEP 66093-020, Belem, Para, Brazil

J Sci Food Agric (2012) www.soci.org c© 2012 Society of Chemical Industry

www.soci.org AM Lima et al.

necessary take into account the potential risks when consideringother existing factors such as hypersensitivity, chronic diseasesand immunosuppression that may cause undesirable episodesof inadequacy.8 – 10 Aspergillus spp., an eventual contaminantof Brazil nut, for example, have the ability to produce naturalcarcinogens known as aflatoxins, making them a potential riskfactor that requires monitoring.11,12 Therefore there is an issueof conflict between Brazil nut as an important product forsustainability and the risk of aflatoxin contamination, whichhas become critical since the reduction of limits of aflatoxinsin Brazil nuts intended for human consumption by the EuropeanCommunity (EC). While the limits recommended by the CodexAlimentarius are 15 µg kg−1 for nuts intended for processing and10 µg kg−1 for ready-to-eat nuts, the values recommended bythe EC are only 4 µg kg−1 for total aflatoxins and 2 µg kg−1

for aflatoxin B1 in ready-to-eat nuts.13,14 The lower limits ofaflatoxins in Brazil nuts adopted by the EC have imposed thenecessity for the qualification of all participants in the productionchain, emphasising the social and environmental impact onnative communities and taking into account the importanceof management and assessment of risks.13,15 According to theOfficial Health Office and Official Agriculture Office, the limits foraflatoxins in Brazil are 30 µg kg−1 for aflatoxin B1 plus aflatoxinG1 and 50 µg kg−1 for total aflatoxins in products for humanand animal consumption. Recently, new legislation has come intoeffect in which the limits recommended are 15 µg kg−1 for nutswithout shell intended for processing, 20 µg kg−1 for ready-to-eat nuts with shell and 10 µg kg−1 for ready-to-eat nuts withoutshell.16 – 18

The production chain of Brazil nuts has well-defined stagesranging from the collectors in the Amazon forest through to theend consumers, who can live nearby in towns or cities surroundedby forest, e.g. Belem in the state of Para, or further away in othertowns or cities or even in other countries. In Belem the nutscollected are usually transported, either with or without shell, bytruck or boat to the marketplace for sale to industry or consumers.However, collectors also store nuts for their own consumptionduring the year without any awareness of the weather or othercontaminant sources, which can represent an additional risk ofAspergillus spp. contamination due the high humidity and elevatedtemperature of tropical forest and the free access of animals andinsects. In processing companies and fairs the nuts can be roasted,peeled and packed into bags for local sale or exportation to otherscountries.

In Brazil a Hazard Analysis and Critical Control Points (HACCP)project was developed to ensure safe food production for con-sumer health. The HACCP system has as prerequisites GoodManufacturing Practices (GMP) and Resolution 275 of the Na-tional Agency for Sanitary Surveillance (ANVISA), which is theregulatory and supervisory agency for health and food prod-uct dealers in Brazil. This resolution deals with the standardoperating procedures of hygiene, identifying potential hazardsto food safety from raw material acquisition to product con-sumption and establishing control and monitoring measuresto ensure at the end of the process a safe and high-qualityfood.19,20

The main objective of this study was to evaluate the potentialhazards, critical control points and key issues in the process designof Brazil nuts produced and marketed in the metropolitan regionof Belem and verify the presence of fungi with the potential toproduce aflatoxins.

EXPERIMENTALStudy sitesHarvesting site of Brazil nuts in forestBrazil nuts were collected in the forest region surrounding themunicipality of Acara, approximately 50 km distant from the fair-market Ver-o-Peso where they are sold commercially.

Selling at fair-market Ver-o-PesoThe fair-market Ver-o-Peso is the place where Brazil nuts from themunicipality of Acara and other collectors distributed throughoutthe state of Para as well as local processing companies are sold.

Material processed and packaged in cans or sealed bagsfrom supermarket chainsThe samples obtained from supermarkets in Belem are processedby companies that supply both these networks and small vendorsin fairs such as the fair-market Ver-o-Peso.

SamplingSamples from the forest, the fair-market Ver-o-Peso and severalsupermarkets were collected during harvest and between harvests.For each sampling site and period, eight samples weighingapproximately 300 g were collected and divided into twoequal portions for fungal culture and aflatoxin determinationsrespectively. The nuts were taken from the top, bottom andcentral parts of the storage container and peeled by the handlersthemselves, then placed in sterile autoclave bags and transportedto the laboratory at 25 ◦C. The time between sample collectionand analysis was 24 h.

Hazards and critical control pointsIn order to determine the reality experienced by workers handlingBrazil nuts, a field research study was conducted throughinterviews and visits to the sites where the product is collected,handled and marketed. A list of questions based on Resolution 218of ANVISA and the Codex Alimentarius, which together providethe technical regulations for sanitary/hygienic procedures in thehandling of foods and beverages prepared with vegetables, waselaborated taking into account the acquisition, receipt and storageof raw materials, ingredients, packaging and supplies as well asaspects associated with Brazil nut handlers and the preparationand exposure to sale of this food.21,22 A total of 16 individuals wereinterviewed, of whom eight were vendors from the traditionalfair-market Ver-o-Peso and eight were Brazil nut gatherers livingin forest areas.

Evaluation of hygienic/sanitary aspectsThe following hygienic/sanitary aspects were observed: cleaningand sanitising locations, instrumentation, the use of masks, glovesand gowns by handlers, waste management, appropriate workingtime and seasonal conditions, temperature and humidity, vectorand pest control and Brazil nut processing conditions.

Characterisation of fungiInitial cultivationFor cultivation of fungi, the packages containing the sampleswere opened in a laminar flow cabinet. The samples were washedwith 20 mL L−1 sodium hypochlorite solution, washed twice withsterile distilled water and cut with a sterile scalpel blade into

wileyonlinelibrary.com/jsfa c© 2012 Society of Chemical Industry J Sci Food Agric (2012)

Brazil nut quality control and Amazon sustainability www.soci.org

pieces of 5 mm length and 5 mm width. The fragments wereplaced in Petri dishes containing Sabouraud agar (SA) and 0.5 g L−1

chloramphenicol and incubated at 25 ◦C for at least 5 days.

Cultivation on microscope slideIn order to identify the fungi growing in the SA culture medium,a clean Petri dish was first sterilised in an autoclave togetherwith a sheet of filter paper on its bottom, a glass holder in thecentre, a microscope slide and a coverslip. A piece of SA mediumapproximately 20 mm thick was cut with a sterile scalpel to asize slightly smaller than the coverslip so that the border of theSA block would not be outside the coverslip. A small portion ofthe desired colony was then inoculated at the border of the SAblock with the aid of a dissecting needle. The coverslip was placedon the SA block and pressed lightly until it was attached, then,using a sterile pipette, a small amount of sterile distilled water wasadded to the filter paper at the bottom of the plate to maintainthe necessary moisture level. Plates were incubated at 25 ◦C for2–5 days depending on the fungus.

StainingTwo drops of blue lactophenol were applied to a clean slidepreviously prepared for staining. The coverslip was carefullyremoved from the culture with the aid of tweezers and placedon the lactophenol so that no air bubbles were formed. Then thespace between the slide and the coverslip was sealed using smalldrops of enamel at each intersection point of the coverslip in orderto restrict movement. The slide used for cultivation, where the SAspot remained and there was also growth of fungal colonies, wasthen used. Two drops of lactophenol were added to the SA spotand a clean, previously prepared, coverslip was fixed with the aid ofenamel. All procedures were performed in a laminar flow cabinetin order to maintain a sterile environment. When ready, the slideswere observed under an optical microscope (magnification 100×)for the identification of fungi through their microscopic structuralform and considering the macroscopic aspects of the colony.

Identification by molecular biologyUsing the loop of Henle, a rebound was made to isolate thefungi that grew on the plates containing the fragments of Brazilnuts to new plates with SA and chloramphenicol in order toisolate and identify the fungi present in all samples throughtheir 5.8S rDNA and internal transcribed spacer 2 (ITS2) sequences.Total DNA was extracted following phenol/chloroform proceduresaccording Sambrook et al.23 The extracted DNA was subjected toamplification with primers ITS3 and ITS4.24 Polymerase chainreaction (PCRs) were conducted in a total volume of 25 µLcontaining 10 ng of genomic DNA, 1× PCR buffer (20 mmolL−1 Tris-HCl, pH 8.4, 50 mmol L−1 KCl), 5 pmol L−1 of eachdNTP (Amersham Biosciences, Piscataway, USA), 3 mmol L−1

MgCl2, 5 pmol of each primer and one unit of Platinum TaqDNA Polymerase (Invitrogen, Carlsbad, USA). Amplifications werecarried out in a Mastercycler gradient thermocycler (EppendorfScientific Inc., Westbury, USA) under the following conditions:denaturation at 95 ◦C for 2 min followed by 33 cycles of annealingat 54.3–61.3 ◦C for 30 s and extension at 72 ◦C for 40 s. Ampliconswere then purified using a QIAquick PCR Purification Kit (Qiagen,Crawley, UK) and sequences were obtained through an ABI3130 genetic analyser (Applied Biosystems, Foster City, CA, USA).Subsequent identity searches were performed with the BLASTprogram available from the National Center for BiotechnologyInformation (NCBI, Bethesda, MD, USA).

Quantification of aflatoxinsAflatoxins were analysed by high-performance liquid chromatog-raphy as follows. Brazil nut slurries with a water/kernel ratio ofapproximately 1 : 1 (w/w) were extracted with methanol/watersolution and the extracts were defatted with hexane. Theextracts were then purified on immunoaffinity columns, andaflatoxins were eluted with methanol. Samples (50 µL injectionvolume) were separated on a VP-ODS analytical column (5 mm,250 mm × 4.6 mm; Shimadzu, Kyoto, Japan). The mobile phasewas acetonitrile/methanol/water (20 : 20:60 v/v/v) at a flow rate of1 mL min−1 and a temperature of 45 ◦C. Post-column derivatisa-tion was performed using saturated iodine solution at a flow rateof 0.3 mL min−1 and a temperature of 70 ◦C. Aflatoxins were de-tected and quantified with a fluorescence detector at wavelengthsof 365 nm (excitation) and 428 nm (emission). Quantitative resultsare reported in µg kg−1. The limit of detection was 0.2 µg kg−1 foreach aflatoxin.

Statistical analysisNormality of data was confirmed by a cumulative normalprobability plot and the Kolmogorov–Smirnov test. Levels ofaflatoxins are expressed as mean ± standard error of mean (SEM),while the proportion of fragments with fungal growth is expressedas a percentage. Statistical analyses were performed by the chi-square test and analysis of variance with Tukey’s a posteriori testusing Statistica Version 6 (StatSoft, Tulsa, OK, USA). All P values weretwo-tailed, and P < 0.05 was considered statistically significant.

RESULTSHazards and critical control points and hygienic/sanitaryaspectsTable 1 shows the hazards and critical control points found inproduction and marketing sites of Brazil nuts. The answers ofthe farmers and traders along with the technical staff assessmentshow that the two sites of nut manipulation, the native forestand the urban fair, need care in order to improve product quality.However, while in the primary sector there is still a great lack ofinformation and basic infrastructure requirements such as accessto clean water, adequate equipment and technical and scientificknowledge, in the tertiary sector, information and infrastructureare present, but it lacks the commitment of manipulators withactions to improve and maintain quality and a greater control ofpublic power for the sanitary/hygienic aspects of the site. Thusthe absence of proper storage facilities and the non-adherence toGood Agricultural Practices (GAP), GMP and the use of personalprotective equipment (PPE) were observed as well as the use ofinappropriate places for the manipulation of food associated withthe lack of basic hygiene by manipulators.

Characterisation of fungiGrowth of fungi was observed in 76 out of 80 fragments (95%) ofBrazil nuts from both the urban fair and the native forest duringthe between-harvest period, while only eight out of 80 fragments(10%) of processed Brazil nuts showed fungal growth (P < 0.001).Growth of fungi during harvest was similar to that during thebetween-harvest period, with 78 out of 80 (P = 0.9987), 75 outof 80 (P = 0.9576) and ten out of 80 (P = 0.8514) fragmentsfrom native forest, urban fair and processed samples respectivelyshowing fungal growth. The macroscopic and microscopic featuresof the fungi found demonstrated the presence of hyaline and

J Sci Food Agric (2012) c© 2012 Society of Chemical Industry wileyonlinelibrary.com/jsfa

www.soci.org AM Lima et al.

Table 1. HACCP in process design of Brazil nuts in Belem, Paraa

Item Collection site (primary sector) Ver-o-peso market (tertiary sector)

Harvest period(conservation)

The harvest season is from December to February whensamples are of better quality. Thus new strategies forconservation are necessary. Families continue toconsume nuts throughout the year, which can behazardous to their health, as confirmed bymicrobiological and toxicological analyses performedon stored samples

Buy nuts from different locations, including companies inBelem, so the harvest period is longer (from December toJune). Although two of the eight vendors interviewedacquire nuts from companies, there is a risk of marketingproducts of poor quality owing to the lack of adequateconservation in market stalls

Equipment andprotective garments

Collectors use a knife and try to protect themselves frompoisonous animals and the sun using boots and a caprespectively. However, they do not have any otherequipment or garment to work

Although some vendors reported owning an apron, goggles,gloves and other garment items, they claimed that theydid not use these protective resources because they hinderthe handling and breaking of nuts

Knowledge of goodpractices (GMP andGAP)

Workers do not know Good Manufacturing Practices(GMP) and Good Agricultural Practices (GAP)

Four of the eight workers interviewed said that they did notknow of the GMP manual and did not have a food handlerlicence

Transportation Directed by middlemen, which results in loss ofprofitability and quality control of the product

Usually, nuts come by truck or boat inside bags sold byresellers

Cleaning The great difficulty regards water quality; communitieshave a great concern with cleaning, but lack technicalknowledge

Nuts are cut and handled in inappropriate places where theconstant presence of rodents, animals such as dogs andcats and pests such as cockroaches and flies can beobserved

Storage There is no care about the weather, because casingscontaining the nuts are ‘left’ under trees with freeaccess to animals and insects

The storage area is protected from the weather. However, thepossibility of free flow of rodents, cockroaches and otherinsects was observed

Access to informationand training

Access to information and training is scarce. There is anapparent organisation and the desire of farmers fortraining courses aimed at improving their quality of lifeand productive capacity, but these actions are still rareand in need of appropriate methodologies

Because the vendors are in a metropolitan area, they areinformed about aspects of maintaining product qualityand how to maintain hygiene and sanitation of thehandling and marketing place of the nuts, but they do notfollow these principles

a Initial approach based on seven principles of HACCP for its implementation: ‘list all potential hazards and perform analysis and control measures’.

(a) (c)(b)

10 µm

Figure 1. Aspects of some pathogenic fungi after initial cultivation and isolation, showing macroscopic and microscopic characteristics. (a) Penicilliumspp.: granular colonies with various shades of green and white border; observe the ‘penicillin’ made by the conidiophore branches. (b) Aspergillusfumigatus: cotton wool-like granular consistency, greyish; conidia are spherical, generally flat and arranged in long chains. (c) Aspergillus niger: colonycovered by a dense cluster of strong-dark black conidia; sporulation is generally abundant, with dense clusters of short chains of strong-dark blackconidia.

dematiaceous fungi of fast or slow growth and multiple species

(Fig. 1). The analysis through molecular biology of isolated samples

of each fungus was used for the confirmation of species and

characterisation of the isolated fungi. Data of five sequences were

obtained after DNA sequencing and identity analysis by the BLAST

program. The sequences were submitted to GenBank (Accessions

JQ791100, JQ791101, JQ791102, JQ791103 and JQ791104). The

presence of several fungi was observed (Table 2), among which

we highlight the genera Aspergillus and Penicillium, which are

capable of producing carcinogenic substances or causing infection

and pathological conditions humans, especially in patients with

immunosuppression.

wileyonlinelibrary.com/jsfa c© 2012 Society of Chemical Industry J Sci Food Agric (2012)

Brazil nut quality control and Amazon sustainability www.soci.org

Table 2. Molecular biology and macroscopic characteristics of fungi found in samples of Brazil nuts in Belem, Paraa

Sample site(% of samples)b

GenBankAccession Fungus found

Querycoveragec (%) E valuec

Maximumidentityc (%) Macroscopic characteristicd

Collection area(95%)

ND Aspergillus flavus – – – Yellow-greenish and grainy colony

JQ791102 Candida vaughaniae 98 3e-114 89 Yeast and white colony

ND Paracoccidioides brasiliensis – – – Light-coloured dimorphic fungus

JQ791101 Penicillium spp. 52 1e-160 99 Green with clear borders

Fair-market (95%) JQ791100 Aspergillus fumigatus 96 1e-147 97 Septate hyphae and dark grey colour

JQ791104 Scedosporium apiospermum 96 3e-174 99 Grey colony with dark grey background

JQ791103 Aspergillus niger 85 3e-116 94 Dense, black colony, grainy to flaky

ND Penicillium spp. – – – Grey colony with white borders

Industriallypackaged (10%)

ND Trichophyton spp. – – – White to yellow colony

ND Bipolaris spp. – – – Dark brown colony

a Using DNA amplification by PCR and sequencing techniques, coupled with macroscopic characterisation.b Percentage of fragments with fungal growth.c Data obtained after DNA sequencing and identity analysis by BLAST program (NCBI).d Growth on Sabouraud agar containing 50 g L−1 chloramphenicol.

Table 3. Aflatoxin levels in Brazil nut samples during off-season

Concentration (µg kg−1)

Mycotoxin Collectors Industry Fair

Aflatoxin B1 446.48 ± 73.35∗∗ 2.29 ± 0.38 3.92 ± 0.85

Aflatoxin B2 0.703 ± 0.15 NQ 0.55 ± 0.20

Aflatoxin G1 30.54 ± 4.71∗∗ 1.4 ± 0.33 1.8 ± 0.63

Aflatoxin G2 NQ 0.34 ± 0.12 0.74 ± 0.26

Total aflatoxins 471.69 ± 72.26∗∗ 4.03 ± 0.76 7.01 ± 1.98

Values are mean ± SEM. Data are representative of one of three experiments with similar results. NQ, not quantified (below quantification limit ofmethod).∗∗ P < 0.01 compared with industry and fair.

Quantification of aflatoxinsThe levels of aflatoxins in the samples studied showed that,although fungal growth was observed in both manipulation sites ofBrazil nuts during both the harvest period and the between-harvestperiod (off-season), only samples from the native forest collectedbetween harvests showed a high risk for consumption, becausetheir levels of aflatoxins were above the limits recommended.During the harvest period the levels of aflatoxins were belowthe limit of detection of the analytical procedure. Urban fairand processed/packaged nuts had significantly lower levelsof aflatoxins compared with native forest nuts (P < 0.01). Itis important to highlight that all off-season samples showedtotal aflatoxin levels higher than the EC recommendations(Table 3).13,16,25,26

DISCUSSION AND CONCLUSIONWhile the idea of HACCP is widely associated with industry, thereare no practical impediments to its use in family agriculture.Many developed countries have used effective technologies forincreasing production associated with improvement of the qualityof products with significant added value, gains in food securityand opening of new markets.27

Our study indicates the main issues related to the process designof Brazil nuts in Belem, supporting the necessity of research on newstrategies to improve the quality of nuts. The most outstandingfeature that differentiates farmers from traders is knowledge andits application, because, while farmers are practically devoid ofknowledge on GAP and adequate infrastructure, traders receiveguidance on Good Food Handling Practices but do not apply theseconcepts in their daily practice.28

The harvest season of Brazil nuts in the sites that were part ofthis study is from December to February. Our study used samplescollected both during the harvest period and during the off-season,which was the determining factor for the degree of contaminationof the nuts, as verified by microbiological analysis and the presenceof aflatoxins. The habit of eating Brazil nuts that have been storedthroughout the year may be a risk to farmers, as there is aconsiderable carcinogenic potential of the aflatoxins, which, whencombined with the usual daily diet, can result in cumulativeand chronic effects.29,30 This fact requires epidemiological studieson the health status of these native communities. In addition,improper storage in open markets implies the risk of contaminationof a larger number of people in the urban environment and thepossibility of increased cases of liver cancer in this population.Although our results have demonstrated lower levels of aflatoxinsin nuts from the fair-market Ver-o-Peso and processing companies

J Sci Food Agric (2012) c© 2012 Society of Chemical Industry wileyonlinelibrary.com/jsfa

www.soci.org AM Lima et al.

compared with nuts from the native forest, the micro-organismsisolated and the levels of aflatoxins in these samples suggest apotential risk of human exposure.

Taken together, our data indicate that the issue of sustainabilityand crop substitution based on the replacement of timberresources by the use of non-timber resources in native forests stillfaces key problems such as the lack of scientific knowledge andadequate technologies. However, the strategy of strengtheningfamily forestry agriculture is feasible and represents a goodalternative if associated with the training of workers. The useof the HACCP system can be an important tool to help this process,generating a higher production of safe food fit for consumption,which in turn brings to the forest people new income sources andimproved quality of life.

ACKNOWLEDGEMENTSThis work was supported by CNPq (477737/2010-2) and MEC(PROEXT-2010), Brazil.

REFERENCES1 Malhi Y, Roberts JT, Betts RA, Killeen TJ, Li W and Nobre CA, Climate

change, deforestation, and the fate of the Amazon. Science319:169–172 (2008).

2 Peres CA, Baider C, Zuidema PA, Wadt LHO, Kainer KA, Gomes-Silva DAP, et al, Demographic threats to the sustainability of Brazilnut exploitation. Science 302:2112–2114 (2003).

3 Clay JW, Implications for biodiversity and conservation, in HarvestingWild Species, ed. by Freese CH. Johns Hopkins University Press,Baltimore, MD, pp. 246–282 (1997).

4 Thomson CD, Crisholm A, McLachlan SK and Campbell JM, Brazil nuts:an effective way to improve selenium status. Am J Clin Nutr87:379–384 (2008).

5 Ministerio do Meio Ambiente, Governo lanca plano para incentivarextrativismo e preservar a floresta. [Online]. Available: http://www.mma.gov.br/[24 July 2009].

6 Becker BK, Ciencia, tecnologia e inovacao para conhecimento e usodo patrimonio natural da Amazonia. Seminarios Tematicos para a 3a

Conferencia Nacional de C, T & I. Revista Parcerias Estrategicas N◦ 20,pp. 621–651 (2005).

7 Eglezos S, Huang B and Stuttard E, A survey of the bacteriologicalquality of preroasted peanut, almond, cashew, hazelnut, andBrazil nut kernels received into three Australian nut-processingfacilities over a period of 3 years. J Food Protect 71:402–404(2008).

8 Senna G, Bonadonna P, Crivellaro M, Schiappoli M and Passalacqua G,Anaphylaxis due to Brazil nut skin testing in a walnut-allergicsubject. J Invest Allergol Clin Immunol 15:225–227 (2005).

9 Bansal AS, Chee R, Nagendran V, Warner A and Hayman G, Dangerousliaison: sexually transmitted allergic reaction to Brazil nuts. J InvestAllergol Clin Immunol 17:189–191 (2007).

10 Ridout S, Mattews S, Gant C, Twiselton R, Dean T and Arshad SH, Thediagnosis of Brazil nut allergy using history, skin prick tests, serum-specific immunoglobulin E and food challenges. Clin Exp Allergy36:226–232 (2006).

11 Midorikawa GEO, Pinheiro MRR, Vidigal BS, Arruda MC, Costa FF,Pappas Jr GJ, et al, Characterization of Aspergillus flavus strains fromBrazilian Brazil nuts and cashew by RAPD and ribosomal DNAanalysis. Lett Appl Microbiol 47:12–18 (2008).

12 Arrus K, Blank G, Abramson D, Clear R and Holley RA, Aflatoxinproduction by Aspergillus flavus in Brazil nuts. J Stored Prod Res41:513–527 (2005).

13 Giorgi L and Tandon A, Putting risk management to the test or why it isineffectual to separate risk assessment from risk management: the

story of maximum levels for aflatoxins. Eur J Risk Regul 1:123–136(2010).

14 Newing H and Harrop S, European health regulations and Brazil nuts:implications for biodiversity conservation and sustainable rurallivelihoods in the Amazon. J Int Wildl Law Pol 3:109–124 (2000).

15 Freitas-Silva O and Venancio A, Brazil nuts: benefits and risksassociated with contamination by fungi and mycotoxins. FoodRes Int 44:1434–1440 (2011).

16 Ministerio da Saude, Leis, decretos, etc. Resolucao N◦ 34/76 daComissao Nacional de Normas e Padroes para Alimentos. DiarioOficial, Brasılia, 19 jan. 1977. Sec. I, pt. I, p.710. Fixa padroes detolerancia para as aflatoxinas em alimentos. [Online]. Available:http://www.saude.gov.br/[20 May 2012].

17 Ministerio da Agricultura, Portaria MA/SNAD/SFA N◦ 07 de 09/11/1988.Diario Oficial, Brasılia, 14 nov. 1988. Baixa os padroes mınimos demateria prima destinada a alimentacao animal. [Online]. Available:http://www.agricultura.gov.br/[20 May 2012].

18 Agencia Nacional de Vigilancia Sanitaria (ANVISA), Resolucao-RDCn◦ . 7, de 18 de fevereiro de 2011. Dispoe sobre limites maximostolerados (LMT) para micotoxinas em alimentos. [Online]. Available:http://www.anvisa.gov.br/[18 May 2012].

19 Agencia Nacional de Vigilancia Sanitaria (ANVISA), Resolucao-RDC n◦ . 275, de 21 de outubro de 2002. Dispoe sobre oregulamento tecnico de procedimentos operacionais padronizadosaplicados aos estabelecimentos produtores/industrializadores dealimentos e a lista de verificacao das boas praticas de fabricacaoem estabelecimentos produtores/industrializadores de alimentos.[Online]. Available: http://www.anvisa.gov.br/[23 May 2012].

20 Agencia Nacional de Vigilancia Sanitaria (ANVISA), Analise deperigos e pontos crıticos de controle. [Online]. Available: http://www.anvisa.gov.br/[23 July 2009].

21 Agencia Nacional de Vigilancia Sanitaria (ANVISA), Resolucao-RDCn◦ . 218, de 29 de junho de 2005. Dispoe sobre o regulamentotecnico de procedimentos higienico-sanitarios para manipulacaode alimentos e bebidas preparados com vegetais. [Online].Available: http://www.anvisa.gov.br/[23 July 2009].

22 Codex Alimentarius Commission, Revised Code of Hygienic Practicefor the Preparation and Sale of Street Foods (Regional Code – LatinAmerica and the Caribbean). CAC/RCP 43–1997, Rev. 1–2001 (2001).

23 Sambrook J, Fritsch EF and Maniatis T, Molecular Cloning. A LaboratoryManual (2nd ed). Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY (1989).

24 White TJ, Brims T, Lee S and Taylor J, Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics,in PCR Protocols: a Guide to Methods and Applications, ed. by Innis M,Gelfand D, Sninsky J and White T. Academic Press, San Diego, CA,pp. 315–322 (1990).

25 European Commission (EC), Commission Regulation (EC) No1881/2006 of 19 December 2006 setting maximum levels for certaincontaminants in foodstuffs. Off J Eur Union L364:5–24 (2006).

26 European Commission (EC), Commission Regulation (EC) No 401/2006of 23 February 2006 laying down the methods of sampling andanalysis for the official control of the levels of mycotoxins infoodstuffs. Off J Eur Union L70:12–34 (2006).

27 Mignouna HD, Abang MM, Omanya G, Nang’ayo F, Bokanga M, Boadi R,et al, Delivery of agricultural technology to resource-poor farmersin Africa. Ann NY Acad Sci 1136:369–376 (2008).

28 Oliveira AC, Seixas AS, Sousa CP and Souza CW, Microbiologicalevaluation of sugarcane juice sold at street stands and juicehandling conditions in Sao Carlos, Sao Paulo, Brazil. Cad SaudePublica 22:1111–1114 (2006).

29 Asim M, Sarma MP, Thayumanavan L and Kar P, Role of aflatoxin B1as a risk for primary liver cancer in North Indian population. ClinBiochem 44:1235–1240 (2011).

30 Busby WF and Wogan GN, Food-borne mycotoxins and alimentarymycotoxicoses, in Food-borne Infections and Intoxications, ed.by Riemann H and Bryan FL. Academic Press, New York, NY,pp. 519–610 (1979).

wileyonlinelibrary.com/jsfa c© 2012 Society of Chemical Industry J Sci Food Agric (2012)