Effect of Oxygen-Reducing Atmospheres on the Safety of Packaged Shelled Brazil Nuts during Storage

This work reports the application of oxygen-(O2-) reducing atmosphere methods on stored shelled Brazil nut (Bertholletia excelsa H.B.K.) packs aiming to evaluate the degree of aflatoxin degradation, nuts lipid oxidative stability, fungi control, and hygienic conditions improvement. The methods applied were (a) ozone: O3, (b) carbon dioxide: CO2, and (c) O2 absorber pads with and without vacuum. From all modified atmospheres evaluated, the best performance was obtained with O3, either with or without vacuum. It was the only nut treatment that was able to degrade aflatoxins. None of the spiked (AFLs: 15 μg·kg−1) nut samples O3- treated had aflatoxins detected up to the LC-MS/MS method LOQ (0.36 μg·kg−1 for total AFLs), thus producing safer nuts. Also it kept the fatty acid oxidation indicator—malondialdehyde stable and improved the sensory attributes for consumer acceptance. In addition, the destruction of fungi and yeast was observed since the O3 application (from 1.8 × 104 cfu/g to NG = no growth). All other treatments stabilized and/or inhibited microorganisms' growth only. By adding CO2 gas also played an important role in the nut quality. Regarding cost, gaseous O3 showed to be of low cost for application in the nut packs.


Introduction
In nature, Brazil nuts (Bertholletia excelsa H.B.K.) that grow in the Amazon forest may get contaminated by fungi and aflatoxins [1][2][3], as other tree nuts. The aflatoxigenic Aspergillus species that have been isolated from Brazil nuts are A. flavus, A. parasiticus, and A. nomius [4][5][6][7]. Their growth is directly related to the climate conditions of that region and to the conditions during their storage, transport, and commercialization, if there is no control of moisture content (m.c.) and temperature. That can also occur if nuts are packaged in a microclimate rich in oxygen (O 2 ) and m.c. enough to allow microorganisms to grow [1,8].
Studies have reported the use of modified atmospheres (MA) in food storage, extensive to packaging, to reduce O 2 concentration by adding gases such as nitrogen, carbon dioxide (CO 2 ), and ozone (O 3 ) which lead to microorganisms (fungi, yeast, and bacteria) inhibition, maintenance of lipid stability, and reduction of grains/nuts/vegetable respiration [9][10][11][12][13][14]. Vacuum also is an alternative for O 2 reduction and in recent years the addition of O 2 absorber pads (which contains a mixture of iron salts) have been the newest alternative in packaged food [15][16][17]. Studies have reported O 3 and CO 2 effect on controlling microorganism growth in several agricultural commodities [13,[18][19][20][21]. CO 2 is a promising and efficient inactivating microorganisms' gas for application on nonthermal sterilization process [22,23]. Maeba et al. (1988) reported the destruction and detoxification of AFB 1 and AFG 1 in agricultural products treated with 1.1 ppm of O 3 during 5 minutes [24]. Aflatoxin degradation in different food products, either fresh or processed at different O 3 concentrations, has been reported by some authors [13,[25][26][27][28]. An advantage of gaseous O 3 , apart from being a powerful disinfectant, oxidant, and aflatoxins degrader, is that it decomposes quite fast into O 2 and does not have toxic effect [29][30][31].  The degradation of mycotoxins by O 3 is found to follow a pseudo-first-order rate, as long as a continuous supply of O 3 is maintained [8]. For aflatoxins, the higher degradation rate for AFB 1 and AFG 1 was attributed to the presence of an 8,9 double bond forming a vinyl ether at the terminal furan ring (Figure 1), which is not present in AFB 2 and AFG 2 . These latter forms require longer exposure due to a possible second mechanism, when the lactone ring is opened during O 3 exposure [29].
High m.c., relative humidity, temperature, and environment rich in O 2 are the main factors for tree nuts to get aflatoxin contaminated and so infected by fungi. During storage and commercialization dry shelled Brazil nut packs need to maintain their safety and quality. Considering that MA in storage (macroenvironment) and packaging (microenvironment) can prolong food shelf life by reducing O 2 concentration, this work reports the application of O 2reducing atmosphere methods (vacuum, CO 2 , O 3 , and O 2 absorber) on fungi reduction, aflatoxin degradation, and lipid stability during storage of snack packs of shelled Brazil nuts.

Shelled Brazil Nut Analysis. (a) Microbiological methods:
for total fungi count the method was of Pitt and Hocking (1997) [33]; the presence of Aspergillus species was checked utilizing the Aspergillus flavus and parasiticus agar (Fluka, St. Gallen, Switzerland) by Pitt et al. (1983) [34]; the identification of fungi in genus and species was carried out according to the keys of Samsom et al. (2004) [35] and Salmonella spp., Staphylococcus spp., and coliforms (45 • C) were checked by APHA (1997) [36]. (b) Aflatoxin determination: was carried out by LC tandem mass spectrometry [37]. Briefly, aflatoxins (Sigma, Zwijndrecht, The Netherlands) were extracted from ground Brazil nuts with acetonitrile : water (HPLC grade, Carlo Erba, Milan, Italy and MilliQ, Millipore, Bedford, MA, USA, resp.) at 80 : 20 v/v, mixed, filtered, and injected into an Waters Alliance 2695 separation module with a 20 µL injection loop (Waters, Milford, USA) and a C 18 column 150 × 3.2 mm, 5 µm (Alltech, Breda, The Netherlands) at 30 • C. Separation was performed utilizing methanol (Carlo Erba) : water (both with 25 mM of ammonium acetate, J. T. Baker, Phillipsburg, NJ, USA) as mobile phase at 1 mL·min −1 of flow rate. The LC system was coupled to a Quatro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK) and toxins were detected and quantified by using atmospheric pressure chemical ionization in the positive   [38]. (d) Fatty acid oxidation was determined by the TBA method of Genot (1996). Extraction with 5% TCA (J. T. Baker) containing freshly prepared BHT in ethanol (J. T. Baker and Carlo Erba, resp.). After filtration, extract was mixed with TBA (J. T. Baker,) and immersed in a 70 • C water bath (Dubnoff Q226D, Quimis, Diadema, Brazil) for 30 min, cooled in ice and the absorbance of the reacted solutions read at 532 nm (spectrophotometer E005, Hitashi, Tokyo, Japan) against a blank containing TCA and TBA reagents. The results expressed as mg of malondialdehyde (MDA) equivalents per kilograms nut sample (LOQ: 0.37 mg·kg −1 ) [39]. (e) Sensory evaluation was based on the descriptive quantitative analysis [40]. Eighteen trained panelists during four sessions (n = 4) described impressions perceived by the hedonic scale of 5 points (1: dislike very much, 2: dislike, 3: neither like nor dislike, 4: like and 5: like very much). Sensory attributes evaluated: nut appearance (AP), color (CO), firmness (FI), resistance to slicing (SR), rancid (RA), and strange (OD) odors.

Statistical Analysis.
The results were expressed as the mean values and standard errors. Statistical analysis was performed by analysis of variance (ANOVA) and included the Tukey's test to evaluate significant differences among the means (P < 0.05). Figure 2 shows the flowchart on the whole study.

Results and Discussion
All the MA-treated shelled Brazil nut packs presented better quality and safety than the MA-untreated nuts (Group I: air) throughout the whole storage period. It was observed different degrees of fungi reduction and in some groups, aflatoxin degradation too. Table 1 shows the safety (fungi load; aflatoxins) and quality (m.c.; fatty acid oxidation; sensory evaluation) data obtained from the different MA-treated nut Groups (I, II, and III).

MA Effects on Shelled Brazil Nuts Microbiological Content
and M.C. As expected, inhibition of microoganisms growth was registered throughout the experiment despite the MA applied.
(a) Total Fungi Load and Aflatoxigenic Strains. A substantial fungi reduction was observed, both with O 2 absorber and      [41] and Food and Drug Administration [42].

MA Effects on Shelled Brazil Nuts Aflatoxin Degradation.
It was possible to observe in the aflatoxin spiked nut samples O 3 treated (Groups II: O 3 and O 3 +vac) that the gas was able to degrade them as none, during the storage period, were detected (LC-MS/MS method LOQ: 0.36 µg·kg −1 ). That was different for the other O 2 reducing atmospheres (CO 2 and O 2 absorber pads with/without vacuum). They were able only to stabilize/reduce the microorganisms growth keeping nuts safe but with aflatoxins. In that sense the packs with O 3 and vacuum applied bring an alternative for aflatoxin degradation and also m.c. reduction, a factor that is directly related to fungi proliferation and development of possible aflatoxigenic strains. Nuts O 3 treated utilized in the study showed to be able for consumption, as no aflatoxin was detected in none of them. Brazil nuts naturally contaminated by O 3 : to make sure O 3 would degrade aflatoxins not only in spiked nuts (i.e., toxins just applied and dried onto nut surfaces), we carried out also an experiment utilizing the special batch of nuts naturally aflatoxin-contaminated (packs with nuts O 3 and O 3 + vacuum treated). Similarly to the nuts spiked, no aflatoxin was detected after O 3 application neither fungi. That was probably due to the fact that Brazil nut has the advantage of its contamination/fungi proliferation to occur mostly on the nut surface/external layer as its structure is completely sealed (Figure 3). In addition, the testae (a pellicle that surrounds the edible part) of the Brazil nut, which is rich in Selenium (antioxidant), can act as a protector [2]. Thus reducing the possibility of easy access by the fungi spores to the nut core, as it occurs in peanuts (loose testae) or shelter fungi spores in-between shell and edible part of pistachios (in-shell), making gaseous O 3 application and action more effective. Currently, there is no available technology to completely eliminate the mycotoxin contamination of food and feed chain. Most of the current strategies for mycotoxin reduction are based on prevention, either pre-or postharvest and detoxification, which are not always effective. From the available tools to ensure food safety, O 3 application may be one of the most promising methods that come to meet the grain producers and food industries needs [43][44][45].

O 2 -Reducing Atmosphere Effect on Nuts Quality.
As far as quality is concerned, the parameters evaluated were the fatty acid oxidation, m.c., and sensorial evaluation which give information on lipid rancidity development, crunchiness texture alterations, and aroma/colour/odor/texture modifications. (a) Fatty acid peroxidation: regarding MDA formation during the nuts storage period and MA applied, no significant changes occurred despite the MA applied except for the O 2 absorber at the end of the storage period. In contrary, the samples packed loose in air (either Control and spiked) had an increase of MDA from 7.24 to 9.98 mg·kg −1 . With regards to CO 2 and O 3 , with and without vacuum, effect on shelled Brazil nuts lipids, it was observed that the values of MDA lowered and kept constant throughout the whole period of storage ( Table 1). The same occurred when Gamli and Hayoǧlu (2007) studied vacuum packaged pistachio [46]. The authors observed no significant difference on the MDA values during the storage period and reported that those results could be attributed to the higher amount of the fatty acid oleic acid (monounsaturated) and less linoleic acid (poliunsaturated) content in that nut. These results can be attributed to the reduction/control in the oxidation rate speed, both, by air withdraw (vacuum) and O 3 treatment (waste from O 2 removal). Similar results occurred in peppers and pistachio after the application of O 3 and vacuum packaging, that is, the effect on lipid oxidation was not apparent, thus could not alter the sensory characteristics [13] which was corroborated with the current data of the Brazil nut experiment. It is different when Rudolph et al. (1992) evaluated the oxidative stability of pecan oil and observed that changes in colour (O 2 effect on carotenoids) followed by a rapid increase of rancidity products (O 2 effect on fatty acids) [47]. However in the case of oil, lipids (fatty acids) present much more intense exposure to air O 2 than when it is protected in the liposomes inside the nut cells (or just the nut damaged surface exposes their lipid content from broken cells/lysosomes. As for Brazil nuts where their oil (lipids) is protected inside the undamaged nut cells or slightly damaged during the industrial nut cracking procedure. (b) Sensory Evaluation: the scores for the sensory attributes tested (nut appearance, strange odor, rancid odor, slicing resistance, and firmness) are shown in Table 1. The O 2 absorber pads applied led to some slight variation with regards to the visual appearance (color) of nuts probably due to its reducing effectiveness in the nuts located away from the pad site, sitting for long time (60 days) during storage. The sensory analysis of the Brazil nuts treated with O 3 and vacuum-packed did not present significant changes among the panelists (P < 0.05). All scores for the O 3 -treated nuts during storage period were between 4 (like) and 5 (like very much). It was verified also that O 3 leaves no residual odor. In fact O 3 with vacuum and vacuum only received the best scores showing that vacuum is still the best choice when preserving sensory characteristics is concerned. Similar thing occurred when Inan et al. (2006) worked with red pepper ozonation [27]. They did not register significant sensory changes after the O 3 application as the peppers were still quite palatable. When Akbas and Ozdemir (2006) studied the quality of pistachio, no significant changes also were observed between sweetness, rancidity, overall appearance, and taste, compared to control samples (no O 3 ) indicating the efficacy of that gas application [13]. Other authors also have reported the efficiency of the O 3 and its low interference in the sensory attributes of quality in several products such as vegetables, fish, birds carcasses, and their byproducts [44,48,49]. In a work carried by Dull and Kays (1988) with pecans, the author reported a slight better sensory quality on the vacuum-treated packaging nuts after 6 months of storage at 24 • C. Other potential uses of O 3 in the food industry include reduction of undesirable volatile metabolites, such as off-flavours or contaminants by their removal during O 3 stream application [50].

General Discussion
All O 2 -reducing atmospheres treated Brazil nut packs presented better nut quality after the period of study. However, the best performance, regarding safety was obtained either with or without vacuum. It was the only nut treatment that was able to degrade aflatoxins. It also led to fungi/yeast destruction, was able to eliminated off-flavours, reduced m.c., and maintained fatty acid oxidative process stable thus leading to safer, cruncher, and of better quality nuts. Next comes vacuum that kept sensory attributes of consumer acceptance, kept controlled lipid oxidation, and microorganisms. All other treatments stabilized and/or inhibited microorganisms' growth only, which also is important regarding safety, microorganisms-wise, and quality nuts. Considering that Brazil nuts can be suitable to aflatoxin contamination, are good fungi substrate, and are rich in oil, as other tree nuts, the best method that could control those parameters and improve consumer acceptance for best and stable dry nut product packs is O 3 with vacuum. Aflatoxin that may still remain in the Brazil nuts at the packaging can be destroyed at that stage. O 3 will be useful for those hard packages (tubs) sold in small portion, that are commonly commercialized in Brazil too-providing a safer product for the national consumers. Regarding the costs and environment impact, O 3 equipments are of low cost and environment friendly, it is fast converted into O 2 .
Our modern days have been emphasizing the importance of additives reduction in food. The use of O 3 features as 8 International Journal of Analytical Chemistry an important technology for food storage and industry as it leaves no residue. Its use has been approved in countries around the world and supported by several international food and health agencies inclusive for use in organically labelled agricultural products, inclusive in medicine. Currently, there is no available technology to completely eliminate the mycotoxins contamination of food and feed chain. Most of the current strategies for mycotoxin reduction are based on prevention, either pre-or postharvest. From the available tools to ensure food safety, O 3 application is one of the most promising methods that come to meet the needs.