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Changes in aflatoxins content during processing of pekmez as a traditional product of grape
LWT - Food Science and Technology 103 (2019) 178–185
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Changes in aflatoxins content during processing of pekmez as a traditional product of grape
T
Ali Heshmatia, Sabah Ghadimia,∗, Akram Ranjbara, Amin Mousavi Khaneghahb,∗∗ a
Department of Nutrition and Food Safety, Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, Caixa Postal: 6121, CEP 13083-862, Campinas, São Paulo, Brazil
b
ARTICLE INFO
ABSTRACT
Keywords: Mycotoxin Fate Clarifiers Grape Contamination Conventional process
The current study was aimed to investigate the changes in aflatoxins (AFs) content during the production of pekmez. In this context, the artificially spiked grape samples in levels of 2.5, 5, and 7.5 μg/kg of aflatoxin B1 (AFB1), and 0.5, 1 and 1.5 μg/kg of aflatoxin B2 (AFB2), G1 (AFG1) and G2 (AFG2) were processed by the conventional procedure and after each step, AFs levels were measured using HPLC-FD. Among different unit operations, the lowest reduction in AFs (8.2, 14.8, 12.4, and 22.7% for AFB1 and AFB2, AFG1, and AFG2, respectively) was obtained for crushing and pressing stages. The corresponded values for reduction average of AFB1, AFB2, AFG1, and AFG2 during washing step was measured as 19.3%, 22.2%, 23.7%, and 34.3%, respectively. Clarification stage was identified as the most effective step in the reduction (63.6%) of AFs. However, the reduction strongly depended on the type and concentration of clarifier as well as initial aflatoxin level. A significant difference between the assessed clarifiers (gelatin, bentonite and white soil) was observed, while the highest efficiency was demonstrated by gelatin. In general, levels of AFB1, AFB2, AFG1, and AFG2 during pekmez production were decreased about 60.4, 76.7, 76.3, and 86.7%, respectively.
1. Introduction
support immediate energy. Therefore, pekmez could play an important role in the diet of different age groups, especially children and athletes (Arslan et al., 2005; Batu, 2005). It is produced in non-industrial scale and rural areas (Batu, 2005; Karababa & Develi Isikli, 2005). In the conventional production method, the hygienic conditions applied during production are weak, and processing control is Inadequate and insufficient in addition to the posed risks due to using of moldy grape for pekmez preparation (Arici et al., 2004). Mycotoxins are one of the main secondary metabolites of the mold which are associated with some issues such as cancer and disorders in animals and humans (Mousavi Khaneghah, Eş, Raeisi, & Fakhri, 2018). Therefore, there are global concerns regarding the contamination of food products by mycotoxin (Campagnollo et al., 2016; Khaneghah, Martins, von Hertwig, Bertoldo, & Sant’Ana, 2018a; Khaneghah, Fakhri, Raeisi, Armoon, & Sant'Ana, 2018b; Khaneghah, Fakhri, & Sant'Ana, 2018c; Amirahmadi, Shoeibi, Rastegar, Elmi, & Mousavi Khaneghah, 2017). More than 300–400 mycotoxins have been identified (Inoue, Nagatomi, Uyama, & Mochizuki, 2013; Majeed, Khaneghah, Kadmic, Khan, & Shariati, 2018; Rastegar et al., 2017). Aflatoxins (AFs) are one
Pekmez as one of the most popular traditional food products in eastern culture could provide some of the essential nutrient elements such as calcium and iron (Arslan, Yener, & Esin, 2005; Batu, 2005; Karababa & Develi Isikli, 2005). It is manufactured by boiling and concentrating of various fruit juices such as grape, fig, raisin, mulberry, apple, sugar beet and most commonly grape juice in open containers or under vacuum conditions (Arici, Gümüs, & Kara, 2004; Arslan et al., 2005; Batu, 2005; Karababa & Develi Isikli, 2005). In this regard, the name of fruit was usually mentioned before “pekmez” to identification such as grape pekmez and mulberry pekmez (Karababa & Develi Isikli, 2005). Grape pekmez contains large amounts of natural sugars (50–80%) including glucose, fructose and sucrose and minerals such as calcium (0.068–0.084%) and iron (0.015–0.01%), so that it can be used to treat anemia, in addition to providing a good source of vitamins (A, C, B2 and B1), organic acids and some antioxidant agents such as phenolic and flavonoids. Due to the high levels of monosaccharides (glucose and fructose), grape pekmez can be digested and adsorbed to
Corresponding author. Department of Nutrition and Food Safety, Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. Corresponding author. Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, Caixa Postal: 6121, Campinas, São Paulo, CEP 13083-862, Brazil. E-mail addresses: [email protected] (S. Ghadimi), [email protected], [email protected] (A.M. Khaneghah). ∗
∗∗
https://doi.org/10.1016/j.lwt.2019.01.001 Received 3 August 2018; Received in revised form 31 December 2018; Accepted 2 January 2019 Available online 04 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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of the most investigated types of mycotoxins which can be produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nemesis (Eslami, Mashak, Heshmati, Shokrzadeh, & Mozaffari Nejad, 2015; Mashak, Sohi, Heshmati, & Nejad, 2016; Mousavi Khaneghah, D Chaves, & Akbarirad, 2017, Mousavi Khaneghah et al., 2018). They could pose some adverse effects such as carcinogenic, mutagenic, estrogenic, thermogenic, teratogenic and immunosuppressive effects on animal and human health (Amirahmadi et al., 2017; Mousavi Khaneghah et al., 2018). Although 18 species of AFs have been identified and reported, most notably reported aflatoxins in nuts, cereals, dried fruit and soybean included Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1) and Aflatoxin G2 (AFG2) (Campagnollo et al., 2016; Yazdanpanah, Mohammadi, Abouhossain, & Cheraghali, 2005). The mycotoxins as toxic compounds were categorized by IARC (International Agency for Research on Cancer) into five groups (Group 1, 2A, 2B, 3 and 4) (Heshmati, Zohrevand, Mousavi Khaneghah, Nejad, & Sant’Ana, 2017; Ostry, Malir, Toman, & Grosse, 2017). In this regard, all aflatoxins including AFB1, AFB2, AFG1, AFG2, and AFM1 are classified as group 1; carcinogenic to humans (Ostry et al., 2017 Khaneghah et al., 2018b; Khaneghah et al., 2018c; Mahmood Fashandi, Abbasi, & Mousavi Khaneghah, 2018). The vast of conducted studies regarding occurrence and contamination of mycotoxins in grape and grape products such as pekmez were focused on ochratoxin A (OTA) and patulin (PAT) (Akdeniz, Ozden, & Alpertunga, 2013; Arici et al., 2004; Oteiza et al., 2017; Tosun, Yıldız, Obuz, & Seçkin, 2014). The information regarding the prevalence of AFs in pekmez is rare. However, the prevalence of these mycotoxins in other grape products has been reported. For instance, the AFs level in the raisin samples collected from different regions in Pakistan was reported as 0.05 ± 0.26 μg AFB1/kg (Asghar, Ahmed, & Iqbal, 2016). Also, according to Kollia, Kanapitsas, and Markaki (2014), the mean concentration of AFB1 in dried vine fruits samples collected from the Greek market was determined as 1.4 μg AFB1/Kg. Due to raised health concerns as a result of consumption of aflatoxin-contaminated pekmez particularly, while moldy grape was utilized, the evaluation of unit impact on the aflatoxin concentration in this product is a matter of concern. The investigations regarding the elimination of mycotoxins by using different techniques as well as the fate of mycotoxins including AFs during food processing have attracted notable attention (Khaneghah, Martins et al., 2018a; Khaneghah, Fakhri et al., 2018b; Campagnollo et al., 2016). In this regard, various studies have been carried out around the world (Arici et al., 2004; Fandohan et al., 2005; Inoue et al., 2013; Kabak, 2009; Kaushik, 2015; Khaneghah et al., 2018a; Mutungi, Lamuka, Arimi, Gathumbi, & Onyango, 2008). However, based on our knowledge, no study was conducted to evaluate the fate of aflatoxins during pekmez production procedure. In this context, the current study was aimed to investigate the effects of the unit operations of the pekmez production on the concentration of AFB1, AFB2, AFG1, and AFG2. Additionally, the effects of different types of clarifiers on removing of AFs were evaluated.
Fig. 1. Schematic illustration for the used procedure.
season (autumn of 2017) and transferred to the laboratory and stored at refrigerator (+4 to +8 °C). In order to confirm that the used grapes were not naturally contaminated, the concentration of AFs in these samples was determined according to 2.3 section. The AFs level in used grape was lower than lower than the limit of detection. 2.2. Pekmez production The schematic diagram for pekmez processing (similar to the conventional procedure) is demonstrated in Fig. 1. The current study was aimed to investigate the impact of processing on the concentration of aflatoxin on its reduction during pekmez and finding of naturally contaminated grape samples with the certain level of aflatoxin was a problem, therefore, inevitably utilized artificially spiked grapes were used. In the first step, stock solutions of AFB1 (1000 ng/mL) and AFB2, AFG1 and AFG2 (200 ng/mL) was prepared. In order to the preparation of artificially contaminated grape samples, 2.5, 5, and 7.5 μg/kg of AFB1 and 0.5, 1 and 1.5 μg/kg of AFB2, AFG1 and AFG2; 250, 500 and 750 μL of the stock solution was spiked into 100 g of grape samples. The samples spiked with AFs remained in refrigerated storage overnight because AFs could penetrate the internal parts of grape samples. Afterward, the samples were washed, crushed and pressed, clarified, and concentrated as the following of conventional procedure. For washing, grape samples (100 g) were immersed in 400 mL of tap water for 5 min and then rinsed with tap water for 10 s. With the aid of a commercial Waring blender (Pars Khazar, Tehran, Iran), the samples were completely crushed. Then crushed grape was also pressed through Whatman No. 2 filter paper to separate the skin and seeds. Obtained juice (80 g) was nominated as “must.” In the traditional production method of pekmez, a calcareous substance called “pekmez soil” or white soil which contains calcium carbonate (ca.90%) was added to “must” to reduces the natural acidity by precipitating inherently existing tartaric and malic acids as calcium tartrate and calcium malate. In this study, in addition to white soil, two other clarifiers, i.e., gelatin and bentonite were used for reduction of acidity as well as clarification.
2. Materials and methods 2.1. Chemical agents and sample collection The AFB1, AFB2, AFG1, and AFG2 standards were purchased from Sigma-Aldrich (St. Louis, MO, US). Methanol, acetonitrile, sodium chloride, phosphate buffer saline (PBS), gelatin and bentonite and other chemicals in analytical grade were obtained from Merck (Darmstadt, Germany). White soil (calcareous substance containing high amounts of CaCO3) with high purity (> 95%) was obtained from the mountains around Malayer (Hamadan, Iran). Immunoaffinity columns (IAC) for clean-up of aflatoxins (Puri-Fast AFLA BG IAC) were provided by Libios (Pontcharra-sur-Turdine, France). Seedless grape (Vitis vinifera) specimens (varieties of Asgari) were gathered from Malayer (Hamadan, Iran) vineyards during harvest 179
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Each clarifier (white soil, gelatin, or bentonite) was added to “must” in two levels of 1.5 g/100 g and 3 g/100 g. After addition of clarifier, “must” samples have remained for 24 h at room temperature. Then samples were filtered to separate liquid phase. In order to increase in the concentration as well as the Brix up to 68–72 °Bx, the obtained clear clarified “must” (65 g) was boiled in an open beaker at 100 °C for 10–15 min. This procedure was carried out in triplicate for each grape sample. In order to have an overview, approximately 22 g pekmez was obtained from 100 g grape.
Table 1 Linearity range, limit of detection (LOD) and limit of quantification (LOQ) for AFs in pekmez.
2.3. AFs determination In order to extraction of AFs, 40 mL of pure water, 160 mL of methanol and 5 g of sodium chloride were added to the homogenized sample grape (50 g), “must” (40 g) or pekmez (20 g) and then were mixed 5 min at high speed (Heshmati et al., 2017; Heshmati & Mozaffari Nejad, 2015). Afterward, the obtained mixture was passed through Whatman No. 4 filter paper. Then, 20 mL of filtrated solution was diluted with 130 mL of phosphate buffered saline (PBS). Diluted solution passed through IAC with a flow rate of 1 mL/min. Before the clean-up process, IAC was conditioned with PBS (10 mL) and placed at room temperature for stabilization. AFs were eluted from IAC with 1.5 mL methanol. Fifty μL of collected elution solution was injected into the HPLC to determine AFs. The total obtained AFs for in grape, “must” and pekmez was expressed in μg/kg dry weight of the sample. A Waters HPLC instrument (Milford, MA, USA) equipped with a fluorescence detector, a binary pump, autosampler and RP C18 analytical column (250 mm 4.6 mm, i.d., 5 mm) was utilized. During the measurement, the column oven temperature was set at 25 °C. The mobile phase included water/methanol/acetonitrile (6:3:2, v/v/v) was applied in the state of isocratic elution at a flow rate of 1 mL/min. The excitation and emission wavelengths of the detector were 365 nm and 333 nm, respectively (Heshmati et al., 2017).
Type of matrix
Type of aflatoxin
Range of linearity (μg/ kg)
R2
LOD (μg/kg)
LOQ (μg/kg)
Pekmez
AFB1 AFB2 AFG1 AFG2
0.8–22 0.3–22 0.5–20 0.2–50
0.993 0.997 0.996 0.993
0.07 0.04 0.05 0.03
0.23 0.13 0.17 0.10
Grape juice (must)
AFB1 AFB2 AFG1 AFG2
0.5–25 0.2–25 0.2–20 0.2–50
0.996 0.998 0.997 0.996
0.05 0.02 0.02 0.02
0.17 0.08 0.08 0.08
Grape
AFB1 AFB2 AFG1 AFG2
1–20 0.5–20 0.5–20 0.2–50
0.991 0.994 0.992 0.990
0.08 0.06 0.07 0.05
0.26 0.20 0.24 0.17
nominated to AFB1, AFB2, AFG1, and AFG2 were reported in Table 2. The mean recovery and the relative standard deviation (%RSD) obtained for different levels of AFs spiked blank pekmez samples were reported as 103.6–110.7% (1.7–2.5%) for AFB1, 88.4–96.1% (1.5–4.0%), for AFB2, 77.0–88.1% (2.7–4.3%) for AFG1, 83.9–89.1% (3.4–4.3%) for AFG2. The data regarding the recovery percentages of AFs from “must,” and grape samples were presented in Table 2. A high coefficient of determination (R2) of measured AFs and good recovery for spiked samples were obtained. In this regard, the established method for AFs analysis had a suitable performance. Similar recovery rates for AFs in mulberry, date, fig, and apricot samples were previously reported by our group (Heshmati et al., 2017). The performance of analysis method used in the current study was confirmed while compared with AOAC, Codex Alimentarius and European Commission guidelines (AOAC International, 2002; Bircan, 2009; Codex Alimentarius, 1995; European Commission, 2006; Ibáñez-Vea, Martínez, González-Peñas, Lizarraga, & de Cerain, 2011).
2.4. Analytical method validation Validation parameters including accuracy, precision, linearity, limit of detection (LOD), and limit of quantification (LOQ) were measured in a similar way of our previous study with brief modifications in the levels of spiked concentrations of AFB1, AFB2, AFG1, and AFG2 (Heshmati et al., 2017). For accuracy assessment, AFB1 in three levels of 2.5, 5 and 7.5 μg/kg and AFB2, AFG1 and AFG2 in three levels of 0.5, 1 and 1.5 μg/ kg was spiked to grape, “must”, and pekmez samples and their recovery percentages, and relative standard deviation percentages (RSD%) were determined. The spiking of AFs and their measurement was repeated for three consecutive days to obtain method precision. The LOD and LOQ were determined as three and ten times of the signal to blank noise, respectively.
3.2. Impact of unit operation During conversion of grape to pekmez, the amount of moisture was reduced. In order to harmonize the effects of each processing step on AFs reduction, the measured concentration of AFs in each stage of pekmez production was reported based on dry weight values. While the dry weight values can be considered as fixed values, therefore, a better judgment on the results can be achieved. The effects of different stages of pekmez production on the levels of AFs were presented in Table 3. Results showed that all stages could reduce the concentrations of AFB1, AFB2, AFG1, and AFG2. Also, significant differences in AFs concentrations were noted among various stages (P < 0.05). Considering to Tables 4 and 5, depending on the initial spiked concentrations of AFB1 (2.5, 5 and 7.5 μg/kg) and AFB2, AFG1 and AFG2 (0.5, 1 and 1.5 μg/kg), their reduction range (average) during washing step were measured as 12.0–28.0 (19.3%), 12.7–34.0% (22.2%), 14.0–30.0% (23.7%) and 26.0–44.0% (34.3%), respectively. This fact that a significant part of AFs remained in the grape following the washing stage implied that the remaining AFs had been infiltrated internal parts of grape. No published information was available regarding the effects of the washing process on the reduction of AFs in grape pekmez. However, some trends were conducted to eliminate aflatoxins and ochratoxin A in black and white pepper by simple washing (Jalili, Jinap, & Son, 2011). Besides, Arici et al. (2004) investigated the fate of ochratoxin A during the pekmez processing; however, according to their results washing has no significant effect on the reduction of mycotoxin in pekmez. The effects of the washing on the removal of patulin
2.5. Statistical analysis Statistical analysis was performed using SPSS Version 16:00 (SPSS Inc., Chicago, IL, USA). Data analysis was done by using One-way analysis of variance (One-way ANOVA) test followed by a Duncan's Multiple Range. Results were considered significant at P < 0.05. 3. Result and discussion 3.1. Method validation The LOD and LOQ for AFB1, AFB2, AFG1, and AFG2 in grape, “must,” and pekmez samples were shown in Table 1. The LODs of the AFB1, AFB2, AFG1, and AFG2 for pekmez samples were reported as 0.07, 0.04, 0.05, and 0.03 μg/kg, respectively. The LOQs of AFB1, AFB2, AFG1, and AFG2 for pekmez samples were determined as 0.23, 0.13, 0.17, and 0.10 μg/kg, respectively. Moreover, the recovery percentages 180
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Table 2 Average recovery percentages for various concentrations of AFs (μg/kg) in pekmez, “must” and grape. Aflatoxin type
Spiked concentration (μg/kg)
Pekmez
“Must”
Grape
Mean (%)
RSD (%)
Mean (%)
RSD (%)
Mean (%)
RSD (%)
AFB1
2.5 5.0 7.5
110.7 103.6 104.0
1.7 2.5 2.3
108.6 102.4 102.1
1.5 2.3 2.1
111.4 101.1 102.5
1.9 3.5 3.4
AFB2
0.5 1.0 1.5
96.1 88.4 89.3
1.5 4.0 2.6
95.4 87.6 85.4
1.4 3.8 2.4
94.2 84.4 87.3
1.8 4.3 2.2
AFG1
0.5 1.0 1.5
88.1 77.0 78.8
4.3 2.7 3.3
87.2 77.6 79.4
4.2 2.2 3.1
88.5 74.6 75.6
4.0 1.5 2.4
AFG2
0.5 1.0 1.5
88.6 83.9 89.1
3.4 4.2 4.3
89.0 83.2 88.0
3.3 4.1 4.1
85.3 82.5 87.4
2.7 4.4 3.3
in apple during juice production in a previously published investigation were remarked (Sant’Ana, Rosenthal, & de Massaguer, 2008). In this context, previous studies showed washing could reduce approximately 80% of patulin during apple juice production (Sant’Ana et al., 2008).
During washing, AFs are transferred into the water and removed (Fandohan et al., 2005). Parameters such as partition coefficient or hydrophobicity (log P) and solubility of AFs could play important roles in washing effectiveness. Hydrophobicity (log P) of AFB1, AFB2, and
Table 3 The changes in AFs concentration (μg/kg) during the pekmez processing stages. Processing stage
AFB1
AFB2
Initial concentration (μg/kg) a
Initial concentration (μg/kg)
a
2.5
5
W W/P W/P/B1.5 W/P/B3 W/P/S1.5 W/P/S3 W/P/Ge1.5 W/P/Ge3 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/Ge1.5/C W/P/Ge3/C
1.8 ± 0.2b 1.6 ± 0.1c 1.2 ± 0.2d 0.8 ± 0.1fg 1.0 ± 0.1e 0.5 ± 0.1hi 0.6 ± 0.1fgh 0.4 ± < 0.1hij 1.0 ± 0.1e 0.6 ± 0.1ghi 0.8 ± 0.1ef 0.4 ± 0.1ij 0.5 ± 0.1hij 0.3 ± 0.1j
4.1 3.8 3.0 2.2 2.9 2.0 2.1 1.9 2.6 1.9 2.6 1.7 1.8 1.6
Processing stage
AFG1
a
7.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2b 0.1c 0.1d 0.1f 0.1d 0.2gh < 0.1fg 0.1gh 0.1e 0.2gh 0.2e 0.1ij 0.1hi 0.1j
6.6 6.3 4.9 4.0 4.2 3.4 3.7 3.2 4.6 3.8 4.0 3.1 3.3 2.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.5b 0.6b 0.6c 0.2de 0.3d 0.3fg 0.1ef 0.1fg 0.1cd 0.1ef 0.1de 0.3fg 0.3fg 0.2g
W W/P W/P/B1.5 W/P/B3 W/P/S1.5 W/P/S3 W/P/Ge1.5 W/P/Ge3 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/Ge1.5/C W/P/Ge3/C
1a
1.5a
0.4 ± < 0.1b 0.3 ± < 0.1 0.2 ± < 0.1 < LOD 0.2 ± < 0.1 < LOD 0.2 ± < 0.1 < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.8 ± 0.1b 0.7 ± 0.1c 0.5 ± 0.1d 0.4 ± < 0.1e 0.4 ± < 0.1de 0.3 ± < 0.1fgh 0.3 ± < 0.1fgh 0.2 ± < 0.1ghi 0.3 ± < 0.1ef 0.3 ± < 0.1fgh 0.3 ± < 0.1fg 0.2 ± < 0.1hi 0.2 ± < 0.1ghi 0.2 ± < 0.1i
1.3 ± < 0.1b 1.2 ± < 0.1c 0.9 ± < 0.1d 0.8 ± < 0.1ef 0.8 ± < 0.1e 0.6 ± < 0.1ghi 0.7 ± < 0.1fg 0.6 ± 0.1hi 0.8 ± < 0.1e 0.7 ± < 0.1fg 0.7 ± < 0.1fg 0.6 ± < 0.1hi 0.6 ± < 0.1gh 0.6 ± < 0.1i
AFG2
Initial concentration (μg/kg) a
0.5a
a
Initial concentration (μg/kgL) a
0.5
1
1.5
0.5a
1a
1.5a
0.4 ± < 0.1b 0.3 ± < 0.1c 0.2 ± < 0.1d < LOD 0.2 ± < 0.1d < LOD 0.2 ± < 0.1d < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.7 ± 0.1b 0.6 ± 0.1c 0.4 ± 0.1d 0.4 ± < 0.1de 0.4 ± < 0.1de 0.3 ± < 0.1fgh 0.3 ± < 0.1fgh 0.2 ± < 0.1ghi 0.3 ± < 0.1ef 0.3 ± < 0.1fgh 0.3 ± < 0.1fg 0.2 ± < 0.1hi 0.2 ± < 0.1ghi 0.2 ± < 0.1i
1.3 ± < 0.1b 1.2 ± < 0.1c 0.9 ± < 0.1d 0.7 ± < 0.1ef 0.8 ± < 0.1e 0.6 ± < 0.1ghi 0.7 ± < 0.1fg 0.6 ± 0.1hi 0.8 ± < 0.1e 0.6 ± < 0.1fg 0.7 ± < 0.1fg 0.6 ± < 0.1hi 0.6 ± < 0.1gh 0.5 ± < 0.1i
0.3 ± < 0.1b 0.2 ± < 0.1c 0.1 ± < 0.1d 0.1 ± < 0.1d 0.1 ± < 0.1d < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.7 ± < 0.1b 0.5 ± < 0.1c 0.3 ± < 0.1d 0.3 ± < 0.1e 0.3 ± < 0.1e 0.2 ± < 0.1h 0.2 ± < 0.1g 0.1 ± < 0.1h 0.2 ± < 0.1f 0.2 ± < 0.1g 0.2 ± < 0.1fg 0.1 ± < 0.1h 0.1 ± < 0.1h < LODi
1.1 ± 1.0 ± 0.7 ± 0.5 ± 0.5 ± 0.4 ± 0.4 ± 0.4 ± 0.6 ± 0.4 ± 0.4 ± 0.3 ± 0.4 ± 0.3 ±
< 0.1b < 0.1c < 0.1d < 0.1e < 0.1e < 0.1gh < 0.1fg < 0.1gh < 0.1e < 0.1f < 0.1f < 0.1ij < 0.1hi < 0.1j
Different letters within each column show significant difference (P < 0.05). W: Washing stage; P: Crushing and pressing stage; B1.5: Bleaching stage by bentonite(1.5 g/100 g); B3: Bleaching stage by bentonite (3 g/100 g); S1.5: Bleaching stage by white soil (1.5 g/100 g); S3: Bleaching stage by white soil (3 g/100 g); 0.5: G1.5: Bleaching stage by gelatin (1.5 g/100 g); G3: Bleaching stage by gelatin (3 g/ 100 g); C:Concentration stage. 181
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Table 4 Impact of each unit operation on the AFs reduction (%) during pekmez processing. The type of applied unit operation
AFB1
AFB2
Initial concentration (μg/kg)
Average
2.5
5
7.5
W P B1.5 B3 S1.5 S3 G1.5 G3 CB1.5 CB3 CS1.5 CS3 CG1.5 CG3
28.0 ± 3.1 11.1 ± 1.7 25.0 ± 3.8 52.5 ± < 0.1 39.4 ± 5.6 66.9 ± < 0.1 61.9 ± 8.02 73.1 ± < 0.1 19.2 ± < 0.1 23.7 ± < 0.1 18.6 ± < 0.1 22.6 ± < 0.1 24.6 ± < 0.1 25.6 ± < 0.1
18.0 ± 1.9 7.3 ± 1.0 21.3 ± 2.8 41.3 ± 4.5 22.1 ± 2.9 47.1 ± 5.3 44.5 ± 5.2 50.0 ± 5.2 12.0 ± 1.4 13.0 ± 1.5 12.5 ± 1.5 14.9 ± 1.7 14.2 ± 1.7 16.8 ± 1.7
12.0 ± 1.3 6.1 ± 1.1 21.6 ± 2.9 35.6 ± 4.2 31.9 ± 4.2 45.6 ± 5.4 41.6 ± 5.2 49.5 ± 5.1 7.2 ± 0.9 8.3 ± 1.0 6.2 ± 0.8 8.38 ± 1.0 8.01 ± 1.0 9.9 ± 1.0
The type of applied unit operation
AFG1
0.5
1
34.0 ± 4.1 27.3 ± 3.7 37.5 ± 6.3 100.0 ± < 0.1 33.3 ± 5.8 100.0 ± < 0.1 41.7 ± 6.7 100.0 ± < 0.1 100.0 ± < 0.1 ND 100.0 ± < 0.1 ND 100.0 ± < 0.1 ND
20.0 12.5 27.1 37.1 44.3 61.4 55.7 68.6 39.2 38.6 28.2 22.2 22.6 13.6
Average 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.7 3.3 4.4 5.4 7.0 6.6 7.4 4.5 4.3 3.2 2.6 2.5 1.4
12.7 ± 1.4 4.6 ± 1.0 28.0 ± 3.6 40.0 ± 4.8 37.6 ± 4.8 48.0 ± 5.7 42.4 ± 5.3 48.8 ± 5.2 13.3 ± 1.5 6.7 ± 0.9 10.3 ± 1.1 4.6 ± 0.7 9.7 ± 1.2 9.4 ± 0.9
22.2 ± 2.4ab 14.8 ± 1.9b 30.9 ± 3.9ab 59.0 ± 6.4a 38.4 ± 4.8ab 69.8 ± 7.6a 46.6 ± 5.6ab 72.5 ± 7.9a 50.9 ± 5.3ab 15.1 ± 2.1b 46.2 ± 4.7ab 9.0 ± 1.5b 44.1 ± 4.5ab 7.97 ± 0.8b
AFG2
Initial concentration (μg/kg)
W P B1.5 B3 S1.5 S3 G1.5 G3 CB1.5 CB3 CS1.5 CS3 CG1.5 CG3
19.3 ± 2.0d 8.2 ± 1.1d 22.6 ± 2.8cd 43.2 ± 4.7ab 31.1 ± 3.8bc 53.2 ± 6.0a 49.3 ± 5.7a 57.6 ± 6.0a 12.8 ± 1.5d 15.0 ± 1.7d 12.4 ± 1.4d 15.3 ± 1.7d 15.6 ± 1.8d 17.4 ± 1.7d
Initial concentration (μg/kg)
0.5
1
30.0 ± 3.4 17.1 ± 2.4 29.2 ± 5.4 100.0 ± < 0.1 29.2 ± 5.4 100.0 ± < 0.1 37.5 ± 6.2 100.0 ± < 0.1 ND ND ND ND ND ND
27.0 13.7 35.7 42.9 42.9 58.6 55.7 64.3 24.4 25.0 20.0 20.7 22.6 20.0
Average 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 1.9 4.2 4.9 5.2 6.7 6.5 6.8 2.8 2.8 2.3 2.4 2.6 2.0
14.0 ± 1.5 6.2 ± 1.3 31.0 ± 4.0 43.2 ± 5.2 40.0 ± 5.1 51.2 ± 6.0 45.6 ± 5.7 53.6 ± 5.6 10.5 ± 1.2 7.0 ± 0.9 10.7 ± 1.2 6.7 ± 0.8 7.4 ± 0.9 6.9 ± 0.7
23.7 ± 2.5cd 12.4 ± 1.6d 32.0 ± 4.0cd 62.0 ± 6.7ab 37.3 ± 4.6bcd 69.9 ± 7.6a 46.3 ± 5.6abc 72.6 ± 7.4a 11.6 ± 1.3d 10.7 ± 1.2d 10.27 ± 1.1d 9.1 ± 1.0d 10.0 ± 1.1d 9.0 ± 0.9d
Initial concentration (μg/kg)
Average
0.5
1
1.5
44.0 ± 4.8 14.0 ± 2.6 47.6 ± 6.1 52.4 ± < 0.1 52.4 ± 7.74 100.0 ± < 0.1 100.0 ± 12.5 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 1000. ± < 0.1 ND ND ND
33.0 ± 3.7 26.0 ± 3.2 40.7 ± 4.8 50 ± 5.7 48.2 ± 5.9 70.4 ± 8.0 63.0 ± 7.4 74.1 ± 7.8 28.1 ± 3.3 29.6 ± 3.3 21.4 ± 2.4 18.8 ± 2.3 30.0 ± 4.5 100.0 ± 10.0
26.0 28.0 28.9 43.3 43.3 59.8 56.7 59.8 15.9 21.8 18.2 18.0 16.7 25.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2 3.4 3.8 5.2 5.6 7.2 6.93 6.3 2.0 2.6 2.2 2.1 2.2 2.6
34.3 22.7 39.1 48.6 47.9 76.7 73.2 78.0 48.0 50.5 46.5 12.2 15.6 41.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.7abc 2.8bc 4.5abc 5.7abc 5.8abc 8.6a 8.3a 8.4a 5.5abc 5.6abc 4.8abc 1.4c 2.1c 4.2abc
W: Washing stage; P: Crushing and pressing stage; B1.5: Bleaching stage by bentonite (1.5 g/100 g); B3: Bleaching stage by bentonite (3 g/100 g); S1.5: Bleaching stage by white soil (1.5 g/100 g); S3: Bleaching stage by white soil (3 g/100 g); 0.5: G1.5: Bleaching stage by gelatin (1.5 g/100 g); G3: Bleaching stage by gelatin (3 g/ 100 g); C:Concentration stage. Different letters indicated significant difference among average value of reduction percentage within each column (P < 0.05). ND: not detected.
AFG1 were reported as 1.73, 1.63 and 1.81 and their solubility in water is 233, 392 and 424 mg/L, respectively (Karlovsky et al., 2016). Based on the findings of the current study, crushing and pressing had a low effect on the removing of AFs. According to Tables 4 and 5, The reduction range (average) of AFB1, AFB2, AFG1 and AFG2 during crushing and pressing processing of pekmez was 6.1–11.1% (8.2%), 4.6–27.3% (14.8%), 6.2–17.1% (12.4%) and 14.0–28.0% (22.7%). Due to some AFs was located in the skin of the grape, it seems that skin removing could result in AFs reduction. The impact of the pressing step on mycotoxin also is varied. In a previously conducted investigation, pressing of pomace after primary alcoholic fermentation during wine production resulted in more than four times reduction in the concentration of ochratoxin A (OTA), mainly due to OTA adsorption by the solid substances (Gil-Serna, Vázquez, González-Jaén, & Patiño, 2018). According to Tables 4 and 5, mean reduction (range) of AFB1, AFB2, AFG1 and AFG2 of pekmez during boiling and concentration stage was measured as 14.8% (6.2–25.6%), 34.6% (4.6–100.0%), 15.1% (6.7–25.0%) and 42.9% (15.6–100.0%), respectively. So far, there is no published information considering the impact of the boiling and concentration process on the reduction of aflatoxins in pekmez. However, Arici et al. (2004) found that boiling and concentration process increased the amount of ochratoxin A, about 5–6 times higher than the
original concentration in grape juice (they did not mention the temperature and length of boiling/concentration and their data was reported based on wet weight), while our results indicated AFs decreased during the boiling and concentration process (our data was reported based dry weight). The effects of the boiling and concentration process on aflatoxin and another mycotoxin in fruit juice and other food products have been studied (Çetin & Sebahattin, 2003; Sant’Ana et al., 2008). For example, according to a study by Çetin and Sebahattin (2003), heat treatment (about 90 and 100 °C for 20 min) reduced 18.81 and 25.99% of patulin concentration in apple juice. The concentrations of patulin were reduced as a result of heat treatment (evaporation) at 70 and 80 °C for 20 min about 9.4% and 14.06%, respectively. The reduction of patulin during the concentration step of apple juice depended on heating temperature and time used (Sant’Ana et al., 2008). Based on the findings of Mutungi et al. (2008), boiling of muthokoi at 98 °C for 150 min resulted in a reduction of 80–93% of aflatoxin. Raters and Matissek (2008) demonstrated that AFB1 could be degraded at temperatures above 160 °C. Meanwhile, several studies have been reported mycotoxin stability during thermal processes (Yazdanpanah et al., 2005). These differences can be associated with the temperature and the heating time applied in preparing of different food products.
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Table 5 The total reduction (%) of AFs during pekmez processing. The whole unit operations applied for pekmez production
AFB1
Average
Initial concentration (μg/kg) 2.5
W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/G1.5/C W/P/G3/C
61.2 76.8 68.4 83.6 81.6 87.2
The whole unit operations applied for pekmez production
AFG1
5 ± ± ± ± ± ±
3.4 2.9 3.2 3.0 5.0 3.0
47.4 61.2 48.2 65.8 63.8 68.4
7.5 ± ± ± ± ± ±
1.4 3.2 2.9 2.6 2.5 1.9
39.9 51.2 47.2 58.8 55.6 62.4
± ± ± ± ± ±
0.4 1.2 0.9 4.2 4.6 2.8
49.5 63.1 54.6 69.4 67.0 72.7
± ± ± ± ± ±
3.5c 6.5b 5.4c 5.4a 3.5ab 4.1a
74.7 ± 65.7 ± 70.3 ± 68.0 ± 76.7 ± 75.7 ±
< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
61.3 48.9 56.2 55.3 61.8 58.2
3.1 4.2 2.5 4.6 2.1 3.5
1
100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 1000.0 ± < 0.1
65.7 70.3 68.0 76.7 75.7 79.7
1.5 ± ± ± ± ± ±
4.2 2.6 4.6 2.1 3.5 1.5
48.9 56.2 55.3 61.8 58.2 63.8
± ± ± ± ± ±
1.0 3.0 2.4 3.4 1.7 2.3
71.5 75.5 74.4 79.5 78.0 81.2
± ± ± ± ± ±
3.5b 6.5ab 5.4ab 5.4a 3.4ab 4.1a
Average
Initial concentration (μg/kg) 1.5
± ± ± ± ± ±
0.5
AFG2
Initial concentration (μg/kg) 1
Average
Initial concentration (μg/kg)
Average
0.5 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/G1.5/C W/P/G3/C
AFB2
61.3 48.9 56.2 55.3 61.8 58.2
± ± ± ± ± ±
4.4 1.0 3.0 2.4 3.4 1.7
78.7 71.5 75.5 74.4 79.5 78.0
± ± ± ± ± ±
4.3a 5.4b 2.7ab 4.3ab 3.4a 4.1a
0.5
1
1.5
77.0 ± < 0.1 83.0 ± < 0.1 80.0 ± < 0.1 89.0 ± < 0.1 87.0 ± < 0.1 100.0 ± < 0.1
64.0 ± 1.5 72.7 ± 2.0 70.0 ± 1.5 79.3 ± 2.1 76.7 ± 1.2 82.0 ± < 0.1
64.0 ± 2.5 72.7 ± 1.4 70.0 ± 0.7 79.3 ± 1.4 76.7 ± 1.7 82.0. ± 1.4
80.3 85.2 83.3 89.4 87.9 94.0
± ± ± ± ± ±
2.5b 2.9ab 4.3ab 3. 9a 4.2a 5.1a
Different letters indicated significant difference among average value of reduction percentage within each column (P < 0.05). W/P/B1.5/C: The stages of washing + crushing and pressing + bleaching by bentonite (1.5 g/100 g) + concentration. W/P/B3/C: The stages of washing + crushing and pressing + bleaching by bentonite (3 g/100 g) + concentration. W/P/S1.5/C: The stages of washing + crushing and pressing + bleaching by white soil (1.5 g/100 g) + concentration. W/P/S3/C: The stages of washing + crushing and pressing + bleaching by white soil (3 g/100 g) + concentration. W/P/G1.5/C: The stages of washing + crushing and pressing + bleaching by gelatin (1.5 g/100 g) + concentration. W/P/G3/C: The stages of washing + crushing and pressing + bleaching by gelatin (3 g/100 g) + concentration.
3.3. Effect of clarifier
commonly used to prevent AFs intestinal absorption (Savari et al., 2013; Thieu & Pettersson, 2008). Bentonite that was often known as montmorillonite is a claylike substance with a volcanic origin (Huwig et al., 2001). It is one of the most used adsorbents for clarification in the beverage industry and decoloration of edible oils (Tahir & Rauf, 2006). Also, the utilization of bentonite resulted in a 3.7% reduction of ochratoxin A in wine (Var et al., 2008). Savari et al. (2013) found that AFs adsorption by clay could be correlated with the formation of binding between β-dicarbonyl part of aflatoxin and metal ions of clay. Calcium carbonate comprises 90% of the white soil and led to the alkalization of “must.” Doyle, Applebaum, Brackett, and Marth (1982) showed that the hydrolysis and oxidation of the lactone ring of aflatoxin are carried out in alkaline medium, which consequently could result in the elimination of aflatoxin. In the current study by increasing the concentrations of clarifiers added to “must,” the absorption and reduction of AFs increased. The inconsistency in density, the size, the diameter of the particles of adsorbents and different mechanism of AFs absorption had the impacts on the capacity of these compounds for AFs removal (Kannewischer, Arvide, White, & Dixon, 2006). Thieu and Pettersson (2008) declared the difference between absorption capacity of various type of zeolite and bentonite in adsorption of aflatoxin B1 can be correlated to the difference in the process and the purification of these materials. On the other hand, the individual adsorbent material obtained from each mine may have a different chemical composition and properties depending on the extraction location.
Among unit processing used during pekmez production, the highest effect on the AFs reduction was correlated with clarification (Tables 4 and 5). The AFs reduction during this stage depended on the type and concentration of clarifying agents as well as initial aflatoxin concentration. By increasing in the clarifier levels from 1 to 3 g/100 g, AFs reduction was significantly raised (P < 0.05). In the samples, which contain 0.5 μg/kg AFB2, AFG1 or AFG2, after incorporation of 3 g gelatin, bentonite, or white soil to 100 g “must,” AFs was lower than LOD. As shown in Tables 4 and 5, the average reduction of AFs during clarification by bentonite, white soil and gelatin was varied; 43.2%, 53.2%, and 57.6 for AFB1; 59.0%, 69.8% and 72.5%, for AFB2, 62.0%, 69.9% and 72.6% for AFG1 and 48.6%, 76.7%, and 78.0% for AFG2, respectively. Therefore, these findings indicated that gelatin had the highest impact on AFS removal while compared with white soil and bentonite. Also, bentonite with a concentration of 1.5 g/100 g showed the lowest effect on the AFs reduction. There is not much information regarding the effects of gelatin in reducing AFs. In a previous study, the utilization of gelatin/bentonite mixture for clarification of apple juice with ultra-filtration processing caused a reduction of about 25% in the patulin level (Acar, Gökmen, & Taydas, 1998). The role and mechanism of the reduction of aflatoxin by gelatin are unclear although it was assumed that gelatin in water formed a gel network which able to trap and further adsorption of AFs. The utilization of commercial adsorbents such as sodium bentonite, cholestyramine crospovidone, montmorillonite, activated carbon (AC), zeolites and hydrated sodium calcium aluminosilicate (HSCAS) for the elimination of mycotoxin is one of the common strategies (Huwig, Freimund, Käppeli, & Dutler, 2001; Var, Kabak, & Erginkaya, 2008). These compounds inhibit the absorption of aflatoxins in the digestive tract and reduce their bioavailability (Ramos, Hernandez, Pla-Delfina, & Merino, 1996; Savari, Dehghanbanadaki, Rezayzady, & Javannikkhah, 2013). Among various adsorbents, bentonite is
4. Conclusions The current study was the first attempt to investigate the fate of aflatoxins during pekmez production processing stages. In order to have accurate results the artificially spiked grape samples in levels of 2.5, 5 and 7.5 μg/kg AFB1 and 0.5, 1 and 1.5 μg/kg of aflatoxin AFB2, AFG1 and AFG2, and the conventional process of pekmez production were 183
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used. The levels of AFB1, AFB2, AFG1, and AFG2 during pekmez production process were decreased by 60.4, 76.7, 76.3 and 86.7%, respectively. Therefore, the highest and lowest reductions during the pekmez production process can be associated with AFG2 and AFB1, respectively. In this regard, all of the steps investigated are effective in the reduction of AFs. However, AFs reduction percentage during pekmez depended on their initial contamination levels and type and content of added clarifier. Among assessed clarifier (gelatin, bentonite and white soil), gelatin demonstrated the highest efficiency in the elimination of AFs from pekmez.
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Conflicts of interest No potential conflict of interest was reported by the authors. Acknowledgements The authors thank Vice-Chancellor of Research and Technology of Hamadan University of Medical Sciences and Health services for financial support from this project No. 9605103036. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.01.001. References Acar, J., Gökmen, V., & Taydas, E. E. (1998). The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production. Zeitschrift für Lebensmittel-Untersuchung und -Forschung A, 207(4), 328–331. Akdeniz, A. S., Ozden, S., & Alpertunga, B. (2013). Ochratoxin A in dried grapes and grape-derived products in Turkey. Food Additives and Contaminants: Part B, 6(4), 265–269. Amirahmadi, M., Shoeibi, S., Rastegar, H., Elmi, M., & Mousavi Khaneghah, A. (2017). Simultaneous analysis of mycotoxins in corn flour using LC/MS-MS combined with a modified QuEChERS procedure. Toxin Reviews, 37(3), 187–195. AOAC International (2002). AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals. AOAC International. Arici, M., Gümüs, T., & Kara, F. (2004). The fate of ochratoxin A during the Pekmez production from mouldy grapes. Food Control, 15(8), 597–600. https://doi.org/10. 1016/j.foodcont.2003.10.001. Arslan, E., Yener, M., & Esin, A. (2005). Rheological characterization of tahin/pekmez (sesame paste/concentrated grape juice) blends. Journal of food engineering, 69(2), 167–172. https://doi.org/10.1016/j.jfoodeng.2004.08.010. Asghar, M. A., Ahmed, A., & Iqbal, J. (2016). Aflatoxins and ochratoxin A in export quality raisins collected from different areas of Pakistan. Food Additives and Contaminants: Part B, 9(1), 51–58. Batu, A. (2005). Production of liquid and white solid pekmez in Turkey. Journal of Food Quality, 28(5‐6), 417–427. Bircan, C. (2009). The incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs. Food and Chemical Toxicology, 47(8), 1996–2001. https://doi. org/10.1016/j.fct.2009.05.008. Campagnollo, F. B., Ganev, K. C., Mousavi Khaneghah, A., Portela, J. B., Cruz, A. G., Granato, D., et al. (2016). The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: A review. Food Control, 68, 310–329. https://doi.org/10.1016/j.foodcont.2016.04.007. Çetin, K., & Sebahattin, N. (2003). Effect of heat treatment and evaporation on patulin and some other properties of apple juice. Journal of the Science of Food and Agriculture, 83(9), 987–990. https://doi.org/10.1002/jsfa.1339. Codex Alimentarius. Codex Standard for Contaminants and Toxins in Food and Feed. Codex standard. (1995). pp. 193e1995. Available at: http://www.codexalimentarius.net// download/standards/17/CXS_193e.pdf AOAC International, 2002. AOAC Guidelines for Single Laborato . Doyle, M., Applebaum, R., Brackett, R., & Marth, E. (1982). Physical, chemical and biological degradation of mycotoxins in foods and agricultural commodities. Journal of Food Protection, 45(10), 964–971. Eslami, M., Mashak, Z., Heshmati, A., Shokrzadeh, M., & Mozaffari Nejad, A. S. (2015). Determination of aflatoxin B1 levels in Iranian rice by ELISA method. Toxin Reviews, 34(3), 125–128. https://doi.org/10.3109/15569543.2015.1074925. European Commission (2006). Commission regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the level. Fandohan, P., Zoumenou, D., Hounhouigan, D., Marasas, W., Wingfield, M., & Hell, K. (2005). Fate of aflatoxins and fumonisins during the processing of maize into food products in Benin. International Journal of Food Microbiology, 98(3), 249–259. Gil-Serna, J., Vázquez, C., González-Jaén, M. T., & Patiño, B. (2018). Wine contamination with ochratoxins: A review. Beverages, 4(1), 6–27.
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37–39. Var, I., Kabak, B., & Erginkaya, Z. (2008). Reduction in ochratoxin A levels in white wine, following treatment with activated carbon and sodium bentonite. Food Control, 19(6), 592–598. https://doi.org/10.1016/j.foodcont.2007.06.013. Yazdanpanah, H., Mohammadi, T., Abouhossain, G., & Cheraghali, A. M. (2005). Effect of roasting on degradation of aflatoxins in contaminated pistachio nuts. Food and Chemical Toxicology, 43(7), 1135–1139.
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Changes in aflatoxins content during processing of pekmez as a traditional product of grape
T
Ali Heshmatia, Sabah Ghadimia,∗, Akram Ranjbara, Amin Mousavi Khaneghahb,∗∗ a
Department of Nutrition and Food Safety, Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, Caixa Postal: 6121, CEP 13083-862, Campinas, São Paulo, Brazil
b
ARTICLE INFO
ABSTRACT
Keywords: Mycotoxin Fate Clarifiers Grape Contamination Conventional process
The current study was aimed to investigate the changes in aflatoxins (AFs) content during the production of pekmez. In this context, the artificially spiked grape samples in levels of 2.5, 5, and 7.5 μg/kg of aflatoxin B1 (AFB1), and 0.5, 1 and 1.5 μg/kg of aflatoxin B2 (AFB2), G1 (AFG1) and G2 (AFG2) were processed by the conventional procedure and after each step, AFs levels were measured using HPLC-FD. Among different unit operations, the lowest reduction in AFs (8.2, 14.8, 12.4, and 22.7% for AFB1 and AFB2, AFG1, and AFG2, respectively) was obtained for crushing and pressing stages. The corresponded values for reduction average of AFB1, AFB2, AFG1, and AFG2 during washing step was measured as 19.3%, 22.2%, 23.7%, and 34.3%, respectively. Clarification stage was identified as the most effective step in the reduction (63.6%) of AFs. However, the reduction strongly depended on the type and concentration of clarifier as well as initial aflatoxin level. A significant difference between the assessed clarifiers (gelatin, bentonite and white soil) was observed, while the highest efficiency was demonstrated by gelatin. In general, levels of AFB1, AFB2, AFG1, and AFG2 during pekmez production were decreased about 60.4, 76.7, 76.3, and 86.7%, respectively.
1. Introduction
support immediate energy. Therefore, pekmez could play an important role in the diet of different age groups, especially children and athletes (Arslan et al., 2005; Batu, 2005). It is produced in non-industrial scale and rural areas (Batu, 2005; Karababa & Develi Isikli, 2005). In the conventional production method, the hygienic conditions applied during production are weak, and processing control is Inadequate and insufficient in addition to the posed risks due to using of moldy grape for pekmez preparation (Arici et al., 2004). Mycotoxins are one of the main secondary metabolites of the mold which are associated with some issues such as cancer and disorders in animals and humans (Mousavi Khaneghah, Eş, Raeisi, & Fakhri, 2018). Therefore, there are global concerns regarding the contamination of food products by mycotoxin (Campagnollo et al., 2016; Khaneghah, Martins, von Hertwig, Bertoldo, & Sant’Ana, 2018a; Khaneghah, Fakhri, Raeisi, Armoon, & Sant'Ana, 2018b; Khaneghah, Fakhri, & Sant'Ana, 2018c; Amirahmadi, Shoeibi, Rastegar, Elmi, & Mousavi Khaneghah, 2017). More than 300–400 mycotoxins have been identified (Inoue, Nagatomi, Uyama, & Mochizuki, 2013; Majeed, Khaneghah, Kadmic, Khan, & Shariati, 2018; Rastegar et al., 2017). Aflatoxins (AFs) are one
Pekmez as one of the most popular traditional food products in eastern culture could provide some of the essential nutrient elements such as calcium and iron (Arslan, Yener, & Esin, 2005; Batu, 2005; Karababa & Develi Isikli, 2005). It is manufactured by boiling and concentrating of various fruit juices such as grape, fig, raisin, mulberry, apple, sugar beet and most commonly grape juice in open containers or under vacuum conditions (Arici, Gümüs, & Kara, 2004; Arslan et al., 2005; Batu, 2005; Karababa & Develi Isikli, 2005). In this regard, the name of fruit was usually mentioned before “pekmez” to identification such as grape pekmez and mulberry pekmez (Karababa & Develi Isikli, 2005). Grape pekmez contains large amounts of natural sugars (50–80%) including glucose, fructose and sucrose and minerals such as calcium (0.068–0.084%) and iron (0.015–0.01%), so that it can be used to treat anemia, in addition to providing a good source of vitamins (A, C, B2 and B1), organic acids and some antioxidant agents such as phenolic and flavonoids. Due to the high levels of monosaccharides (glucose and fructose), grape pekmez can be digested and adsorbed to
Corresponding author. Department of Nutrition and Food Safety, Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. Corresponding author. Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Rua Monteiro Lobato, 80, Caixa Postal: 6121, Campinas, São Paulo, CEP 13083-862, Brazil. E-mail addresses: [email protected] (S. Ghadimi), [email protected], [email protected] (A.M. Khaneghah). ∗
∗∗
https://doi.org/10.1016/j.lwt.2019.01.001 Received 3 August 2018; Received in revised form 31 December 2018; Accepted 2 January 2019 Available online 04 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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of the most investigated types of mycotoxins which can be produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nemesis (Eslami, Mashak, Heshmati, Shokrzadeh, & Mozaffari Nejad, 2015; Mashak, Sohi, Heshmati, & Nejad, 2016; Mousavi Khaneghah, D Chaves, & Akbarirad, 2017, Mousavi Khaneghah et al., 2018). They could pose some adverse effects such as carcinogenic, mutagenic, estrogenic, thermogenic, teratogenic and immunosuppressive effects on animal and human health (Amirahmadi et al., 2017; Mousavi Khaneghah et al., 2018). Although 18 species of AFs have been identified and reported, most notably reported aflatoxins in nuts, cereals, dried fruit and soybean included Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1) and Aflatoxin G2 (AFG2) (Campagnollo et al., 2016; Yazdanpanah, Mohammadi, Abouhossain, & Cheraghali, 2005). The mycotoxins as toxic compounds were categorized by IARC (International Agency for Research on Cancer) into five groups (Group 1, 2A, 2B, 3 and 4) (Heshmati, Zohrevand, Mousavi Khaneghah, Nejad, & Sant’Ana, 2017; Ostry, Malir, Toman, & Grosse, 2017). In this regard, all aflatoxins including AFB1, AFB2, AFG1, AFG2, and AFM1 are classified as group 1; carcinogenic to humans (Ostry et al., 2017 Khaneghah et al., 2018b; Khaneghah et al., 2018c; Mahmood Fashandi, Abbasi, & Mousavi Khaneghah, 2018). The vast of conducted studies regarding occurrence and contamination of mycotoxins in grape and grape products such as pekmez were focused on ochratoxin A (OTA) and patulin (PAT) (Akdeniz, Ozden, & Alpertunga, 2013; Arici et al., 2004; Oteiza et al., 2017; Tosun, Yıldız, Obuz, & Seçkin, 2014). The information regarding the prevalence of AFs in pekmez is rare. However, the prevalence of these mycotoxins in other grape products has been reported. For instance, the AFs level in the raisin samples collected from different regions in Pakistan was reported as 0.05 ± 0.26 μg AFB1/kg (Asghar, Ahmed, & Iqbal, 2016). Also, according to Kollia, Kanapitsas, and Markaki (2014), the mean concentration of AFB1 in dried vine fruits samples collected from the Greek market was determined as 1.4 μg AFB1/Kg. Due to raised health concerns as a result of consumption of aflatoxin-contaminated pekmez particularly, while moldy grape was utilized, the evaluation of unit impact on the aflatoxin concentration in this product is a matter of concern. The investigations regarding the elimination of mycotoxins by using different techniques as well as the fate of mycotoxins including AFs during food processing have attracted notable attention (Khaneghah, Martins et al., 2018a; Khaneghah, Fakhri et al., 2018b; Campagnollo et al., 2016). In this regard, various studies have been carried out around the world (Arici et al., 2004; Fandohan et al., 2005; Inoue et al., 2013; Kabak, 2009; Kaushik, 2015; Khaneghah et al., 2018a; Mutungi, Lamuka, Arimi, Gathumbi, & Onyango, 2008). However, based on our knowledge, no study was conducted to evaluate the fate of aflatoxins during pekmez production procedure. In this context, the current study was aimed to investigate the effects of the unit operations of the pekmez production on the concentration of AFB1, AFB2, AFG1, and AFG2. Additionally, the effects of different types of clarifiers on removing of AFs were evaluated.
Fig. 1. Schematic illustration for the used procedure.
season (autumn of 2017) and transferred to the laboratory and stored at refrigerator (+4 to +8 °C). In order to confirm that the used grapes were not naturally contaminated, the concentration of AFs in these samples was determined according to 2.3 section. The AFs level in used grape was lower than lower than the limit of detection. 2.2. Pekmez production The schematic diagram for pekmez processing (similar to the conventional procedure) is demonstrated in Fig. 1. The current study was aimed to investigate the impact of processing on the concentration of aflatoxin on its reduction during pekmez and finding of naturally contaminated grape samples with the certain level of aflatoxin was a problem, therefore, inevitably utilized artificially spiked grapes were used. In the first step, stock solutions of AFB1 (1000 ng/mL) and AFB2, AFG1 and AFG2 (200 ng/mL) was prepared. In order to the preparation of artificially contaminated grape samples, 2.5, 5, and 7.5 μg/kg of AFB1 and 0.5, 1 and 1.5 μg/kg of AFB2, AFG1 and AFG2; 250, 500 and 750 μL of the stock solution was spiked into 100 g of grape samples. The samples spiked with AFs remained in refrigerated storage overnight because AFs could penetrate the internal parts of grape samples. Afterward, the samples were washed, crushed and pressed, clarified, and concentrated as the following of conventional procedure. For washing, grape samples (100 g) were immersed in 400 mL of tap water for 5 min and then rinsed with tap water for 10 s. With the aid of a commercial Waring blender (Pars Khazar, Tehran, Iran), the samples were completely crushed. Then crushed grape was also pressed through Whatman No. 2 filter paper to separate the skin and seeds. Obtained juice (80 g) was nominated as “must.” In the traditional production method of pekmez, a calcareous substance called “pekmez soil” or white soil which contains calcium carbonate (ca.90%) was added to “must” to reduces the natural acidity by precipitating inherently existing tartaric and malic acids as calcium tartrate and calcium malate. In this study, in addition to white soil, two other clarifiers, i.e., gelatin and bentonite were used for reduction of acidity as well as clarification.
2. Materials and methods 2.1. Chemical agents and sample collection The AFB1, AFB2, AFG1, and AFG2 standards were purchased from Sigma-Aldrich (St. Louis, MO, US). Methanol, acetonitrile, sodium chloride, phosphate buffer saline (PBS), gelatin and bentonite and other chemicals in analytical grade were obtained from Merck (Darmstadt, Germany). White soil (calcareous substance containing high amounts of CaCO3) with high purity (> 95%) was obtained from the mountains around Malayer (Hamadan, Iran). Immunoaffinity columns (IAC) for clean-up of aflatoxins (Puri-Fast AFLA BG IAC) were provided by Libios (Pontcharra-sur-Turdine, France). Seedless grape (Vitis vinifera) specimens (varieties of Asgari) were gathered from Malayer (Hamadan, Iran) vineyards during harvest 179
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Each clarifier (white soil, gelatin, or bentonite) was added to “must” in two levels of 1.5 g/100 g and 3 g/100 g. After addition of clarifier, “must” samples have remained for 24 h at room temperature. Then samples were filtered to separate liquid phase. In order to increase in the concentration as well as the Brix up to 68–72 °Bx, the obtained clear clarified “must” (65 g) was boiled in an open beaker at 100 °C for 10–15 min. This procedure was carried out in triplicate for each grape sample. In order to have an overview, approximately 22 g pekmez was obtained from 100 g grape.
Table 1 Linearity range, limit of detection (LOD) and limit of quantification (LOQ) for AFs in pekmez.
2.3. AFs determination In order to extraction of AFs, 40 mL of pure water, 160 mL of methanol and 5 g of sodium chloride were added to the homogenized sample grape (50 g), “must” (40 g) or pekmez (20 g) and then were mixed 5 min at high speed (Heshmati et al., 2017; Heshmati & Mozaffari Nejad, 2015). Afterward, the obtained mixture was passed through Whatman No. 4 filter paper. Then, 20 mL of filtrated solution was diluted with 130 mL of phosphate buffered saline (PBS). Diluted solution passed through IAC with a flow rate of 1 mL/min. Before the clean-up process, IAC was conditioned with PBS (10 mL) and placed at room temperature for stabilization. AFs were eluted from IAC with 1.5 mL methanol. Fifty μL of collected elution solution was injected into the HPLC to determine AFs. The total obtained AFs for in grape, “must” and pekmez was expressed in μg/kg dry weight of the sample. A Waters HPLC instrument (Milford, MA, USA) equipped with a fluorescence detector, a binary pump, autosampler and RP C18 analytical column (250 mm 4.6 mm, i.d., 5 mm) was utilized. During the measurement, the column oven temperature was set at 25 °C. The mobile phase included water/methanol/acetonitrile (6:3:2, v/v/v) was applied in the state of isocratic elution at a flow rate of 1 mL/min. The excitation and emission wavelengths of the detector were 365 nm and 333 nm, respectively (Heshmati et al., 2017).
Type of matrix
Type of aflatoxin
Range of linearity (μg/ kg)
R2
LOD (μg/kg)
LOQ (μg/kg)
Pekmez
AFB1 AFB2 AFG1 AFG2
0.8–22 0.3–22 0.5–20 0.2–50
0.993 0.997 0.996 0.993
0.07 0.04 0.05 0.03
0.23 0.13 0.17 0.10
Grape juice (must)
AFB1 AFB2 AFG1 AFG2
0.5–25 0.2–25 0.2–20 0.2–50
0.996 0.998 0.997 0.996
0.05 0.02 0.02 0.02
0.17 0.08 0.08 0.08
Grape
AFB1 AFB2 AFG1 AFG2
1–20 0.5–20 0.5–20 0.2–50
0.991 0.994 0.992 0.990
0.08 0.06 0.07 0.05
0.26 0.20 0.24 0.17
nominated to AFB1, AFB2, AFG1, and AFG2 were reported in Table 2. The mean recovery and the relative standard deviation (%RSD) obtained for different levels of AFs spiked blank pekmez samples were reported as 103.6–110.7% (1.7–2.5%) for AFB1, 88.4–96.1% (1.5–4.0%), for AFB2, 77.0–88.1% (2.7–4.3%) for AFG1, 83.9–89.1% (3.4–4.3%) for AFG2. The data regarding the recovery percentages of AFs from “must,” and grape samples were presented in Table 2. A high coefficient of determination (R2) of measured AFs and good recovery for spiked samples were obtained. In this regard, the established method for AFs analysis had a suitable performance. Similar recovery rates for AFs in mulberry, date, fig, and apricot samples were previously reported by our group (Heshmati et al., 2017). The performance of analysis method used in the current study was confirmed while compared with AOAC, Codex Alimentarius and European Commission guidelines (AOAC International, 2002; Bircan, 2009; Codex Alimentarius, 1995; European Commission, 2006; Ibáñez-Vea, Martínez, González-Peñas, Lizarraga, & de Cerain, 2011).
2.4. Analytical method validation Validation parameters including accuracy, precision, linearity, limit of detection (LOD), and limit of quantification (LOQ) were measured in a similar way of our previous study with brief modifications in the levels of spiked concentrations of AFB1, AFB2, AFG1, and AFG2 (Heshmati et al., 2017). For accuracy assessment, AFB1 in three levels of 2.5, 5 and 7.5 μg/kg and AFB2, AFG1 and AFG2 in three levels of 0.5, 1 and 1.5 μg/ kg was spiked to grape, “must”, and pekmez samples and their recovery percentages, and relative standard deviation percentages (RSD%) were determined. The spiking of AFs and their measurement was repeated for three consecutive days to obtain method precision. The LOD and LOQ were determined as three and ten times of the signal to blank noise, respectively.
3.2. Impact of unit operation During conversion of grape to pekmez, the amount of moisture was reduced. In order to harmonize the effects of each processing step on AFs reduction, the measured concentration of AFs in each stage of pekmez production was reported based on dry weight values. While the dry weight values can be considered as fixed values, therefore, a better judgment on the results can be achieved. The effects of different stages of pekmez production on the levels of AFs were presented in Table 3. Results showed that all stages could reduce the concentrations of AFB1, AFB2, AFG1, and AFG2. Also, significant differences in AFs concentrations were noted among various stages (P < 0.05). Considering to Tables 4 and 5, depending on the initial spiked concentrations of AFB1 (2.5, 5 and 7.5 μg/kg) and AFB2, AFG1 and AFG2 (0.5, 1 and 1.5 μg/kg), their reduction range (average) during washing step were measured as 12.0–28.0 (19.3%), 12.7–34.0% (22.2%), 14.0–30.0% (23.7%) and 26.0–44.0% (34.3%), respectively. This fact that a significant part of AFs remained in the grape following the washing stage implied that the remaining AFs had been infiltrated internal parts of grape. No published information was available regarding the effects of the washing process on the reduction of AFs in grape pekmez. However, some trends were conducted to eliminate aflatoxins and ochratoxin A in black and white pepper by simple washing (Jalili, Jinap, & Son, 2011). Besides, Arici et al. (2004) investigated the fate of ochratoxin A during the pekmez processing; however, according to their results washing has no significant effect on the reduction of mycotoxin in pekmez. The effects of the washing on the removal of patulin
2.5. Statistical analysis Statistical analysis was performed using SPSS Version 16:00 (SPSS Inc., Chicago, IL, USA). Data analysis was done by using One-way analysis of variance (One-way ANOVA) test followed by a Duncan's Multiple Range. Results were considered significant at P < 0.05. 3. Result and discussion 3.1. Method validation The LOD and LOQ for AFB1, AFB2, AFG1, and AFG2 in grape, “must,” and pekmez samples were shown in Table 1. The LODs of the AFB1, AFB2, AFG1, and AFG2 for pekmez samples were reported as 0.07, 0.04, 0.05, and 0.03 μg/kg, respectively. The LOQs of AFB1, AFB2, AFG1, and AFG2 for pekmez samples were determined as 0.23, 0.13, 0.17, and 0.10 μg/kg, respectively. Moreover, the recovery percentages 180
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Table 2 Average recovery percentages for various concentrations of AFs (μg/kg) in pekmez, “must” and grape. Aflatoxin type
Spiked concentration (μg/kg)
Pekmez
“Must”
Grape
Mean (%)
RSD (%)
Mean (%)
RSD (%)
Mean (%)
RSD (%)
AFB1
2.5 5.0 7.5
110.7 103.6 104.0
1.7 2.5 2.3
108.6 102.4 102.1
1.5 2.3 2.1
111.4 101.1 102.5
1.9 3.5 3.4
AFB2
0.5 1.0 1.5
96.1 88.4 89.3
1.5 4.0 2.6
95.4 87.6 85.4
1.4 3.8 2.4
94.2 84.4 87.3
1.8 4.3 2.2
AFG1
0.5 1.0 1.5
88.1 77.0 78.8
4.3 2.7 3.3
87.2 77.6 79.4
4.2 2.2 3.1
88.5 74.6 75.6
4.0 1.5 2.4
AFG2
0.5 1.0 1.5
88.6 83.9 89.1
3.4 4.2 4.3
89.0 83.2 88.0
3.3 4.1 4.1
85.3 82.5 87.4
2.7 4.4 3.3
in apple during juice production in a previously published investigation were remarked (Sant’Ana, Rosenthal, & de Massaguer, 2008). In this context, previous studies showed washing could reduce approximately 80% of patulin during apple juice production (Sant’Ana et al., 2008).
During washing, AFs are transferred into the water and removed (Fandohan et al., 2005). Parameters such as partition coefficient or hydrophobicity (log P) and solubility of AFs could play important roles in washing effectiveness. Hydrophobicity (log P) of AFB1, AFB2, and
Table 3 The changes in AFs concentration (μg/kg) during the pekmez processing stages. Processing stage
AFB1
AFB2
Initial concentration (μg/kg) a
Initial concentration (μg/kg)
a
2.5
5
W W/P W/P/B1.5 W/P/B3 W/P/S1.5 W/P/S3 W/P/Ge1.5 W/P/Ge3 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/Ge1.5/C W/P/Ge3/C
1.8 ± 0.2b 1.6 ± 0.1c 1.2 ± 0.2d 0.8 ± 0.1fg 1.0 ± 0.1e 0.5 ± 0.1hi 0.6 ± 0.1fgh 0.4 ± < 0.1hij 1.0 ± 0.1e 0.6 ± 0.1ghi 0.8 ± 0.1ef 0.4 ± 0.1ij 0.5 ± 0.1hij 0.3 ± 0.1j
4.1 3.8 3.0 2.2 2.9 2.0 2.1 1.9 2.6 1.9 2.6 1.7 1.8 1.6
Processing stage
AFG1
a
7.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2b 0.1c 0.1d 0.1f 0.1d 0.2gh < 0.1fg 0.1gh 0.1e 0.2gh 0.2e 0.1ij 0.1hi 0.1j
6.6 6.3 4.9 4.0 4.2 3.4 3.7 3.2 4.6 3.8 4.0 3.1 3.3 2.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.5b 0.6b 0.6c 0.2de 0.3d 0.3fg 0.1ef 0.1fg 0.1cd 0.1ef 0.1de 0.3fg 0.3fg 0.2g
W W/P W/P/B1.5 W/P/B3 W/P/S1.5 W/P/S3 W/P/Ge1.5 W/P/Ge3 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/Ge1.5/C W/P/Ge3/C
1a
1.5a
0.4 ± < 0.1b 0.3 ± < 0.1 0.2 ± < 0.1 < LOD 0.2 ± < 0.1 < LOD 0.2 ± < 0.1 < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.8 ± 0.1b 0.7 ± 0.1c 0.5 ± 0.1d 0.4 ± < 0.1e 0.4 ± < 0.1de 0.3 ± < 0.1fgh 0.3 ± < 0.1fgh 0.2 ± < 0.1ghi 0.3 ± < 0.1ef 0.3 ± < 0.1fgh 0.3 ± < 0.1fg 0.2 ± < 0.1hi 0.2 ± < 0.1ghi 0.2 ± < 0.1i
1.3 ± < 0.1b 1.2 ± < 0.1c 0.9 ± < 0.1d 0.8 ± < 0.1ef 0.8 ± < 0.1e 0.6 ± < 0.1ghi 0.7 ± < 0.1fg 0.6 ± 0.1hi 0.8 ± < 0.1e 0.7 ± < 0.1fg 0.7 ± < 0.1fg 0.6 ± < 0.1hi 0.6 ± < 0.1gh 0.6 ± < 0.1i
AFG2
Initial concentration (μg/kg) a
0.5a
a
Initial concentration (μg/kgL) a
0.5
1
1.5
0.5a
1a
1.5a
0.4 ± < 0.1b 0.3 ± < 0.1c 0.2 ± < 0.1d < LOD 0.2 ± < 0.1d < LOD 0.2 ± < 0.1d < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.7 ± 0.1b 0.6 ± 0.1c 0.4 ± 0.1d 0.4 ± < 0.1de 0.4 ± < 0.1de 0.3 ± < 0.1fgh 0.3 ± < 0.1fgh 0.2 ± < 0.1ghi 0.3 ± < 0.1ef 0.3 ± < 0.1fgh 0.3 ± < 0.1fg 0.2 ± < 0.1hi 0.2 ± < 0.1ghi 0.2 ± < 0.1i
1.3 ± < 0.1b 1.2 ± < 0.1c 0.9 ± < 0.1d 0.7 ± < 0.1ef 0.8 ± < 0.1e 0.6 ± < 0.1ghi 0.7 ± < 0.1fg 0.6 ± 0.1hi 0.8 ± < 0.1e 0.6 ± < 0.1fg 0.7 ± < 0.1fg 0.6 ± < 0.1hi 0.6 ± < 0.1gh 0.5 ± < 0.1i
0.3 ± < 0.1b 0.2 ± < 0.1c 0.1 ± < 0.1d 0.1 ± < 0.1d 0.1 ± < 0.1d < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD
0.7 ± < 0.1b 0.5 ± < 0.1c 0.3 ± < 0.1d 0.3 ± < 0.1e 0.3 ± < 0.1e 0.2 ± < 0.1h 0.2 ± < 0.1g 0.1 ± < 0.1h 0.2 ± < 0.1f 0.2 ± < 0.1g 0.2 ± < 0.1fg 0.1 ± < 0.1h 0.1 ± < 0.1h < LODi
1.1 ± 1.0 ± 0.7 ± 0.5 ± 0.5 ± 0.4 ± 0.4 ± 0.4 ± 0.6 ± 0.4 ± 0.4 ± 0.3 ± 0.4 ± 0.3 ±
< 0.1b < 0.1c < 0.1d < 0.1e < 0.1e < 0.1gh < 0.1fg < 0.1gh < 0.1e < 0.1f < 0.1f < 0.1ij < 0.1hi < 0.1j
Different letters within each column show significant difference (P < 0.05). W: Washing stage; P: Crushing and pressing stage; B1.5: Bleaching stage by bentonite(1.5 g/100 g); B3: Bleaching stage by bentonite (3 g/100 g); S1.5: Bleaching stage by white soil (1.5 g/100 g); S3: Bleaching stage by white soil (3 g/100 g); 0.5: G1.5: Bleaching stage by gelatin (1.5 g/100 g); G3: Bleaching stage by gelatin (3 g/ 100 g); C:Concentration stage. 181
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Table 4 Impact of each unit operation on the AFs reduction (%) during pekmez processing. The type of applied unit operation
AFB1
AFB2
Initial concentration (μg/kg)
Average
2.5
5
7.5
W P B1.5 B3 S1.5 S3 G1.5 G3 CB1.5 CB3 CS1.5 CS3 CG1.5 CG3
28.0 ± 3.1 11.1 ± 1.7 25.0 ± 3.8 52.5 ± < 0.1 39.4 ± 5.6 66.9 ± < 0.1 61.9 ± 8.02 73.1 ± < 0.1 19.2 ± < 0.1 23.7 ± < 0.1 18.6 ± < 0.1 22.6 ± < 0.1 24.6 ± < 0.1 25.6 ± < 0.1
18.0 ± 1.9 7.3 ± 1.0 21.3 ± 2.8 41.3 ± 4.5 22.1 ± 2.9 47.1 ± 5.3 44.5 ± 5.2 50.0 ± 5.2 12.0 ± 1.4 13.0 ± 1.5 12.5 ± 1.5 14.9 ± 1.7 14.2 ± 1.7 16.8 ± 1.7
12.0 ± 1.3 6.1 ± 1.1 21.6 ± 2.9 35.6 ± 4.2 31.9 ± 4.2 45.6 ± 5.4 41.6 ± 5.2 49.5 ± 5.1 7.2 ± 0.9 8.3 ± 1.0 6.2 ± 0.8 8.38 ± 1.0 8.01 ± 1.0 9.9 ± 1.0
The type of applied unit operation
AFG1
0.5
1
34.0 ± 4.1 27.3 ± 3.7 37.5 ± 6.3 100.0 ± < 0.1 33.3 ± 5.8 100.0 ± < 0.1 41.7 ± 6.7 100.0 ± < 0.1 100.0 ± < 0.1 ND 100.0 ± < 0.1 ND 100.0 ± < 0.1 ND
20.0 12.5 27.1 37.1 44.3 61.4 55.7 68.6 39.2 38.6 28.2 22.2 22.6 13.6
Average 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.7 3.3 4.4 5.4 7.0 6.6 7.4 4.5 4.3 3.2 2.6 2.5 1.4
12.7 ± 1.4 4.6 ± 1.0 28.0 ± 3.6 40.0 ± 4.8 37.6 ± 4.8 48.0 ± 5.7 42.4 ± 5.3 48.8 ± 5.2 13.3 ± 1.5 6.7 ± 0.9 10.3 ± 1.1 4.6 ± 0.7 9.7 ± 1.2 9.4 ± 0.9
22.2 ± 2.4ab 14.8 ± 1.9b 30.9 ± 3.9ab 59.0 ± 6.4a 38.4 ± 4.8ab 69.8 ± 7.6a 46.6 ± 5.6ab 72.5 ± 7.9a 50.9 ± 5.3ab 15.1 ± 2.1b 46.2 ± 4.7ab 9.0 ± 1.5b 44.1 ± 4.5ab 7.97 ± 0.8b
AFG2
Initial concentration (μg/kg)
W P B1.5 B3 S1.5 S3 G1.5 G3 CB1.5 CB3 CS1.5 CS3 CG1.5 CG3
19.3 ± 2.0d 8.2 ± 1.1d 22.6 ± 2.8cd 43.2 ± 4.7ab 31.1 ± 3.8bc 53.2 ± 6.0a 49.3 ± 5.7a 57.6 ± 6.0a 12.8 ± 1.5d 15.0 ± 1.7d 12.4 ± 1.4d 15.3 ± 1.7d 15.6 ± 1.8d 17.4 ± 1.7d
Initial concentration (μg/kg)
0.5
1
30.0 ± 3.4 17.1 ± 2.4 29.2 ± 5.4 100.0 ± < 0.1 29.2 ± 5.4 100.0 ± < 0.1 37.5 ± 6.2 100.0 ± < 0.1 ND ND ND ND ND ND
27.0 13.7 35.7 42.9 42.9 58.6 55.7 64.3 24.4 25.0 20.0 20.7 22.6 20.0
Average 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 1.9 4.2 4.9 5.2 6.7 6.5 6.8 2.8 2.8 2.3 2.4 2.6 2.0
14.0 ± 1.5 6.2 ± 1.3 31.0 ± 4.0 43.2 ± 5.2 40.0 ± 5.1 51.2 ± 6.0 45.6 ± 5.7 53.6 ± 5.6 10.5 ± 1.2 7.0 ± 0.9 10.7 ± 1.2 6.7 ± 0.8 7.4 ± 0.9 6.9 ± 0.7
23.7 ± 2.5cd 12.4 ± 1.6d 32.0 ± 4.0cd 62.0 ± 6.7ab 37.3 ± 4.6bcd 69.9 ± 7.6a 46.3 ± 5.6abc 72.6 ± 7.4a 11.6 ± 1.3d 10.7 ± 1.2d 10.27 ± 1.1d 9.1 ± 1.0d 10.0 ± 1.1d 9.0 ± 0.9d
Initial concentration (μg/kg)
Average
0.5
1
1.5
44.0 ± 4.8 14.0 ± 2.6 47.6 ± 6.1 52.4 ± < 0.1 52.4 ± 7.74 100.0 ± < 0.1 100.0 ± 12.5 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 1000. ± < 0.1 ND ND ND
33.0 ± 3.7 26.0 ± 3.2 40.7 ± 4.8 50 ± 5.7 48.2 ± 5.9 70.4 ± 8.0 63.0 ± 7.4 74.1 ± 7.8 28.1 ± 3.3 29.6 ± 3.3 21.4 ± 2.4 18.8 ± 2.3 30.0 ± 4.5 100.0 ± 10.0
26.0 28.0 28.9 43.3 43.3 59.8 56.7 59.8 15.9 21.8 18.2 18.0 16.7 25.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2 3.4 3.8 5.2 5.6 7.2 6.93 6.3 2.0 2.6 2.2 2.1 2.2 2.6
34.3 22.7 39.1 48.6 47.9 76.7 73.2 78.0 48.0 50.5 46.5 12.2 15.6 41.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.7abc 2.8bc 4.5abc 5.7abc 5.8abc 8.6a 8.3a 8.4a 5.5abc 5.6abc 4.8abc 1.4c 2.1c 4.2abc
W: Washing stage; P: Crushing and pressing stage; B1.5: Bleaching stage by bentonite (1.5 g/100 g); B3: Bleaching stage by bentonite (3 g/100 g); S1.5: Bleaching stage by white soil (1.5 g/100 g); S3: Bleaching stage by white soil (3 g/100 g); 0.5: G1.5: Bleaching stage by gelatin (1.5 g/100 g); G3: Bleaching stage by gelatin (3 g/ 100 g); C:Concentration stage. Different letters indicated significant difference among average value of reduction percentage within each column (P < 0.05). ND: not detected.
AFG1 were reported as 1.73, 1.63 and 1.81 and their solubility in water is 233, 392 and 424 mg/L, respectively (Karlovsky et al., 2016). Based on the findings of the current study, crushing and pressing had a low effect on the removing of AFs. According to Tables 4 and 5, The reduction range (average) of AFB1, AFB2, AFG1 and AFG2 during crushing and pressing processing of pekmez was 6.1–11.1% (8.2%), 4.6–27.3% (14.8%), 6.2–17.1% (12.4%) and 14.0–28.0% (22.7%). Due to some AFs was located in the skin of the grape, it seems that skin removing could result in AFs reduction. The impact of the pressing step on mycotoxin also is varied. In a previously conducted investigation, pressing of pomace after primary alcoholic fermentation during wine production resulted in more than four times reduction in the concentration of ochratoxin A (OTA), mainly due to OTA adsorption by the solid substances (Gil-Serna, Vázquez, González-Jaén, & Patiño, 2018). According to Tables 4 and 5, mean reduction (range) of AFB1, AFB2, AFG1 and AFG2 of pekmez during boiling and concentration stage was measured as 14.8% (6.2–25.6%), 34.6% (4.6–100.0%), 15.1% (6.7–25.0%) and 42.9% (15.6–100.0%), respectively. So far, there is no published information considering the impact of the boiling and concentration process on the reduction of aflatoxins in pekmez. However, Arici et al. (2004) found that boiling and concentration process increased the amount of ochratoxin A, about 5–6 times higher than the
original concentration in grape juice (they did not mention the temperature and length of boiling/concentration and their data was reported based on wet weight), while our results indicated AFs decreased during the boiling and concentration process (our data was reported based dry weight). The effects of the boiling and concentration process on aflatoxin and another mycotoxin in fruit juice and other food products have been studied (Çetin & Sebahattin, 2003; Sant’Ana et al., 2008). For example, according to a study by Çetin and Sebahattin (2003), heat treatment (about 90 and 100 °C for 20 min) reduced 18.81 and 25.99% of patulin concentration in apple juice. The concentrations of patulin were reduced as a result of heat treatment (evaporation) at 70 and 80 °C for 20 min about 9.4% and 14.06%, respectively. The reduction of patulin during the concentration step of apple juice depended on heating temperature and time used (Sant’Ana et al., 2008). Based on the findings of Mutungi et al. (2008), boiling of muthokoi at 98 °C for 150 min resulted in a reduction of 80–93% of aflatoxin. Raters and Matissek (2008) demonstrated that AFB1 could be degraded at temperatures above 160 °C. Meanwhile, several studies have been reported mycotoxin stability during thermal processes (Yazdanpanah et al., 2005). These differences can be associated with the temperature and the heating time applied in preparing of different food products.
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Table 5 The total reduction (%) of AFs during pekmez processing. The whole unit operations applied for pekmez production
AFB1
Average
Initial concentration (μg/kg) 2.5
W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/G1.5/C W/P/G3/C
61.2 76.8 68.4 83.6 81.6 87.2
The whole unit operations applied for pekmez production
AFG1
5 ± ± ± ± ± ±
3.4 2.9 3.2 3.0 5.0 3.0
47.4 61.2 48.2 65.8 63.8 68.4
7.5 ± ± ± ± ± ±
1.4 3.2 2.9 2.6 2.5 1.9
39.9 51.2 47.2 58.8 55.6 62.4
± ± ± ± ± ±
0.4 1.2 0.9 4.2 4.6 2.8
49.5 63.1 54.6 69.4 67.0 72.7
± ± ± ± ± ±
3.5c 6.5b 5.4c 5.4a 3.5ab 4.1a
74.7 ± 65.7 ± 70.3 ± 68.0 ± 76.7 ± 75.7 ±
< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
61.3 48.9 56.2 55.3 61.8 58.2
3.1 4.2 2.5 4.6 2.1 3.5
1
100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 100.0 ± < 0.1 1000.0 ± < 0.1
65.7 70.3 68.0 76.7 75.7 79.7
1.5 ± ± ± ± ± ±
4.2 2.6 4.6 2.1 3.5 1.5
48.9 56.2 55.3 61.8 58.2 63.8
± ± ± ± ± ±
1.0 3.0 2.4 3.4 1.7 2.3
71.5 75.5 74.4 79.5 78.0 81.2
± ± ± ± ± ±
3.5b 6.5ab 5.4ab 5.4a 3.4ab 4.1a
Average
Initial concentration (μg/kg) 1.5
± ± ± ± ± ±
0.5
AFG2
Initial concentration (μg/kg) 1
Average
Initial concentration (μg/kg)
Average
0.5 W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/G1.5/C W/P/G3/C
AFB2
61.3 48.9 56.2 55.3 61.8 58.2
± ± ± ± ± ±
4.4 1.0 3.0 2.4 3.4 1.7
78.7 71.5 75.5 74.4 79.5 78.0
± ± ± ± ± ±
4.3a 5.4b 2.7ab 4.3ab 3.4a 4.1a
0.5
1
1.5
77.0 ± < 0.1 83.0 ± < 0.1 80.0 ± < 0.1 89.0 ± < 0.1 87.0 ± < 0.1 100.0 ± < 0.1
64.0 ± 1.5 72.7 ± 2.0 70.0 ± 1.5 79.3 ± 2.1 76.7 ± 1.2 82.0 ± < 0.1
64.0 ± 2.5 72.7 ± 1.4 70.0 ± 0.7 79.3 ± 1.4 76.7 ± 1.7 82.0. ± 1.4
80.3 85.2 83.3 89.4 87.9 94.0
± ± ± ± ± ±
2.5b 2.9ab 4.3ab 3. 9a 4.2a 5.1a
Different letters indicated significant difference among average value of reduction percentage within each column (P < 0.05). W/P/B1.5/C: The stages of washing + crushing and pressing + bleaching by bentonite (1.5 g/100 g) + concentration. W/P/B3/C: The stages of washing + crushing and pressing + bleaching by bentonite (3 g/100 g) + concentration. W/P/S1.5/C: The stages of washing + crushing and pressing + bleaching by white soil (1.5 g/100 g) + concentration. W/P/S3/C: The stages of washing + crushing and pressing + bleaching by white soil (3 g/100 g) + concentration. W/P/G1.5/C: The stages of washing + crushing and pressing + bleaching by gelatin (1.5 g/100 g) + concentration. W/P/G3/C: The stages of washing + crushing and pressing + bleaching by gelatin (3 g/100 g) + concentration.
3.3. Effect of clarifier
commonly used to prevent AFs intestinal absorption (Savari et al., 2013; Thieu & Pettersson, 2008). Bentonite that was often known as montmorillonite is a claylike substance with a volcanic origin (Huwig et al., 2001). It is one of the most used adsorbents for clarification in the beverage industry and decoloration of edible oils (Tahir & Rauf, 2006). Also, the utilization of bentonite resulted in a 3.7% reduction of ochratoxin A in wine (Var et al., 2008). Savari et al. (2013) found that AFs adsorption by clay could be correlated with the formation of binding between β-dicarbonyl part of aflatoxin and metal ions of clay. Calcium carbonate comprises 90% of the white soil and led to the alkalization of “must.” Doyle, Applebaum, Brackett, and Marth (1982) showed that the hydrolysis and oxidation of the lactone ring of aflatoxin are carried out in alkaline medium, which consequently could result in the elimination of aflatoxin. In the current study by increasing the concentrations of clarifiers added to “must,” the absorption and reduction of AFs increased. The inconsistency in density, the size, the diameter of the particles of adsorbents and different mechanism of AFs absorption had the impacts on the capacity of these compounds for AFs removal (Kannewischer, Arvide, White, & Dixon, 2006). Thieu and Pettersson (2008) declared the difference between absorption capacity of various type of zeolite and bentonite in adsorption of aflatoxin B1 can be correlated to the difference in the process and the purification of these materials. On the other hand, the individual adsorbent material obtained from each mine may have a different chemical composition and properties depending on the extraction location.
Among unit processing used during pekmez production, the highest effect on the AFs reduction was correlated with clarification (Tables 4 and 5). The AFs reduction during this stage depended on the type and concentration of clarifying agents as well as initial aflatoxin concentration. By increasing in the clarifier levels from 1 to 3 g/100 g, AFs reduction was significantly raised (P < 0.05). In the samples, which contain 0.5 μg/kg AFB2, AFG1 or AFG2, after incorporation of 3 g gelatin, bentonite, or white soil to 100 g “must,” AFs was lower than LOD. As shown in Tables 4 and 5, the average reduction of AFs during clarification by bentonite, white soil and gelatin was varied; 43.2%, 53.2%, and 57.6 for AFB1; 59.0%, 69.8% and 72.5%, for AFB2, 62.0%, 69.9% and 72.6% for AFG1 and 48.6%, 76.7%, and 78.0% for AFG2, respectively. Therefore, these findings indicated that gelatin had the highest impact on AFS removal while compared with white soil and bentonite. Also, bentonite with a concentration of 1.5 g/100 g showed the lowest effect on the AFs reduction. There is not much information regarding the effects of gelatin in reducing AFs. In a previous study, the utilization of gelatin/bentonite mixture for clarification of apple juice with ultra-filtration processing caused a reduction of about 25% in the patulin level (Acar, Gökmen, & Taydas, 1998). The role and mechanism of the reduction of aflatoxin by gelatin are unclear although it was assumed that gelatin in water formed a gel network which able to trap and further adsorption of AFs. The utilization of commercial adsorbents such as sodium bentonite, cholestyramine crospovidone, montmorillonite, activated carbon (AC), zeolites and hydrated sodium calcium aluminosilicate (HSCAS) for the elimination of mycotoxin is one of the common strategies (Huwig, Freimund, Käppeli, & Dutler, 2001; Var, Kabak, & Erginkaya, 2008). These compounds inhibit the absorption of aflatoxins in the digestive tract and reduce their bioavailability (Ramos, Hernandez, Pla-Delfina, & Merino, 1996; Savari, Dehghanbanadaki, Rezayzady, & Javannikkhah, 2013). Among various adsorbents, bentonite is
4. Conclusions The current study was the first attempt to investigate the fate of aflatoxins during pekmez production processing stages. In order to have accurate results the artificially spiked grape samples in levels of 2.5, 5 and 7.5 μg/kg AFB1 and 0.5, 1 and 1.5 μg/kg of aflatoxin AFB2, AFG1 and AFG2, and the conventional process of pekmez production were 183
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used. The levels of AFB1, AFB2, AFG1, and AFG2 during pekmez production process were decreased by 60.4, 76.7, 76.3 and 86.7%, respectively. Therefore, the highest and lowest reductions during the pekmez production process can be associated with AFG2 and AFB1, respectively. In this regard, all of the steps investigated are effective in the reduction of AFs. However, AFs reduction percentage during pekmez depended on their initial contamination levels and type and content of added clarifier. Among assessed clarifier (gelatin, bentonite and white soil), gelatin demonstrated the highest efficiency in the elimination of AFs from pekmez.
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