Article pubs.acs.org/JAFC
Effect of Almond Processing on Levels and Distribution of Aflatoxins in Finished Products and Byproducts Rosanna Zivoli, Lucia Gambacorta, Giancarlo Perrone, and Michele Solfrizzo* Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Via Amendola 122/O, 70126 Bari, Italy ABSTRACT: The fate of aflatoxins during processing of contaminated almonds into nougat, pastries, and almond syrup was evaluated by testing the effect of each processing step (blanching, peeling, roasting, caramelization, cooking, and water infusion) on the distribution and levels of aflatoxins. Blanching and peeling did not reduce total aflatoxins that were distributed between peeled almonds (90−93%) and skins (7−10%). Roasting of peeled almonds reduced up to 50% of aflatoxins. Up to 70% reduction of aflatoxins was observed during preparation and cooking of almond nougat in caramelized sugar. Aflatoxins were substantially stable during preparation and cooking of almond pastries. The whole process of almond syrup preparation produced a marked increase of total aflatoxins (up to 270%) that were distributed between syrup (18−25%) and spent almonds (75−82%). The increase of total aflatoxins was probably due to the activation of almond enzymes during the infusion step that released free aflatoxins from masked aflatoxins. KEYWORDS: aflatoxin, almond, processing, pastries, nougat, syrup
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INTRODUCTION Almonds are the edible seeds of the almond tree (Prunus dulcis) originating from regions of Central Asia and currently cultivated around the world in dry and temperate climates. The world production in 2012 was mainly distributed among the United States (mainly California, 37.2%), Spain (11.1%), Australia (7.4%), Iran (5.2%), Morocco (5.1%), Italy (4.6%), and the Syrian Arabic Republic (4.5%). The remaining production (24.9%) is distributed in other countries, each of them producing 0.05) when compared to initial levels (Table 2). This was probably due to the inhomogeneous distribution of aflatoxins in chopped almonds that produced high values of standard deviation for replicate analyses that made problematic the statistical comparison of the mean levels measured before and after roasting. No aflatoxin reduction was observed for almonds roasted at 120 °C for 30 min, whereas mean reductions of 54−58% for AFB1 and 24− 30% for AFB2 were observed after 60−120 min, although these reductions were not statistically significant. Similar mean reductions were observed at 120 °C for 120 min (54% for AFB1, 24% for AFB2) and at 150 °C for 30 min (55% for AFB1, 20% for AFB2) (Table 2). Taken together, these data suggest that AFB2 may be more resistant to roasting because a lower reduction, compared to AFB1, was observed at both 120 and 150 °C. However, the percent reduction may also be a function
to retain aflatoxins, which belong to the less polar mycotoxins. In fact, when aflatoxin-contaminated ginger tea, a fat-free matrix, was steeped in boiling water for 5−10 min, 30−40% of aflatoxins passed in water.21 On the other hand, a mean aflatoxin reduction of 27% was observed during blanching and peeling of roasted peanuts.22 Sorting and sizing processes are used to separate aflatoxin-contaminated almonds from uncontaminated ones. In particular, up to 97% of total aflatoxins were found in almonds damaged by insects or mold or mechanically injured, whereas 3% of total aflatoxins were found in high-quality almonds that accounted for 84% of the total kernel mass.13 Roasting. The results of different roasting conditions on aflatoxin levels in peeled almonds are shown in Table 2. For almonds roasted at 150 °C, mean AFB1 and AFB2 reductions ranged between 55 and 84% and between 20 and 69%, respectively, after 30, 60, or 120 min. A lower reduction of 5711
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Figure 3. HPLC chromatograms of sugar spiked with AFB1 (394.7 μg/kg) and AFB2 (31.7 μg/kg) (- - -), sugar caramelized and then spiked with AFB1 (394.7 μg/kg) and AFB2 (31.7 μg/kg) (), and sugar spiked with AFB1 (394.7 μg/kg) and AFB2 (31.7 μg/kg) and then caramelized (- · - ·).
possible toxicity or harmlessness of degradation products and to define the roasting conditions that inactivate aflatoxins.23−26 Almond Nougat. The results of aflatoxin levels and aflatoxin mass balance in roasted almonds and nougat obtained from contaminated almonds of batches 1 and 3 and cooked with caramelized sugar or caramelized sugar−honey are reported in Figure 2. Clear reductions of AFB1 and AFB2 levels were observed in both types of nougat. The mass balance of toxins confirmed these reductions. For batch 1, the average reductions were 54 and 70% for AFB1 and 83 and 93% for AFB2 in sugar nougat and sugar−honey nougat, respectively. Similar reductions were observed for batch 3 (Figure 2B). The contact of almonds with hot caramel may have produced the reduction of aflatoxins. To verify this hypothesis, additional experiments were performed in the absence of almonds. In particular, aliquots of sugar were spiked with AFB1 (394.7 μg/ kg) and AFB2 (31.7 μg/kg) and then caramelized in a pot and successively analyzed for aflatoxins. For comparison, aliquots of sugar were spiked with the same levels of AFB1 and AFB2 and successively analyzed for aflatoxins. The second control was constituted of aliquots of caramelized sugar that were spiked with AFB1 and AFB2 and successively analyzed for aflatoxins. As shown in Figure 3 the peaks of AFB1 and AFB2 in the chromatogram of spiked sugar submitted to caramelization are markedly lower than the corresponding peaks of spiked sugar and spiked caramelized sugar with an average reduction of 82% for both AFB1 and AFB2. This reduction is quantitatively equivalent to the reduction observed during nougat preparation; therefore, the caramelization of sugar may be responsible for aflatoxin reduction. However, mixing almonds with sugar during its caramelization should partially explain the big reduction of aflatoxin observed during nougat preparation because only the surface of almonds is affected by sugar caramelization. On the other hand, almonds contain high levels of sucrose (11−22%) followed by raffinose (0.7−2.1%), glucose (0.4−1.5%), and fructose (0.1−0.6%)27 that should be caramelized during nougat preparation. Caramelization occurs when food surfaces are heated strongly.28 Thermally induced
of aflatoxin concentration; that is, the higher the concentration, the higher the percent reduction, because the levels of AFB2 tested in this study were always lower than those of AFB1 (Tables 2 and 3). Although 150 °C for 120 min produced the highest reduction of aflatoxins, these conditions seem to be impracticable because roasted almonds appeared blackened/ burned and most probably lost their organoleptic characteristics, making them inedible. The results of roasting experiments conducted at 150 °C with blank almonds spiked with the four aflatoxins at two levels are shown in Table 3. After 120 min, the AFB1 reductions were 39 and 48% for the two spiking levels, whereas for AFB2 the reductions were 25 and 31%, respectively. Lower reductions were observed after 30 min, those being 11 and 23% for AFB1 and 6 and 14% for AFB2 for the two spiking levels. As observed in the previous experiments, a higher reduction was observed for AFB1 as compared to AFB2, and roasting times >30 min (60−120 min) irreversibly altered the characteristics of almonds (color, smell etc.), making them unfit for consumption. Although no data are available in the literature on the effect of roasting on aflatoxins in almonds, our results are comparable with those published for other nuts. Yazdanpanah et al.23 reported that roasting pistachios at 90, 120, and 150 °C for 30, 60, and 120 min reduced the content of aflatoxins at levels ranging between 17 and 95%, and the maximum reduction was obtained at higher temperature and longer roasting time. Mean AFB1 reductions of 78−80% were reported for spiked and naturally contaminated peanuts roasted at 150 °C for 120 min.24 A similar reduction of AFB1 (79%) was observed for peanuts inoculated with A. f lavus and roasted at 150 °C for 30 min.25 Roasting naturally contaminated and spiked peanuts at 150 °C for 30 min produced 48−61 and 30−44% AFB1 reductions, respectively.26 No degradation products of aflatoxins have been isolated and chemically characterized from roasted nuts so far, and no attempts were performed in this study. Further studies are necessary to obtain these data that are necessary to establish the 5712
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Figure 4. (A) Effect of almond pastry preparation and cooking conditions on aflatoxin levels; (B) relevant mass balance of aflatoxins. G.P.A., ground peeled almonds; P, pastries.
Figure 5. (A) Effect of almond syrup preparation on aflatoxin levels; (B) relevant mass balance of aflatoxins. G.P.A., ground peeled almonds; A.S., almond syrup; S.A., spent almonds.
caramelization of sugars contained in almonds may therefore contribute to the aflatoxin reduction occurring during nougat preparation. Caramelization of almond sugars may also occur during roasting of almonds and may explain the reduction of aflatoxins observed during almond roasting (Tables 2 and 3). Further investigations are necessary to understand if this reduction is a result of degradation or conjugation of aflatoxins with reactive sugar degradation products such as hydroxymethylfurfural, hydroxyacetylfuran, and hydroxydimethylfuranone that have been reported as degradation products formed during sugar caramelization.28 Almond Pastries. The results of aflatoxin levels and aflatoxin mass balance in ground peeled almonds and pastries
obtained from contaminated almonds of batches 1−3 and cooked at three different temperatures and times are reported in Figure 4. The grinding of peeled almonds allowed a good aflatoxin homogenization as demonstrated by the low SD values for triplicate analyses performed for each batch. In general, mean levels of AFB1 and AFB2 in cooked pastries were lower than those measured in ground peeled almonds. However, these reductions were mainly due to the dilution effect of ingredients (sugar and egg white meringue) used for pastry preparation. The whole results of aflatoxin levels and relevant mass balance did not result in a clear trend related to cooking conditions. In particular, the increase of cooking temperature from 140 to 180 °C did not result in a clear 5713
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of processing such as caramelization in reducing aflatoxin levels in almonds and the increase of aflatoxin content in spent almonds after infusion.
reduction of aflatoxins also because the increase of temperatures paralleled the reduction in cooking time. Altogether these results suggest that aflatoxins remained substantially stable during the cooking conditions tested in this study, probably because the maximum inner temperature measured in pastries was 98 °C and sugar caramelization should be excluded. The stability of aflatoxins during cooking and baking has been reported by various studies on different matrices. A substantial stability of aflatoxins during cooking of ground peanuts has been reported.29 Experiments with microwave (700 W for 6 min) and home cooking (177 °C for 45 min) of spice sauces did not produce a significant decrease of aflatoxin levels in the sauce.30 Baking of biscuits (10 min at 250 °C) produced only a 17% reduction of AFB1, whereas a greater reduction (89%) was attributed to the mixing of contaminated wheat flour with leavening agents.31 A significant reduction of AFB1 was observed during the kneading in preparing bread, whereas no further reduction of AFB1 was observed during baking.32 On the other hand, a 34% reduction of AFB1 was observed during cooking (20 min at 160 °C) of polished rice.33 We did not use leavening agents for the preparation of almond pastries because they are not required by the recipe. Almond Syrup. Almond syrup can be prepared by infusing ground peeled almonds with water or with sugared water. After infusion, spent almonds are separated from the infuse, which is boiled until reaching typical syrup consistency. For the infusion conducted only with water, the sugar is added to the infusion after separation of spent almonds. In Figure 5 are shown the results of aflatoxin levels and aflatoxin mass balances in ground peeled almonds, almond syrup, and spent ground peeled almonds obtained from contaminated almonds of batches 1−3. Surprisingly, the levels of AFB1 and AFB2 in spent almonds were significantly higher than those measured in ground peeled almonds. These results were confirmed with the mass balance of aflatoxins. On average, for the three batches, the sum of AFB1 measured in the syrup and spent almonds was more than twice the amount the AFB1 measured in the initial ground peeled almonds. Similar results were obtained for AFB2 (Figure 5B). With regard to the passage of aflatoxins from almonds to the syrup, on average, for the three batches, the percentage of total AFB1 that passed into the syrup was 18%, whereas for AFB2 it was 25%. These percentages increased to 39 and 46% for AFB1 and AFB2, respectively, when they were calculated with respect to aflatoxin levels measured in the initial ground peeled almonds. These results were obtained by infusing ground peeled almonds with water that was successively sugared after separation from spent almonds. The same results were obtained when ground peeled almonds were infused in sugared water. A similar mean percentage of AFB1 (14%) was transferred into the liquid phase during mashing of naturally contaminated maize grits and malted barley.34 This is the first study that has monitored aflatoxins during water infusion of contaminated tree nuts. A possible explanation of this robust increment of aflatoxins during water infusion may be the presence of masked aflatoxins in almonds that were released by enzymatic activity because almonds are rich sources of various glycosidases.35,36 Several masked mycotoxins have been reported to occur in plant-based foods and feeds, but no masked aflatoxins have been identified to date.37 Further investigations are necessary to confirm the increase of aflatoxin levels during water infusion of naturally contaminated almonds and to isolate and chemically characterize the masked aflatoxins. In conclusion, the main outcomes of this study are the efficacy
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AUTHOR INFORMATION
Corresponding Author
*(M.S.) E-mail:
[email protected]. Phone: +39 080 5929367. Fax: +39 080 5929374. Funding
This research was supported by EU-FPVII Project MYCORED (KBBE-2007-222690). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Filomena Epifani for her valuable technical assistance. ABBREVIATIONS USED AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxin G2; AFtot, total aflatoxins; ISPA, Institute of Sciences of Food Production
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