The main production areas of apricots (Prunus armeniaca
) are the Mediterranean and Middle East. The top five producers of apricots (fresh fruit) in 2013 were Turkey (811,609 tonnes), Iran (457,308 tonnes), Uzbekistan (430,000 tonnes), Algeria (319,784 tonnes), and Italy (198,290 tonnes) [1
] (FAOSTAT). A significant portion of apricots are used to produce apricot stone/pit and its kernel. Apricot kernels are byproducts of the apricot processing industry [2
]. In Turkey, 10% of the apricots are used as fresh product, the rest of the product is traditionally stored in sacks with a 20% moisture level after harvesting, sulphuring, drying and stone/pit separation processes [3
]. Apricot stones/pits are separated from apricot pulp and processed into shells, mainly used as fuel, and kernels that are exported worldwide, mainly to European countries [4
]. Two main varieties of apricot kernels can be easily differentiated, sweet and bitter. Bitter kernels are a good source of amygdaline, which is about 4.5%–6.5% of dry kernels [5
]. The oil of bitter kernels (53%) is used in cosmetics and aroma perfume [6
] or as a cheaper substitute of bitter almond oil. Apricot kernels can also be of interest as a food or feed ingredient because of their high crude protein content (20%–25% w
, dry weight basis) [2
]. Sweet apricot kernels can be added to bakery products as whole or ground kernels, as well as consumed as appetizers [7
]. Bitter apricot kernels can be used as a substitute of bitter almonds, a more expensive kernel, to produce “persipan” a material used in confectionery and bakery products [5
]. The use of persipan as an ingredient of marzipan, made by almond and sugar, at a level >0.5% is considered an adulteration [8
]. Moreover, apricot kernels are an important ingredient of the Italian biscuit “amaretti”, whereas the Italian liqueur amaretto is flavoured with extract of these kernels.
Apricot kernels and almonds are at high risk of aflatoxin contamination but they are poorly studied, especially apricot kernels. The first notification of aflatoxins in apricot kernels was published in 1999 in the European Rapid Alert System for Food and Feed (RASFF) network. Between 1999 and 2015 a total of 28 notifications (six alerts, 13 border rejections, nine information) were reported for imported apricot kernels, or derived products produced in Europe, that were found contaminated with high levels of aflatoxins [9
]. From 2010 the European maximum levels of aflatoxin B1
) and total aflatoxins (AFs) in apricot kernels intended for further processing (12 µg/kg for AFB1
and 15 µg/kg for total AFs) and ready-to-eat (8 µg/kg for AFB1
and 10 µg/kg for total AFs) have been aligned to those of Codex Alimentarius after a positive opinion of the European Food Safety Authority (EFSA) [10
Aflatoxins contamination in maize, peanuts, almonds, pistachios, and Brazil nuts have an extremely uneven distribution [14
]. Often the contaminated lots have few kernels contaminated with high levels of aflatoxins, whereas most of kernels have low or no detectable contamination. Sampling plans have been developed to establish the true levels of contamination in lots, but a real correspondence between true levels and results obtained by applying the sampling plans is often not obtained [18
]. The uneven distribution of aflatoxins prompt to the development of strategies of segregation of contaminated kernels from healthy ones, because their removal can drastically reduce aflatoxin contamination of the entire lot. Moreover, it is known that changes of intrinsic characteristics of nuts, like discoloration and staining of skins or kernels, appearance of fluorescent material, changes of size or density, are caused by fungal growth [19
]. Some technologies able to detect changes in nut characteristics, such as hand-picking, electronic color sorting (sometimes combined with blanching), size separation, and flotation have been applied to different kind of nuts. Several studies were carried out for peanuts, pistachios, walnuts, and Brazil nuts [21
]. Few data are available on almonds [20
], whereas apricot kernels have not been studied yet.
The objective of this study was to reduce aflatoxin content in naturally-contaminated apricot kernels by using electronic and manual color sorting as a tool to separate contaminated kernels from healthy ones. The mass balance approach was used to quantitatively determine the distribution of aflatoxins in final and rejected products during sorting processes. The occurrence of aflatoxins was also evaluated on commercial products containing apricot kernels and/or almonds and commercially available in Italy.
3. Experimental Section
Apricot kernels. Samples of apricot kernels (20 kg) originating from Turkey were provided by a local importer (Bari, Italy) and used for electronic and manual color sorting experiments.
Commercial products. Forty-seven commercial products containing apricot kernels and/or almonds were analysed for aflatoxins content (AFB1, AFB2, AFG1, and AFG2). In particular, seven samples of almonds, six samples of peeled almonds, two samples of roasted almonds, one sample of bakery pastries, five samples of Cantucci pastries, eight samples of amaretti mini biscuits, seven bakery products containing peeled almonds (four) or almonds (three); six samples of almond nougat, and five other products (almond flour, chopped almonds, smoked almonds, sugared almonds praline, and mixed nuts snack). The seven bakery products (“panettone” and “colomba”) containing embedded whole almonds were crumbled to pick up the almonds that were analyzed separately. Each sample was finely ground by blending and analysed for aflatoxins.
3.2. Electronic Color Sorting of Apricot Kernels
The e-sorting experiments were performed in triplicate by using an optical sorter machine (Bühler Z+) (Bühler, Brescia, Italy). In particular, 15 kg of apricot kernels naturally contaminated with aflatoxins were manually mixed and divided in 3 × 5 kg aliquots that were singularly submitted to e-sorting. For each of the three aliquots the machine separated the kernels in two fractions called “final” and “reject”. Final and reject fractions of the 3 × 5 kg aliquots were singularly collected, weighted, slurried, or homogenized and analyzed for their aflatoxin content. With this approach, the initial aflatoxin concentration of the 15 kg of kernels was deduced from the results of the analysis of the whole “final” and “reject” fractions. Fractions weighting ≥4 kg were slurried (1:1.5 with water) using the slurry mixer Silverson (Silverson Machines Ltd., Waterside, Chesham, UK), whereas the fractions that could not be slurried, because of low amount (<1 kg), were homogenized by blending (DitoSama, Model F23200, Aubusson, France).
3.3. Blanching, Peeling, and Manual Sorting of Apricot Kernels
Steam blanching was carried out in a steamer (model HD 9140, Philips Electronics, Eindhoven, The Netherlands) for 40 min on 3 × 500 g of apricot kernels naturally contaminated with aflatoxins. After steam blanching, apricot kernels were manually peeled to separate peeled kernels from skins. Peeled kernels were further manually sorted to separate discolored kernels (brown/dark and spotted ones) from healthy ones. Samples of skins and peeled kernels (discolored and healthy) were separately freeze-dried, weighed, homogenized, and analyzed for their aflatoxin content for a total of nine samples. Each sample was analyzed in triplicate for a total of 27 analyses. The analytical method for aflatoxin determination is describe below.
3.4. Determination of AFB1, AFB2, AFG1 and AFG2
The HPLC-FLD method previously described [28
] was used herein with some changes depending of the nature of samples analysed.
Aflatoxin determination in samples of apricot kernels and skins derived from e-sorting and manual sorting. Aflatoxins were extracted from 10 g of dry sample by sonication for 30 min with 100 mL of acetone:water (85:15 v/v) in 250 mL Pyrex screw-capped glass flasks. Extracts were filtered on filter paper (No. 4, Whatman, Maidstone, UK) and 5 mL of filtered extract were diluted with 75 mL of ultrapure water and filtered through glass microfilter (GF/A, Whatman, Maidstone, UK). A 40 mL volume of filtered diluted extract (equivalent to 0.25 g of matrix) was passed through the immunoaffinity column (IAC) AflaTest (Vicam, Milford, MA, USA) at the flow rate of 1–2 drops/second. Then the column was washed with 2 × 10 mL of ultrapure water that was discarded. Aflatoxins were eluted from the column by passing 3 × 0.5 mL of methanol. The eluates were collected and diluted with ultrapure water up to 5 mL in a volumetric flask. Volume of 100–500 μL (equivalent to 5–25 mg of matrix) were injected in the HPLC-FLD apparatus.
Aflatoxin determination in discolored apricot kernels. These samples were supposed to contain high levels of aflatoxins therefore, the sample extracts were diluted and directly analysed by HPLC-FLD without using the immunoaffinity clean-up. In particular, 10 g were extracted with acetone:water (85:15 v/v), and 0.5 mL of the filtered extract was diluted with 30 mL of MeCN:H2O (30:70 v/v), vortexed, filtered through a 0.45 μm PTFE filter (Sartorius Stedim Italy SpA, Bagno a Ripoli, Firenze, Italy) and injected into the HPLC apparatus (the injection volume was 100 μL equivalent to 1.7 mg of matrix).
Analysis of commercial products. To obtain low limits of detection the method was modified to get a more concentrated final extracts for HPLC analysis. Briefly, 10 g of dry ground sample was weighted in a 250 mL Pyrex screw-capped glass flask and extracted by sonication for 30 min with 50 mL of extraction mixture of acetone:water (85:15 v/v). After filtration on filter paper (No. 4, Whatman, Maidstone, UK) 10 mL of filtered extract were diluted with 140 mL of ultrapure water and filtered with a GF/A glass microfiber (Whatman, Maidstone, UK). The immunoaffinity clean-up was performed by passing 113 mL of filtered diluted extract (equivalent to 1.5 g of matrix) through the IAC column at the flow rate of 1–2 drops/min. After washing the IAC column with 2 × 10 mL of ultrapure water, aflatoxins were eluted with 0.75 mL methanol, and after 1 min eluted again with 0.5 mL methanol. The collected eluates were diluted with ultrapure water up to 5 mL in a volumetric flask and analyzed by HPLC-FLD. Injection volumes were 100–500 μL equivalent to 30–150 mg of matrix.
3.5. Chemicals and Reagents
Methanol (MeOH), acetonitrile (MeCN) and acetone were purchased from Sigma Aldrich (Milan, Italy). Ultrapure water was produced with a Milli-Q system (Millipore, Bedford, MA, USA). AflaTest WB wide bore affinity columns were purchased from Vicam L.P. (Watertown, MA, USA). The mixed aflatoxins standard solution (purity 99% ± 1%), prepared in acetonitrile and containing 2.00 µg/mL AFB1, 2.02 µg/mL AFG1, 0.50 µg/mL AFB2 and 0.50 µg/mL AFG2, was purchased from Romer Labs Diagnostic (Tulln, Austria). This solution was used to prepare calibration solutions for HPLC-FLD determinations and recovery experiments.
3.6. HPLC-FLD Apparatus and Conditions
The HPLC analyses of aflatoxins were performed by using a mixture of acetonitrile: water (30:70) as mobile phase. A Perkin Elmer Series 200 binary pump was used at a flow rate of 0.8 mL/min (the run time was 20 min). Sample extracts were injected into the HPLC apparatus via a Rheodyne 7125 manual injection valve equipped with a 500 μL sample loop. The apparatus was equipped with a chromatographic data handling software for Microsoft Windows XP (PerkinElmer TotalChrom Workstation version 6.3.1) (Perkin Elmer, Waltham, MA, USA). The separation of aflatoxins was obtained with a Luna® analytical column PFP (2) (pentafluorophenyl propyl), 150 × 4.6 mm i.d., 3 μm, 100 Å (Phenomenex, Torrance, CA, USA), preceded by a security guard cartridge (4 × 3 mm, 5 μm) (Phenomenex). Aflatoxin detection was carried out using a Jasco FP-2020 plus fluorescence detector set at 365 nm (λex) and 435 nm (λem). A photochemical post-column derivatisation (UVE™ system, LCTech, Dorfen, Germany) was used to enhance the fluorescence of AFB1 and AFG1.
3.7. Calibration Curves
A mixed aflatoxins stock solution was prepared by diluting 500 μL of commercial standard solution (2 µg/mL AFB1 and AFG1, 0.5 µg/mL AFB2 and AFG2) to 10 mL with acetonitrile in a volumetric flask. The five HPLC calibration solutions of combined aflatoxins (AFB1 + AFB2 + AFG1 + AFG2) were prepared by diluting appropriate volumes of the stock solution with MeOH:H2O (40:60 v/v). The toxin concentration ranges of the five calibration solutions were 0.10–3.64 ng/mL for AFB1 and AFG1, 0.025–0.90 ng/mL for AFB2 and AFG2.
3.8. Recovery Experiments
The performances of the improved HPLC/FLD method were assessed by performing recovery and repeatability experiments. Blank samples of almonds, peeled almonds, amaretti, cantucci, and almond nougat were spiked with mixtures of the four aflatoxins and analyzed in triplicate. Two spiking levels of aflatoxins were tested (0.2 μg/kg and 1 μg/kg for AFB1; 0.5 μg/kg and 2.5 μg/kg for total AFs). In particular, for recovery experiments at spiking levels of 0.2 μg/kg of AFB1 and 0.5 μg/kg for total AFs, 3 × 10 g of samples were spiked with 3 × 20 µL of the spiking solution. For experiments at spiking levels of 1 μg/kg of AFB1 and 2.5 μg/kg of total AFs, 3 × 100 μL of the same spiking solution were added to 3 × 10 g of samples. Spiked samples were left overnight in the dark at room temperature to allow solvent evaporation. The values of LOD and LOQ were calculated as signal-to-noise ratio of three and six, respectively.
3.9. Statistical Analysis
Mean and standard deviation (SD) of data were calculated with a SigmaPlot for Windows version 12.0 statistical software package (Sistat, Software, Inc., Chicago, IL, USA). SigmaPlot was also used to perform one-way analysis of variance (ANOVA) followed by a Tukey pairwise multiple comparison test, and a least significant difference (LSD) test at 95% confidence levels (p = 0.05) to identify significant differences among groups.