- Email: [email protected]
Cistus incanus L. extract inhibits Aflatoxin B1 production by Aspergillus parasiticus in macadamia nuts
Industrial Crops & Products 111 (2018) 63–68
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Research Paper
Cistus incanus L. extract inhibits Aflatoxin B1 production by Aspergillus parasiticus in macadamia nuts Venetia Kalli, Eleni Kollia, Anna Roidaki, Charalampos Proestos, Panagiota Markaki
MARK
⁎
Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15784 Athens, Greece
A R T I C L E I N F O
A B S T R A C T
Keywords: Aflatoxin B1 Aspergillus parasiticus Cistus incanus L. Macadamia
Aflatoxins are secondary metabolites, with aflatoxin B1 being the most common, reported as carcinogenic, teratogenic and genotoxic. This study investigates the antiaflatoxigenic efficacy of the herbaceous plant Cistus incanus L. against Aspergillus parasiticus in two substrates, yeast extract sucrose medium and macadamia nuts. The methanolic extract of Cistus incanus showed pronounced antiaflatoxigenic ability, inhibiting aflatoxin B1 production in both substrates. AFB1 production was decreased significantly in a percentage of 87.1–90.1% after Cistus incanus extract addition in YES medium. The extract effectiveness was also observed in macadamia nuts, where the AFB1 production by Aspergillus parasiticus was reduced in a percentage of 72.5–85.9%. Moreover, the risk assessment was estimated taking into account the maximum amounts of AFB1 produced in inoculated samples with and without Cistus incanus addition. It was revealed that Cistus incanus presence leads to a lesser exposure of AFB1 to consumers.
1. Introduction
et al., 2017; Rengel et al., 2015). Generally, nuts rich in lipids are known to be possibly contaminated with aflatoxins and many studies have shown that contamination often exceeds the legal threshold in commercial samples (Siciliano et al., 2017). The genus Cistus belongs to the family of Cistaceae, which is one of the characteristic genera of the Mediterranean region and comprises several medicinal plants of perennial shrubs. These Mediterranean shrubs species, such as C. incanus, are naturally rich in polyphenols. C. incanus is a thermophilic plant which requires much light and for this reason it’s growing in warm and temperate places (Gori et al., 2016). Studies showed that the extract of C. incanus has antifungal, antibacterial and antiviral properties, because of its constituents which includes tannins, flavonoids and other bioactive compounds (Attaguille et al., 2000; Riehle et al., 2013; Roidaki et al., 2016) In traditional medicine, C. incanus has been used in anti-inflammatory, anti-allergic, antiulcerogenic, wound healing, antimicrobial, cytotoxic and vasodilator remedies (Riehle et al., 2013). In recent years, the scientific community has focused on the use of natural products to control the mold growth and inactivate mycotoxins. The reason for this trend of scientific research is that most plants produce naturally, antimicrobial secondary metabolites, during their development or in response to stress conditions (infection, wounding etc.) (Aly et al., 2016). These secondary metabolites are usually bioactive compounds such as phenolics that are known to exhibit antioxidant, anti-inflammatory, antihepatotoxic, antitumor, antimicrobial and
Aflatoxins (AFs) are secondary metabolites, produced by certain Aspergillus sp. such as Aspergillus flavus and Aspergillus parasiticus. The most common aflatoxins are aflatoxin B1, B2, G1, G2 and are characterized as carcinogenic, teratogenic and genotoxic, while aflatoxin B1 (AFB1), is the most prevalent of the AFs and has been characterized as human carcinogen belonging in Group I (International Agency for Research on Cancer (IARC), 2002). Because aflatoxigenic fungal contamination is unavoidable, numerous crops (cereals, sesame seed, dried fruits and other nuts, peanuts, pistachios etc.) are lost annually (Kollia et al., 2016a; Kollia et al., 2014; Villa and Markaki, 2009). The fungal and aflatoxin contamination that cause quantitative and qualitative losses of food commodities can also potentially induce various health problems to consumers (Kedia et al., 2014). Such losses of foodstuffs and their importance in international food trade, require the necessity for suitable and effective control actions. According to literature, it is pointed out that, the presence of lipids in foodstuffs stimulates biosynthesis of AFB1 (Fanelli and Fabbri, 1989; Kollia et al., 2016a,b; Rajasekaran et al., 2017). Macadamia nuts are originated from eastern Australia and they are rich in lipids which are representing more than 70% of their weight. Moreover, macadamia nuts are also rich in trace elements (calcium, phosphorus, iron etc.) and vitamins (thiamine, riboflavin, retinol and niacin) (Acquino-Bolanos
⁎
Corresponding author. E-mail address: [email protected] (P. Markaki).
http://dx.doi.org/10.1016/j.indcrop.2017.10.003 Received 21 June 2017; Received in revised form 29 August 2017; Accepted 2 October 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
2.3. Reagents
antifungal activities (Kollia et al., 2016b; Tzanidi et al., 2012). Many studies have demonstrated a strong correlation between the phenolic/ antioxidant compounds of plants and their antifungal/antiaflatoxigenic activities. Consequently plants’ extracts could potentially provide protection against aflatoxigenic fungus and aflatoxins. Redox reactions are fundamental to cellular catabolism and anabolism processes. Antioxidants could interfere, reducing the pool of nicotinamide adenine dinucleotide phosphate (reduced form) available for AF biosynthetic reactions (Maggio-Hall et al., 2005). Furthermore, data from the literature indicate an association of the antioxidant profile of plants with their possibly antimicrobial, antifungal and anti-aflatoxigenic efficiency (Kollia et al., 2017; Kollia et al., 2016b; , Loizzo et al., 2013; Tzanidi et al., 2012). Previously many plant extracts have been investigated for their capacity to inhibit aflatoxin production not only in culture medium conditions but also in food commodities. Particularly Equisetum arvense, Stevia rebaudiana, Zingiber officinalis, Oxalis corniculata and Trigonella foenum-graecum have been reported to have antifungal and antiaflatoxigenic properties (Garcia et al., 2012; Krishna Reddy et al., 2011). Moreover Yazdani et al. (2013) showed that Piper betle L. was able to reduce the aflatoxin production by A. flavus. Due to these facts, the scientific community is searching for new natural bioactive compounds, which can possibly act as suppressor of aflatoxins. In this work macadamia nuts were evaluated as natural substrate for AFB1 production by A. parasiticus and compared with synthetic medium yeast extract sucrose (YES). C. incanus plant was examined for the first time to our knowledge, for its effect on AFB1 biosynthesis by A. parasiticus on both substrates. In addition, the efficiency of a routine analysis by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector for the determination of AFB1 in macadamia nut was evaluated. In the present study, the risk for the consumers is also assessed, before and after addition of C. incanus in inoculated nuts.
Aflatoxin B1 standard was obtained from Sigma (st. Louis, MO, USA). The Aflatest immunoaffinity columns were purchased from Vicam (Watertown, MA, USA). All reagents were analytical grade. The HPLC solvents were bought from Fischer Chemical (Fischer Scientific, Loughborough, Leicestershire, UK) while hexane and methanol (pro analysis grade) were from Merck (Darmstadt, Germany) and trifluoroacetic acid was from Fluka (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). 2.4. Culture media For the preparation of Aspergillus Flavus Parasiticus Agar (AFPA), 2 g of yeast extract (Oxoid, Basingstoke, Hampshire, UK), 1 g of bacteriological peptone (Oxoid), 0.05 g of ferric ammonium citrate (Merck, Germany), 0.1 mL of Dichloran 0.2% in ethanol (Fluka Steinheim, The Netherlands), 0.01 g of chloramphenicol (Oxoid), and 1.5 g of agar (Oxoid) were dissolved per 100 mL of distilled water. The final pH was adjusted to 6.0–6.5. 2.5. Experimental design The experimental design of this study is shown in Table 1. All samples were examined for AFB1 production on days 0, 3, 7, 9, 12, and 15 of incubation. The experiments were repeated thrice. 2.6. Extraction of Cistus incanus For the preparation of C. incanus extract, two different extraction procedures were combined. A conventional extraction procedure was performed, followed by an ultrasound-assisted extraction. Specifically, one gram of the dried ground sample was placed in a beaker and mixed with 30 mL of methanol:water (80:20 v/v) solution acidified with 0.1% HCl. After 24 h in the dark, at room temperature the beakers were sonicated thrice (10 mL of solvent each time) for 15 min in the ultrasonic device. After the extraction, filtration took place using Buchner funnel. The extracts were evaporated to dryness, were redissolved to 5 mL of MeOH and kept at −20 °C until analysis. The extraction procedures were performed in triplicate (Roidaki et al., 2016).
2. Materials and methods 2.1. Sampling and treatment Macadamia nuts were purchased from a local market in Athens (Greece), weighting approximately 3 kg. Macadamia nuts were washed with water, dried and homogenized forming a paste. Then macadamia nuts were sterilized at 110 °C for 2 min, in order to minimize the natural microbial load before inoculation with A. parasiticus. An amount of 10 g of treated macadamia were transferred in flasks under aseptic conditions, in such a way as to create a single layer. Cistus incanus in dried form was purchased from a local farmer in Chalkidiki (North Greece) during winter of 2015.
2.7. Preparation of spore inoculum The strain A. parasiticus speare (IMI 283883) used during this work was obtained from the International Mycological Institute (Engham Surrey, UK). The isolate was subcultured on potato dextrose agar slants and incubated at 30 °C for 7 days. Inoculum suspensions were prepared from the 7 days culture mentioned above. Spores were harvested aseptically using 10 mL of sterile 0.01% v/v Tween 80 solution. Aflatoxin B1 carried over from the initial growth
2.2. Apparatus The extraction of C. incanus plant was performed using an ultrasonic bath device (Elmasonic S, Elma Schmidbauer GmbH, Germany) at a frequency of 37 kHz. The extract was evaporated to dryness using a rotary evaporator (Heidolph, Laborota 4000 efficient, WB eco). In addition High-Performance Liquid Chromatography (HPLC) analysis was done using a Hewlett-Packard 1050 (Hewlett-Packard, Waldborn, Germany) liquid chromatograph with a JASCO FP-920 (Jasco Ltd., Tokyo, Japan) fluorescence detector and an HP integrator 3395.The HPLC column was a C18 Nova-Pak (4.6 × 250 mm, 4 μm, 60 Å, WatersMillipore, Milford, MA, USA). For the filtration of the mobile phase (water: acetonitrile: methanol [20:4:3 v/v/v]) for AFB1 determination, Millipore HA-VLP (0.45 μm) filters were used. AFB1 was derivatized to its hemiacetal (AFB2a) and was detected at λex = 365 nm/ λem = 425 nm. The retention time was 13.58 ± 0.38 min, while flow rate was 1 mL min−1.
Table 1 Groups of inoculated samples. YESa (10 mL) + 102 conidia of A. parasiticus + 100 μL methanol YES (10 mL) + 102 conidia of A. parasiticus without methanol addition YES (10 mL) + 102 conidia of A. parasiticus + 100 μL of C. incanus extractb Macadamia paste (10 g) + 102 conidia of A. parasiticus + 100 μL methanol Macadamia paste (10 g) + 102 conidia of A. parasiticus + 100 μL C. incanus extractb Macadamia paste (10 g) + 102 conidia of A. parasiticus Macadamia paste (10 g) (control)c Three flasks were used for each case and for each day of observation. a Yeast Extract Sucrose Medium. b Concentration of extract: 0.2 g of C. incanus fresh weight mL−1 methanol. c Non-inoculated, without C. incanus addition.
64
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
was reduced by centrifuging the spore suspension (2500 rpm for 10 min) and dissolving the biomass in 10 mL of sterile Tween 80 solution twice. Dilutions (10−1, 10−2, 10−3, 10−4) from the initial spore suspension in sterile tubes containing 10 mL of Tween 80 0.05% v/v were made. The spore concentration was determined by the spread plate surface count technique using 0.1 mL of each dilution on four AFPA plates incubated at 30 °C for 48 h. To accomplish an inoculum of 100 conidia, petri dishes (AFPA) with 10–100 colony-forming units (cfu) were chosen and the number of 100 spores was estimated and used in the specific experiment (Kostarellou et al., 2014; Vergopoulou et al., 2001).
Table 2 Accuracy of the method for AFB1a determination applied in macadamia nuts. AFB1 (ng) spike in ng g−1 macadamia nuts 5
2.8. Inoculation of YES medium and macadamia nuts In Table 1 is shown that for each day of incubation, three flasks containing 10 mL YES medium and three flasks containing 10 g of macadamia nuts, were inoculated with 100 conidia of A. parasiticus per flask. Then 100 μL of C. incanus methanolic extract were added in the inoculated flasks. For the control cultures, 100 μL of methanol alone (without the addition of C. incanus extract) were added in the inoculated with the fungus samples. All flasks were incubated at stationary conditions, at 30 °C and AFB1 production was examined on days 0, 3, 7, 9, 12, and 15.
a
20
40
50
Measured AFB1 (ng) in ng g−1 macadamia nuts
Samples 1 2 3 4 Mean SD RSD%
10
4.17 3.74 5.05 4.36 4.33 0.55 12.60
9.84 8.84 11.8 10.27 10.19 1.23 12.08
23.29 20.22 19.66 23.68 21.71 2.07 9.51
42.15 37.86 49.59 43.41 43.26 4.85 11.21
61.19 54.95 55.98 48.57 55.17 5.19 9.41
Aflatoxin B1.
AFB1 g−1). The reproducibility was examined by spiking with AFB1 (100 ng), four samples (10 g) of macadamia nuts in several time-periods, under the same conditions. The coefficient of variation (RSD) was found to be 4.51% (mean: 10.27 ng AFB1 g−1). Furthermore, the repeatability (r) and reproducibility (R) limits were calculated as following: r = 2.8SDr and R = 2.8SDR and they were found to be r = 12.98 and R = 18.54, respectively. In the present study, accuracy was estimated by analyzing samples of macadamia paste (10 g) spiked with different amounts of AFB1. The regression coefficient r was 0.9984 and the mean recovery was found to be 112.10% (RSD = 8.47%) [y = 1.1201( ± 0.0931)x − 1.0695 ( ± 0.63806)] (Table 2). A Fisher test was applied in order to examine the reliability of the accuracy linear regression analysis. The F (1, 15) ratio 11742.5 was greater than the critical Fisher value of 16.587 at an alpha risk of 0.1% for 1 and 5° of freedom. Consequently, the regression model is satisfactory. In addition, the lack of fit of the model was found to be low, as the experimental F ratio (0.635) was found less than the critical value F = 9.335 with a risk a = 0.1% and df = 3,15. Therefore, the linearity is acceptable. The limits of determination (LOD) and quantification (LOQ) were calculated using the following equations
2.9. AFB1 determination and HPLC analysis Ten grams of grounded macadamia nuts was mixed with 30 mL methanol/water (80:20 v/v) and shaken well for 10 min. After filtration, an aliquot of 1 mL was used for AFB1 analysis. For the clean-up procedure, 1 mL from the filtrate was diluted with 10 mL distilled water for 1 min. The mixture was loaded into an Aflatest immunoaffinity column (flow rate 3 mL min−1) and washed two times with 10 mL of distilled water. Then the moisture was removed from the column by passing air. Finally AFB1 was eluted with 2 mL of acetonitrile in dark glass vials and the solvent was evaporated under a mild stream of nitrogen. The derivatization of AFB1 to AFB2a (hemiacetical of AFB1) was followed. A mixture of 200 μL hexane and 200 μL of trifluoroacetic acid were added to the evaporated AFB1 solution. The derivatization was completed by heating at 40 °C in a water bath for 10 min. After that the solution was evaporated to aridity, dissolved in 200 μL water:acetonitrile (9:1 v/v) and analyzed by HPLC with fluorescence detector (volume injected = 40 μL) (Daradimos et al., 2000).
LOD = {b0 + 3S(b0)}/b1, LOQ = {b0 + 10 (b0)}/b1 where b0 is the response of the blank, S(b0) is the standard deviation of the blank and b1 is the sensitivity. The LOD and LOQ were found to be 1.17 ng g−1 and 3.56 ng g−1 respectively. Previously in the literature it was reported that the recovery in a study for AFB1 determination in several nuts, was estimated at 102.0% while LOD and LOQ were 0.08 ng g−1 and 0.15 ng g−1 respectively (Chun et al., 2007). In another study, Kabak (2014) reported recoveries from 95.7% to 96.4% in walnuts and LOD and LOQ at 0.02 ng g−1 and 0.07 ng g−1 respectively. Moreover Masood et al. (2015) in a study concerning the natural occurrence of aflatoxins in edible nuts found that the recoveries of the fortified samples were from 83% to 90% while the LOD for AFB1 was 0.04 ng g−1and the LOQ 0.12 ng g−1. In the present study, the LOD (1.17 ng g−1), LOQ (3.56 ng g−1) and the accuracy (recovery factor: 112.10%) of the method mentioned above, are in agreement with the requirements of the Commission Regulation (EC) No 401/2006 (European Commission, 2006). Consequently, the HPLC method developed in the present study, for the determination of AFB1 in macadamia nuts, is sensitive, selective, efficient, and accurate and it can be used for research purposes or as a routine analysis method as well.
3. Results and discussion 3.1. Development and validation of AFB1 determination method in macadamia nuts A sensitive and selective HPLC method was developed and characterized in-house, for the determination of AFB1 in macadamia nuts. The method was validated observing parameters of the compliance criteria. Analytical parameters were investigated including linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), repeatability (r) and reproducibility (R). A solution at concentration of 5 μg AFB1 mL−1 MeOH was prepared from the initial stock solution of AFB1 (1 mg mL−1) by diluting 100 μL in 9.900 μL of methanol. A series of standards in the final concentration of AFB1 corresponding to 20, 10, 5, 2,5, 2 and 1 ng AFB1 mL was prepared in mobile phase (water: acetonitrile: methanol [20:4:3 v/v/v]) and 40 μL of each standard were injected into the column. The regression equation of the standard curve was: y = 2 × 107x + 520087 r2 = 0.996. Repeatability was examined by analyzing four sub-samples (10 g) of the matrix spiked with 100 ng of AFB1 corresponding to 10 ng g−1. The coefficient of variation (RSD) was found to be 6.59% (mean: 10.05 ng
3.2. Evaluation of C. incanus extract as AFB1 suppressor in YES medium As mentioned earlier, in this work C. incanus was selected to study 65
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
for its effect on AFB1 production by A. parasiticus. To the best of our knowledge, so far there has not been any relevant study on the effectiveness of C. incanus extract against AFB1 production by A. parasiticus. In a previous study of our laboratory, C. incanus was investigated for its antioxidant and antifungal activity (Roidaki et al., 2016). The analysis revealed that among a variety of plants, C. incanus possessed significant antioxidant capacity and high total phenolic content. Moreover, the extract of C. incanus (concentration 0.2 g mL−1) showed the strongest antifungal activity, inhibiting the A. parasiticus growth in a percentage of 45.91%. Therefore in the present study, the same extract in the same concentration was evaluated as AFB1 suppressor in YES medium. Yeast Extract Sucrose (YES) was chosen as the culture medium which is beneficial for development and AFB1 biosynthesis of A. parasiticus and A. flavus. AFB1 was studied as it is the most potent carcinogenic mycotoxin and it is usually produced at high levels by the fungus A. parasiticus (Davis et al., 1966; Luchese and Harrigan, 1993). The inoculum of 100 spores was chosen for the inoculation of the samples, as it is the minimum amount of spores found in the literature that produce high levels of AFB1 by A. parasiticus (Vergopoulou et al., 2001). The analytical method for AFB1 determination in YES medium has been previously developed in our laboratory. The recovery of the method was found 99.2% while the detection limit of the derivatized AFB1 (AFB2a) was 0.02 ng AFB1 mL−1 (Leontopoulos et al., 2003). AFB1 production in YES medium inoculated with A. parasiticus and supplemented with C. incanus extract, was continuing, until the last day (15th) of observation where the production of AFB1 was 0.532 μg g−1. In control samples (without the extract addition) the AFB1 production was also increasing until the 15th day (maximum production 5.143 μg g−1). The addition of C. incanus extract (100 μL of 0.2 g mL−1 MeOH) in YES medium cultures inoculated with A. parasiticus showed a significant inhibition of AFB1 production compared with control cultures (df = 10, Texp = 2.888, Ttheor = 2.228, p < 0.05). In the specific study, C. incanus extract was suppressed significantly the AFB1 production by A. parasiticus in YES medium. The AFB1 reduction amounted from 87.1% to 90.1% after the 3rd day of observation (Table 3, Fig. 1).
Fig. 1. C. incanus extract inhibited AFB1 production by A. parasiticus in YES medium in comparison to AFB1 production in control YES cultures.
Table 4 AFB1a production (μg g−1) by A. parasiticus in inoculated macadamia nuts with and without Cistus incanus extract addition. Days
Macadamia nuts + A. parasiticus + 100 μL MeOHb (control)
Macadamia nuts + A. parasiticus + 100 μL C. incanus extractc
0 3 7 9 12 15
0 0.0040 0.0195 0.0359 0.3270 0.3010
0 0.0011 0.0024 0.0075 0.0461 0.0458
a b c
± ± ± ± ±
0.01 0.01 0.02 0.01 0.01
± ± ± ± ±
0.00 0.00 0.01 0.12 0.10
Aflatoxin B1. Methanol. Concentration of extract: 0.2 g fresh weight mL−1 methanol.
3.3. Evaluation of C. incanus extract as AFB1 suppressor in macadamia In this work, the extract of C. incanus was found efficacious inhibiting aflatoxin B1 production by A. parasiticus in macadamia paste. The declining trend of the AFB1 production in macadamia treated with C. incanus was estimated from 72.5% to 85.9%. In Table 4 is shown that maximum AFB1 production was observed on 12th day of incubation for both inoculated macadamia samples spiked with C. incanus extract and macadamia control samples (0.0461 μg g−1 0.327 μg g−1, respectively) (Τable 4, Fig. 2). Additionally, in order to evaluate the findings of the present study, macadamia nuts were examined as substrate for AFB1 production by A. parasiticus in comparison with YES medium. As mentioned previously YES medium is a conductive substrate that supports AF production. As
Fig. 2. C. incanus extract inhibited the AFB1 production by A. parasiticus in macadamia nuts in comparison to AFB1 production in control macadamia cultures.
Table 3 AFB1a production (μg g−1) by A. parasiticus in YESb medium in comparison with AFB1 production in YESb with Cistus incanus extract addition. Days
YES + A. parasiticus + 100 μL MeOHc (control)
YES + A. parasiticus + 100 μL of C. incanus.d
0 3 7 9 12 15
0 1.012 2.150 3.369 4.786 5.143
0 0.093 0.256 0.435 0.491 0.532
a b c d
± ± ± ± ±
1.80 2.07 1.78 2.24 2.06
± ± ± ± ±
0.16 0.42 0.54 0.33 0.42
Fig. 3. AFB1 production by A. parasiticus in YES medium and in macadamia nuts. The AFB1 production in macadamia substrate is significantly lower if compared with AFB1 production in the favorable microbiological yeast extract sucrose medium (YES).
shown in Fig. 3, the AFB1 production in YES medium was increased until the 15th day of observation. However, AFB1 production in inoculated with A .parasiticus macadamia was increased until the 12th
Aflatoxin B1. Yeast extract sucrose medium. Methanol. Concentration of extract: 0.2 g fresh weight mL−1 methanol.
66
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
sample (maximum: 327 ng AFB1 g−1) was about 46.7 fold higher than the PMTDI. As for inoculated macadamia nuts spiked with C. incanus extract (maximum: 46.1 ng AFB1 g−1), the exposure was 6.6 fold higher than the PMTDI. It is obvious that C. incanus extract inhibited the AFB1 production in macadamia nuts leading to a reduction of the daily exposure. Even though the daily intake was reduced, it was still higher than the PMTDI. However, this reduction is important because in cases of natural contamination, the AFB1 amounts will be probably lower than these produced in artificially inoculated macadamia (spore inoculum: 100 conidia). On the other hand, even negligible AFB1 quantities are undesirable, since it may contribute to the total daily intake of AFB1. To our knowledge in the literature, there are no studies concerning the occurrence of AFB1 in macadamia nuts. Due to this lack of the scientific research, it is obvious that macadamia nuts must be studied more, since they are used widely in pastry products, cereal breakfast, vegetarian dishes, etc. This type of nut is also used as source for edible oil and for cosmetics products as well. Therefore, there is a need for occurrence studies that will indicate the presence and levels of AFB1 in macadamia nuts while these data could be used for purposes of risk assessment and exposure.
day of observation where it was the maximum AFB1 production. T-test (p < 0.05) analysis showed that there is significant difference between YES medium and macadamia paste substrates for the whole period of observation (df = 10, Texp = 1.812, Ttheor = 3.118). The amounts of AFB1 produced in inoculated macadamia were from 4 μg kg−1 to 327 μg kg−1 (0.0040 μg g−1–0.3270 μg g−1). These levels were above the limit of 2 μg kg−1 as established by EU legislation (Commission Regulation (EC) No. 1881/2006). This fact reveals that macadamia is a substrate that can be easily contaminated with AFB1, even though it is not a favorable substrate for AFB1 production if compared with YES medium, as it was shown in the present study. The production of AFB1 was investigated previously in several substrates. In olives of two different black varieties and in caper the production was not significantly higher against control (YES medium). Although in green olives was stimulated (Ghitakou et al., 2006; Leontopoulos et al., 2003; Meimaroglou et al., 2009). The AFB1 production in YES medium was found to be approximately higher compared with the production in currants originated from Crete and Corinth (Kostarellou et al., 2014). In addition, the bee pollen and sesame seeds’ paste are favorable substrates for AFB1 production when invasion by an aflatoxigenic fungi such as A. parasiticus occurs (Kollia et al., 2016a,b; Pitta and Markaki 2010). Aflatoxin production and fungi’s growth are greatly affected by the identity and concentration of available nutritional factors. Macadamia nuts contain many nutritional components such as trace metals, vitamins, sugars and lipids. The total sugar content of macadamia was estimated to 8.2% while lipids are representing the 70% of their weight (Acquino-Bolanos et al., 2017; Jamieson, 1943; Rengel et al., 2015). Sucrose was identified as the main sugar constituent in macadamia nuts whereas traces of glucose were also detected (Fourie and Basson, 1990). Buchanan and Stahl, (1984) showed that among other sugars, glucose and sucrose supported high levels of aflatoxin production. Moreover, many studies report that the existence of lipids or their derivatives are essential for the fungus to produce aflatoxin (Fabbri et al., 1983; Fanelli and Fabbri, 1989; Meimaroglou et al., 2009). Consequently, macadamia nuts contain several major constituents that are potential fungal nutrient sources and possible stimulators of aflatoxin biosynthesis. Furthemore, the pre-harvest conditions, combined with delayed harvest during rainy seasons, increase the natural microflora of the raw macadamia kernels. So contamination with fungi may result to the mycotoxin occurrence in the final product (Wall, 2013). Another important factor is the moisture content of the nuts during storage (Beuchat, 1978). Meanwhile the nuts are often mixed with other foods without receiving any sterilization treatments, therefore the occurrence of aflatoxin-producing molds on them are of serious concern (Weinert, 1993).
4. Conclusions The present work comprises the first report on antiaflatoxigenic mode of action of C. incanus. It was shown that C. incanus extract displayed a significant inhibition of AFB1 production both in inoculated YES medium (87.1–90.1%) and macadamia nuts (72.5–85.9%). The C. incanus extract was found to inhibit aflatoxin B1 production effectively and it may be able to provide protection of food commodities, enhancing their shelf life. The anti-aflatoxigenic C. incanus extract can be possibly used for reducing or preventing pre/post-harvest AF production and combined with good agricultural, manufacturing and storage practices could lead to a product of guaranteed quality and safety. Aknowledgement This work was supported by the National and Kapodistrian University of Athens, Special Account for Research Grants (11400). References Acquino-Bolanos, E.N., Mapel-Velazco, L., Martin-del-Campo, S.T., Martinez, A.J., Verdalet-Guzman, I., 2017. Fatty acids profile of oil from nine varieties of macadamia nut. Int. J. Food Prop. 20 (6), 1262–1269. Aly, S., Sabry Shaheen, B.M., Hathout, A., 2016. Assessment of antimycotoxigenic and antioxidant activity of star anise (Illicium verum) in vitro. J. Saudi Soc. Agric. Sci. 15 (1), 20–27. Attaguille, G., Campisi, A., Savoca, F., Acquaviva, R., Ragusa, N., Vanella, A., 2000. Antioxidant activity and protective efect on DNA cleavage of extracts from Cistus incanus L. And Cistus monspeliensis L. Cell Biol. Toxicol. 16 (2), 83–90. Beuchat, L.R., 1978. Relationship of water activity to moisture content in tree nuts. J. Food Sci. 43 (3), 754–755. Buchanan, R.L., Stahl, H.G., 1984. Ability of various carbon sources to induce and support aflatoxin synthesis by Aspergillus parasiticus. J. Food Saf. 6, 59–67. Chun, H.S., Kim, H.J., Ok, H.E., Hwang, J.B., Chung, D.H., 2007. Determination of aflatoxin levels in nuts and their products consumed in South Korea. Food Chem. 102, 385–391. Daradimos, E., Markaki, P., Koupparis, M., 2000. Evaluation validation of two fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil. Food Addit. Contam. 17 (4), 65–73. Davis, N.D., Diener, U.L., Eldridge, D.W., 1966. Production of aflatoxin B1 and G1 by Aspergillus flavus in a semisynthetic medium. Appl. Microbiol. 14, 378–380. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed – Council statement. EFSA Panel on Contaminants in the Food Chain, 2007. Opinion of the scientific panel on contaminants in the food chain [CONTAM] related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and derived products. EFSA J. 5 (3), 127–446. European Commission, 2006. Commission Regulation 2006/1881/EC of 19 December 2006 replacing Regulation (EC) 466/2001 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. Union 49 L364/5-24. Fabbri, A.A., Fanelli, C., Panfili, G., Passi, S., Fasella, P., 1983. Lipoperoxidation and
3.4. Risk assessment The most significant aspects in risk analysis of chemical substances are to estimate the degree of human exposure (World Health Organization, 2002). As already stated, AFB1 is a genotoxic carcinogen and a strong acute toxin and for this reason is the only mycotoxin with maximum permitted levels (MPLs) set under Directive 2002/32/EC. Therefore the MPLs estimated for AFB1 have been set as low as reasonably achievable (ALARA) in order to protect public health (EFSA, 2007). Taking into account AFB1 toxicity, the authorities have not established a tolerable daily intake (TDI). Nevertheless, Kuiper-Goodman (1998) assessed a provisional maximum tolerable daily intake (PMTDI) of 1.0 ng AFB1 kg−1 body weight−1 for adults and children without hepatitis B. The established PMTDI has been used in this study for the assessment of aflatoxin ingestion. For the risk analysis, only the maximum amounts of AFB1 produced in each sample were used, while the risk assessment was estimated for an adult (70 kg) consuming approximately 10 g of macadamia nuts, daily. The daily exposure of an adult to AFB1 from inoculated macadamia 67
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al. aflatoxin biosynthesis by Aspergillus parasiticus and A. flavus. J. Gen. Microbiol. 129, 3447–3453. Fanelli, C., Fabbri, A.A., 1989. Relationship between lipids and aflatoxin biosynthesis. Mycopathologia 107, 115–120. Fourie, P.C., Basson, D.S., 1990. Sugar content of almond, pecan, and macadamia nut. J. Agric. Food Chem. 38 (1), 101–104. Garcia, D., Ramos, A.J., Sanchis, V., Marín, S., 2012. Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity. Int. J. Food Microbiol. 153, 21–27. Ghitakou, S., Koutras, K., Kanellou, E., Markaki, P., 2006. Study of aflatoxin B1 and ochratoxin A production by natural microflora and Aspergillus parasiticus in black and green olives of Greek origin. Food Microbiol. 23, 612–621. Gori, A., Ferrini, F., Marzano, M.C., Tattini, M., Centritto, M.C., Pogni, R., Brunetti, C., 2016. Characterisation and antioxidant activity of crude extract and polyphenolic rich fractions from C. incanus leaves. Int. J. Mol. Sci. 17 (8), 1344. International Agency for Research on Cancer (IARC), 2002. Aflatoxins, IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, vol. 82. IARC World Health Organization, Lyon, France, pp. 171–300. Jamieson, G.S., 1943. Vegetable Fats and Oils: Their Chemistry, Production and Utilization for Edible, Medicinal and Technical Purposes, second edition. Reinhold, New York. Kabak, B., 2014. Quantitation of Aflatoxins in walnut kernels by high-performance Liquid Chromatography with Fluorescence detection. Food Addit. Contam. Part B Surveill. 7 (4), 288–294. Kedia, A., Bhanu, P., Prashant, K.M., Dubey, N.K., 2014. Antifungal and antiaflatoxigenic properties of Cuminum cyminum (L.) seed essential oil and its efficacy as a preservative in stored commodities. Int. J. Food Microbiol. 1–7, 168–169. Kollia, E., Kanapitsas, A., Markaki, P., 2014. Occurrence of aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market. Food Addit. Contam.: Part B Surveill. 7 (1), 11–16. Kollia, E., Tsourouflis, K., Markaki, P., 2016a. Aflatoxin B1 in sesame seeds and sesame products from the Greek market. Food Addit Contam.: Part B 9 (3), 1–6. Kollia, E., Proestos, C., Zoumpoulakis, P., Markaki, P., 2016b. Inhibitory effect of Cynara cardunculus L. extract on aflatoxin B1 production by Aspergillus parasiticus in sesame (Sesamum indicum L.). Int. J. Food Prop. 20 (6), 1270–1279. Kollia, E., Proestos, C., Zoumpoulakis, P., Markaki, P., 2017. Comparison of different extraction methods for the determination of the antioxidant and antifungal activity of Cynara scolymus and C. cardunculus extracts and infusions. Nat. Prod. Commun. 12 (3), 423–426. Kostarellou, P., Kanapitsas, A., Pyrri, I., Kapsanaki-Gotsi, E., Markaki, P., 2014. Aflatoxin B1 production by Aspergillus parasiticus strains of Aspergillus section Nigri in currants of greek origin. Food control 43, 121–128. Krishna Reddy, V., Srinivas, M., Rajender Reddy, A., Srujana, G., Surekha, M., Reddy, S.M., 2011. Plant extracts in the management of aflatoxin production by aspergillus flavus. Int. J. Pharm. Bio Sci. 2 (2), B492–B498. Kuiper-Goodman, T., 1998. Food safety, mycotoxins and phycotoxins in perspective. In: Miraglia, M., van Egmond, H.P., Brera, C., Gilbert, J. (Eds.), Mycotoxins and Phycotoxins/developments in Chemistry. Toxicol. food Saf. Fort Collins (CO),
Alakens, pp. 25–48. Leontopoulos, D., Siafaka, A., Markaki, P., 2003. Black olives as substrate for Aspergillus parasiticus growth and aflatoxin B1 production. Food Microbiol. 20, 119–126. Loizzo, M.R., Ben Jemia, M., Senatore, F., Bruno, M., Menichini, F., Tundis, R., 2013. Chemistry and functional properties in prevention of neurodegenerative disorders of five Cistus species essential oils. Food Chem. Toxicol. 59, 586–594. Luchese, R.H., Harrigan, W.F., 1993. Biosynthesis of aflatoxin—the role of nutritional factors. J. Appl. Bacteriol. 74, 5–14. Maggio-Hall, L.A., Wilson, R.A., Keller, N.P., 2005. Fun-damental contribution of β oxidation to polyketide my-cotoxin production in planta. Mol. Plant-Microb. Int. 18 (8), 783–793. Masood, M., Iqbal, S.Z., Asi, M.R., Malik, N., 2015. Natural occurrence of aflatoxins in dry fruits and edible nuts. Food Control 55, 62–65. Meimaroglou, D.M., Galanopoulou, D., Markaki, P., 2009. Study of the effect of methyl jasmonate concentration on aflatoxin B1 biosynthesis by Aspergillus parasiticus in yeast extract sucrose medium. Int. J. Microbiol. 1–7. Pitta, M., Markaki, P., 2010. Study of Aflatoxin B1 production by Aspergillus parasiticus in bee pollen of Greek origin. Mycotox Res. 26 (4), 229–234. Rajasekaran, G., Ford Sethumadhavan, K., Carter-Wienties, C., Bland, J., Cao, H., 2017. Aspergillus flavus growth and aflatoxin production as influenced by total lipid content during growth and development of cottonseed. J. Crop Improv. 31 (1), 91–99. Rengel, A., Pérez, E., Piombo, G., Ricci, J., Servent, R., Tapia, M.S., Gibert, O., Montet, D., 2015. Lipid profile and antioxidant activity of macadamia nuts (Macadamia integrifolia) cultivated in Venezuela. Nat. Sci. 7, 535–547. Riehle, P., Volmer, M., Rohn, S., 2013. Phenolic compounds in Cistus incanus herbal infusions-antioxidant capacity and thermal stability during the brewing process. Food Res. Int. 53 (2), 891–899. Roidaki, A., Kollia, E., Chiou, A., Varzakas, T., Markaki, P., Proestos, C., 2016. Superfoods and superherbs: antioxidant and antifungal activity. Nutr. Food Sci. 4 (2), 138–145. Siciliano, I., Dal Bello, B., Zeppa, G., Spadaro, D., Gullino, M.L., 2017. Static hot air and infrared roasting are efficient methods for aflatoxin decontamination on hazelnuts. Toxins 9 (2), 72. Tzanidi, C., Proestos, C., Markaki, P., 2012. Saffron (Crocus sativus L.) inhibits aflatoxin B1 production by aspergillus parasiticus. Adv. Microbiol. 2, 310–316. Vergopoulou, S., Galanopoulou, D., Markaki, P., 2001. Methyl jasmonate stimulates aflatoxin B1 biosynthesis by Aspergillus parasiticus. J. Agric. Food Chem. 7, 3494–3498. Villa, P., Markaki, P., 2009. Aflatoxin B1 and ochratoxin A in breakfast cereals from Athens market: occurrence and risk assessment. Food Control 20 (5), 455–461. Wall, M.M., 2013. Improving the quality and safety of macadamia nuts. In: Harris, L.J. (Ed.), Improving the Safety and Quality of Nuts. Woodhead Publishing, pp. 274–296. Weinert, A.G., 1993. Macadamia Nut Processing. The Southern African Macadamia Growers Association Yearbook, pp. 54–75. World Health Organization, 2002. Evaluation of Certain Mycotoxins in Food. World Health Organization (WHO technical report series, 906), Geneva. Yazdani, D., Mior Ahmad, Z.A., Yee How, T., Jaganath, I.B., Shahnazi, S., 2013. Inhibition of aflatoxin biosynthesis in Aspergillus flavus by phenolic compounds extracted of Piper betle L. Iran J. Microbiol. 5 (4), 428–433.
68
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Research Paper
Cistus incanus L. extract inhibits Aflatoxin B1 production by Aspergillus parasiticus in macadamia nuts Venetia Kalli, Eleni Kollia, Anna Roidaki, Charalampos Proestos, Panagiota Markaki
MARK
⁎
Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15784 Athens, Greece
A R T I C L E I N F O
A B S T R A C T
Keywords: Aflatoxin B1 Aspergillus parasiticus Cistus incanus L. Macadamia
Aflatoxins are secondary metabolites, with aflatoxin B1 being the most common, reported as carcinogenic, teratogenic and genotoxic. This study investigates the antiaflatoxigenic efficacy of the herbaceous plant Cistus incanus L. against Aspergillus parasiticus in two substrates, yeast extract sucrose medium and macadamia nuts. The methanolic extract of Cistus incanus showed pronounced antiaflatoxigenic ability, inhibiting aflatoxin B1 production in both substrates. AFB1 production was decreased significantly in a percentage of 87.1–90.1% after Cistus incanus extract addition in YES medium. The extract effectiveness was also observed in macadamia nuts, where the AFB1 production by Aspergillus parasiticus was reduced in a percentage of 72.5–85.9%. Moreover, the risk assessment was estimated taking into account the maximum amounts of AFB1 produced in inoculated samples with and without Cistus incanus addition. It was revealed that Cistus incanus presence leads to a lesser exposure of AFB1 to consumers.
1. Introduction
et al., 2017; Rengel et al., 2015). Generally, nuts rich in lipids are known to be possibly contaminated with aflatoxins and many studies have shown that contamination often exceeds the legal threshold in commercial samples (Siciliano et al., 2017). The genus Cistus belongs to the family of Cistaceae, which is one of the characteristic genera of the Mediterranean region and comprises several medicinal plants of perennial shrubs. These Mediterranean shrubs species, such as C. incanus, are naturally rich in polyphenols. C. incanus is a thermophilic plant which requires much light and for this reason it’s growing in warm and temperate places (Gori et al., 2016). Studies showed that the extract of C. incanus has antifungal, antibacterial and antiviral properties, because of its constituents which includes tannins, flavonoids and other bioactive compounds (Attaguille et al., 2000; Riehle et al., 2013; Roidaki et al., 2016) In traditional medicine, C. incanus has been used in anti-inflammatory, anti-allergic, antiulcerogenic, wound healing, antimicrobial, cytotoxic and vasodilator remedies (Riehle et al., 2013). In recent years, the scientific community has focused on the use of natural products to control the mold growth and inactivate mycotoxins. The reason for this trend of scientific research is that most plants produce naturally, antimicrobial secondary metabolites, during their development or in response to stress conditions (infection, wounding etc.) (Aly et al., 2016). These secondary metabolites are usually bioactive compounds such as phenolics that are known to exhibit antioxidant, anti-inflammatory, antihepatotoxic, antitumor, antimicrobial and
Aflatoxins (AFs) are secondary metabolites, produced by certain Aspergillus sp. such as Aspergillus flavus and Aspergillus parasiticus. The most common aflatoxins are aflatoxin B1, B2, G1, G2 and are characterized as carcinogenic, teratogenic and genotoxic, while aflatoxin B1 (AFB1), is the most prevalent of the AFs and has been characterized as human carcinogen belonging in Group I (International Agency for Research on Cancer (IARC), 2002). Because aflatoxigenic fungal contamination is unavoidable, numerous crops (cereals, sesame seed, dried fruits and other nuts, peanuts, pistachios etc.) are lost annually (Kollia et al., 2016a; Kollia et al., 2014; Villa and Markaki, 2009). The fungal and aflatoxin contamination that cause quantitative and qualitative losses of food commodities can also potentially induce various health problems to consumers (Kedia et al., 2014). Such losses of foodstuffs and their importance in international food trade, require the necessity for suitable and effective control actions. According to literature, it is pointed out that, the presence of lipids in foodstuffs stimulates biosynthesis of AFB1 (Fanelli and Fabbri, 1989; Kollia et al., 2016a,b; Rajasekaran et al., 2017). Macadamia nuts are originated from eastern Australia and they are rich in lipids which are representing more than 70% of their weight. Moreover, macadamia nuts are also rich in trace elements (calcium, phosphorus, iron etc.) and vitamins (thiamine, riboflavin, retinol and niacin) (Acquino-Bolanos
⁎
Corresponding author. E-mail address: [email protected] (P. Markaki).
http://dx.doi.org/10.1016/j.indcrop.2017.10.003 Received 21 June 2017; Received in revised form 29 August 2017; Accepted 2 October 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
2.3. Reagents
antifungal activities (Kollia et al., 2016b; Tzanidi et al., 2012). Many studies have demonstrated a strong correlation between the phenolic/ antioxidant compounds of plants and their antifungal/antiaflatoxigenic activities. Consequently plants’ extracts could potentially provide protection against aflatoxigenic fungus and aflatoxins. Redox reactions are fundamental to cellular catabolism and anabolism processes. Antioxidants could interfere, reducing the pool of nicotinamide adenine dinucleotide phosphate (reduced form) available for AF biosynthetic reactions (Maggio-Hall et al., 2005). Furthermore, data from the literature indicate an association of the antioxidant profile of plants with their possibly antimicrobial, antifungal and anti-aflatoxigenic efficiency (Kollia et al., 2017; Kollia et al., 2016b; , Loizzo et al., 2013; Tzanidi et al., 2012). Previously many plant extracts have been investigated for their capacity to inhibit aflatoxin production not only in culture medium conditions but also in food commodities. Particularly Equisetum arvense, Stevia rebaudiana, Zingiber officinalis, Oxalis corniculata and Trigonella foenum-graecum have been reported to have antifungal and antiaflatoxigenic properties (Garcia et al., 2012; Krishna Reddy et al., 2011). Moreover Yazdani et al. (2013) showed that Piper betle L. was able to reduce the aflatoxin production by A. flavus. Due to these facts, the scientific community is searching for new natural bioactive compounds, which can possibly act as suppressor of aflatoxins. In this work macadamia nuts were evaluated as natural substrate for AFB1 production by A. parasiticus and compared with synthetic medium yeast extract sucrose (YES). C. incanus plant was examined for the first time to our knowledge, for its effect on AFB1 biosynthesis by A. parasiticus on both substrates. In addition, the efficiency of a routine analysis by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector for the determination of AFB1 in macadamia nut was evaluated. In the present study, the risk for the consumers is also assessed, before and after addition of C. incanus in inoculated nuts.
Aflatoxin B1 standard was obtained from Sigma (st. Louis, MO, USA). The Aflatest immunoaffinity columns were purchased from Vicam (Watertown, MA, USA). All reagents were analytical grade. The HPLC solvents were bought from Fischer Chemical (Fischer Scientific, Loughborough, Leicestershire, UK) while hexane and methanol (pro analysis grade) were from Merck (Darmstadt, Germany) and trifluoroacetic acid was from Fluka (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). 2.4. Culture media For the preparation of Aspergillus Flavus Parasiticus Agar (AFPA), 2 g of yeast extract (Oxoid, Basingstoke, Hampshire, UK), 1 g of bacteriological peptone (Oxoid), 0.05 g of ferric ammonium citrate (Merck, Germany), 0.1 mL of Dichloran 0.2% in ethanol (Fluka Steinheim, The Netherlands), 0.01 g of chloramphenicol (Oxoid), and 1.5 g of agar (Oxoid) were dissolved per 100 mL of distilled water. The final pH was adjusted to 6.0–6.5. 2.5. Experimental design The experimental design of this study is shown in Table 1. All samples were examined for AFB1 production on days 0, 3, 7, 9, 12, and 15 of incubation. The experiments were repeated thrice. 2.6. Extraction of Cistus incanus For the preparation of C. incanus extract, two different extraction procedures were combined. A conventional extraction procedure was performed, followed by an ultrasound-assisted extraction. Specifically, one gram of the dried ground sample was placed in a beaker and mixed with 30 mL of methanol:water (80:20 v/v) solution acidified with 0.1% HCl. After 24 h in the dark, at room temperature the beakers were sonicated thrice (10 mL of solvent each time) for 15 min in the ultrasonic device. After the extraction, filtration took place using Buchner funnel. The extracts were evaporated to dryness, were redissolved to 5 mL of MeOH and kept at −20 °C until analysis. The extraction procedures were performed in triplicate (Roidaki et al., 2016).
2. Materials and methods 2.1. Sampling and treatment Macadamia nuts were purchased from a local market in Athens (Greece), weighting approximately 3 kg. Macadamia nuts were washed with water, dried and homogenized forming a paste. Then macadamia nuts were sterilized at 110 °C for 2 min, in order to minimize the natural microbial load before inoculation with A. parasiticus. An amount of 10 g of treated macadamia were transferred in flasks under aseptic conditions, in such a way as to create a single layer. Cistus incanus in dried form was purchased from a local farmer in Chalkidiki (North Greece) during winter of 2015.
2.7. Preparation of spore inoculum The strain A. parasiticus speare (IMI 283883) used during this work was obtained from the International Mycological Institute (Engham Surrey, UK). The isolate was subcultured on potato dextrose agar slants and incubated at 30 °C for 7 days. Inoculum suspensions were prepared from the 7 days culture mentioned above. Spores were harvested aseptically using 10 mL of sterile 0.01% v/v Tween 80 solution. Aflatoxin B1 carried over from the initial growth
2.2. Apparatus The extraction of C. incanus plant was performed using an ultrasonic bath device (Elmasonic S, Elma Schmidbauer GmbH, Germany) at a frequency of 37 kHz. The extract was evaporated to dryness using a rotary evaporator (Heidolph, Laborota 4000 efficient, WB eco). In addition High-Performance Liquid Chromatography (HPLC) analysis was done using a Hewlett-Packard 1050 (Hewlett-Packard, Waldborn, Germany) liquid chromatograph with a JASCO FP-920 (Jasco Ltd., Tokyo, Japan) fluorescence detector and an HP integrator 3395.The HPLC column was a C18 Nova-Pak (4.6 × 250 mm, 4 μm, 60 Å, WatersMillipore, Milford, MA, USA). For the filtration of the mobile phase (water: acetonitrile: methanol [20:4:3 v/v/v]) for AFB1 determination, Millipore HA-VLP (0.45 μm) filters were used. AFB1 was derivatized to its hemiacetal (AFB2a) and was detected at λex = 365 nm/ λem = 425 nm. The retention time was 13.58 ± 0.38 min, while flow rate was 1 mL min−1.
Table 1 Groups of inoculated samples. YESa (10 mL) + 102 conidia of A. parasiticus + 100 μL methanol YES (10 mL) + 102 conidia of A. parasiticus without methanol addition YES (10 mL) + 102 conidia of A. parasiticus + 100 μL of C. incanus extractb Macadamia paste (10 g) + 102 conidia of A. parasiticus + 100 μL methanol Macadamia paste (10 g) + 102 conidia of A. parasiticus + 100 μL C. incanus extractb Macadamia paste (10 g) + 102 conidia of A. parasiticus Macadamia paste (10 g) (control)c Three flasks were used for each case and for each day of observation. a Yeast Extract Sucrose Medium. b Concentration of extract: 0.2 g of C. incanus fresh weight mL−1 methanol. c Non-inoculated, without C. incanus addition.
64
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
was reduced by centrifuging the spore suspension (2500 rpm for 10 min) and dissolving the biomass in 10 mL of sterile Tween 80 solution twice. Dilutions (10−1, 10−2, 10−3, 10−4) from the initial spore suspension in sterile tubes containing 10 mL of Tween 80 0.05% v/v were made. The spore concentration was determined by the spread plate surface count technique using 0.1 mL of each dilution on four AFPA plates incubated at 30 °C for 48 h. To accomplish an inoculum of 100 conidia, petri dishes (AFPA) with 10–100 colony-forming units (cfu) were chosen and the number of 100 spores was estimated and used in the specific experiment (Kostarellou et al., 2014; Vergopoulou et al., 2001).
Table 2 Accuracy of the method for AFB1a determination applied in macadamia nuts. AFB1 (ng) spike in ng g−1 macadamia nuts 5
2.8. Inoculation of YES medium and macadamia nuts In Table 1 is shown that for each day of incubation, three flasks containing 10 mL YES medium and three flasks containing 10 g of macadamia nuts, were inoculated with 100 conidia of A. parasiticus per flask. Then 100 μL of C. incanus methanolic extract were added in the inoculated flasks. For the control cultures, 100 μL of methanol alone (without the addition of C. incanus extract) were added in the inoculated with the fungus samples. All flasks were incubated at stationary conditions, at 30 °C and AFB1 production was examined on days 0, 3, 7, 9, 12, and 15.
a
20
40
50
Measured AFB1 (ng) in ng g−1 macadamia nuts
Samples 1 2 3 4 Mean SD RSD%
10
4.17 3.74 5.05 4.36 4.33 0.55 12.60
9.84 8.84 11.8 10.27 10.19 1.23 12.08
23.29 20.22 19.66 23.68 21.71 2.07 9.51
42.15 37.86 49.59 43.41 43.26 4.85 11.21
61.19 54.95 55.98 48.57 55.17 5.19 9.41
Aflatoxin B1.
AFB1 g−1). The reproducibility was examined by spiking with AFB1 (100 ng), four samples (10 g) of macadamia nuts in several time-periods, under the same conditions. The coefficient of variation (RSD) was found to be 4.51% (mean: 10.27 ng AFB1 g−1). Furthermore, the repeatability (r) and reproducibility (R) limits were calculated as following: r = 2.8SDr and R = 2.8SDR and they were found to be r = 12.98 and R = 18.54, respectively. In the present study, accuracy was estimated by analyzing samples of macadamia paste (10 g) spiked with different amounts of AFB1. The regression coefficient r was 0.9984 and the mean recovery was found to be 112.10% (RSD = 8.47%) [y = 1.1201( ± 0.0931)x − 1.0695 ( ± 0.63806)] (Table 2). A Fisher test was applied in order to examine the reliability of the accuracy linear regression analysis. The F (1, 15) ratio 11742.5 was greater than the critical Fisher value of 16.587 at an alpha risk of 0.1% for 1 and 5° of freedom. Consequently, the regression model is satisfactory. In addition, the lack of fit of the model was found to be low, as the experimental F ratio (0.635) was found less than the critical value F = 9.335 with a risk a = 0.1% and df = 3,15. Therefore, the linearity is acceptable. The limits of determination (LOD) and quantification (LOQ) were calculated using the following equations
2.9. AFB1 determination and HPLC analysis Ten grams of grounded macadamia nuts was mixed with 30 mL methanol/water (80:20 v/v) and shaken well for 10 min. After filtration, an aliquot of 1 mL was used for AFB1 analysis. For the clean-up procedure, 1 mL from the filtrate was diluted with 10 mL distilled water for 1 min. The mixture was loaded into an Aflatest immunoaffinity column (flow rate 3 mL min−1) and washed two times with 10 mL of distilled water. Then the moisture was removed from the column by passing air. Finally AFB1 was eluted with 2 mL of acetonitrile in dark glass vials and the solvent was evaporated under a mild stream of nitrogen. The derivatization of AFB1 to AFB2a (hemiacetical of AFB1) was followed. A mixture of 200 μL hexane and 200 μL of trifluoroacetic acid were added to the evaporated AFB1 solution. The derivatization was completed by heating at 40 °C in a water bath for 10 min. After that the solution was evaporated to aridity, dissolved in 200 μL water:acetonitrile (9:1 v/v) and analyzed by HPLC with fluorescence detector (volume injected = 40 μL) (Daradimos et al., 2000).
LOD = {b0 + 3S(b0)}/b1, LOQ = {b0 + 10 (b0)}/b1 where b0 is the response of the blank, S(b0) is the standard deviation of the blank and b1 is the sensitivity. The LOD and LOQ were found to be 1.17 ng g−1 and 3.56 ng g−1 respectively. Previously in the literature it was reported that the recovery in a study for AFB1 determination in several nuts, was estimated at 102.0% while LOD and LOQ were 0.08 ng g−1 and 0.15 ng g−1 respectively (Chun et al., 2007). In another study, Kabak (2014) reported recoveries from 95.7% to 96.4% in walnuts and LOD and LOQ at 0.02 ng g−1 and 0.07 ng g−1 respectively. Moreover Masood et al. (2015) in a study concerning the natural occurrence of aflatoxins in edible nuts found that the recoveries of the fortified samples were from 83% to 90% while the LOD for AFB1 was 0.04 ng g−1and the LOQ 0.12 ng g−1. In the present study, the LOD (1.17 ng g−1), LOQ (3.56 ng g−1) and the accuracy (recovery factor: 112.10%) of the method mentioned above, are in agreement with the requirements of the Commission Regulation (EC) No 401/2006 (European Commission, 2006). Consequently, the HPLC method developed in the present study, for the determination of AFB1 in macadamia nuts, is sensitive, selective, efficient, and accurate and it can be used for research purposes or as a routine analysis method as well.
3. Results and discussion 3.1. Development and validation of AFB1 determination method in macadamia nuts A sensitive and selective HPLC method was developed and characterized in-house, for the determination of AFB1 in macadamia nuts. The method was validated observing parameters of the compliance criteria. Analytical parameters were investigated including linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), repeatability (r) and reproducibility (R). A solution at concentration of 5 μg AFB1 mL−1 MeOH was prepared from the initial stock solution of AFB1 (1 mg mL−1) by diluting 100 μL in 9.900 μL of methanol. A series of standards in the final concentration of AFB1 corresponding to 20, 10, 5, 2,5, 2 and 1 ng AFB1 mL was prepared in mobile phase (water: acetonitrile: methanol [20:4:3 v/v/v]) and 40 μL of each standard were injected into the column. The regression equation of the standard curve was: y = 2 × 107x + 520087 r2 = 0.996. Repeatability was examined by analyzing four sub-samples (10 g) of the matrix spiked with 100 ng of AFB1 corresponding to 10 ng g−1. The coefficient of variation (RSD) was found to be 6.59% (mean: 10.05 ng
3.2. Evaluation of C. incanus extract as AFB1 suppressor in YES medium As mentioned earlier, in this work C. incanus was selected to study 65
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
for its effect on AFB1 production by A. parasiticus. To the best of our knowledge, so far there has not been any relevant study on the effectiveness of C. incanus extract against AFB1 production by A. parasiticus. In a previous study of our laboratory, C. incanus was investigated for its antioxidant and antifungal activity (Roidaki et al., 2016). The analysis revealed that among a variety of plants, C. incanus possessed significant antioxidant capacity and high total phenolic content. Moreover, the extract of C. incanus (concentration 0.2 g mL−1) showed the strongest antifungal activity, inhibiting the A. parasiticus growth in a percentage of 45.91%. Therefore in the present study, the same extract in the same concentration was evaluated as AFB1 suppressor in YES medium. Yeast Extract Sucrose (YES) was chosen as the culture medium which is beneficial for development and AFB1 biosynthesis of A. parasiticus and A. flavus. AFB1 was studied as it is the most potent carcinogenic mycotoxin and it is usually produced at high levels by the fungus A. parasiticus (Davis et al., 1966; Luchese and Harrigan, 1993). The inoculum of 100 spores was chosen for the inoculation of the samples, as it is the minimum amount of spores found in the literature that produce high levels of AFB1 by A. parasiticus (Vergopoulou et al., 2001). The analytical method for AFB1 determination in YES medium has been previously developed in our laboratory. The recovery of the method was found 99.2% while the detection limit of the derivatized AFB1 (AFB2a) was 0.02 ng AFB1 mL−1 (Leontopoulos et al., 2003). AFB1 production in YES medium inoculated with A. parasiticus and supplemented with C. incanus extract, was continuing, until the last day (15th) of observation where the production of AFB1 was 0.532 μg g−1. In control samples (without the extract addition) the AFB1 production was also increasing until the 15th day (maximum production 5.143 μg g−1). The addition of C. incanus extract (100 μL of 0.2 g mL−1 MeOH) in YES medium cultures inoculated with A. parasiticus showed a significant inhibition of AFB1 production compared with control cultures (df = 10, Texp = 2.888, Ttheor = 2.228, p < 0.05). In the specific study, C. incanus extract was suppressed significantly the AFB1 production by A. parasiticus in YES medium. The AFB1 reduction amounted from 87.1% to 90.1% after the 3rd day of observation (Table 3, Fig. 1).
Fig. 1. C. incanus extract inhibited AFB1 production by A. parasiticus in YES medium in comparison to AFB1 production in control YES cultures.
Table 4 AFB1a production (μg g−1) by A. parasiticus in inoculated macadamia nuts with and without Cistus incanus extract addition. Days
Macadamia nuts + A. parasiticus + 100 μL MeOHb (control)
Macadamia nuts + A. parasiticus + 100 μL C. incanus extractc
0 3 7 9 12 15
0 0.0040 0.0195 0.0359 0.3270 0.3010
0 0.0011 0.0024 0.0075 0.0461 0.0458
a b c
± ± ± ± ±
0.01 0.01 0.02 0.01 0.01
± ± ± ± ±
0.00 0.00 0.01 0.12 0.10
Aflatoxin B1. Methanol. Concentration of extract: 0.2 g fresh weight mL−1 methanol.
3.3. Evaluation of C. incanus extract as AFB1 suppressor in macadamia In this work, the extract of C. incanus was found efficacious inhibiting aflatoxin B1 production by A. parasiticus in macadamia paste. The declining trend of the AFB1 production in macadamia treated with C. incanus was estimated from 72.5% to 85.9%. In Table 4 is shown that maximum AFB1 production was observed on 12th day of incubation for both inoculated macadamia samples spiked with C. incanus extract and macadamia control samples (0.0461 μg g−1 0.327 μg g−1, respectively) (Τable 4, Fig. 2). Additionally, in order to evaluate the findings of the present study, macadamia nuts were examined as substrate for AFB1 production by A. parasiticus in comparison with YES medium. As mentioned previously YES medium is a conductive substrate that supports AF production. As
Fig. 2. C. incanus extract inhibited the AFB1 production by A. parasiticus in macadamia nuts in comparison to AFB1 production in control macadamia cultures.
Table 3 AFB1a production (μg g−1) by A. parasiticus in YESb medium in comparison with AFB1 production in YESb with Cistus incanus extract addition. Days
YES + A. parasiticus + 100 μL MeOHc (control)
YES + A. parasiticus + 100 μL of C. incanus.d
0 3 7 9 12 15
0 1.012 2.150 3.369 4.786 5.143
0 0.093 0.256 0.435 0.491 0.532
a b c d
± ± ± ± ±
1.80 2.07 1.78 2.24 2.06
± ± ± ± ±
0.16 0.42 0.54 0.33 0.42
Fig. 3. AFB1 production by A. parasiticus in YES medium and in macadamia nuts. The AFB1 production in macadamia substrate is significantly lower if compared with AFB1 production in the favorable microbiological yeast extract sucrose medium (YES).
shown in Fig. 3, the AFB1 production in YES medium was increased until the 15th day of observation. However, AFB1 production in inoculated with A .parasiticus macadamia was increased until the 12th
Aflatoxin B1. Yeast extract sucrose medium. Methanol. Concentration of extract: 0.2 g fresh weight mL−1 methanol.
66
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al.
sample (maximum: 327 ng AFB1 g−1) was about 46.7 fold higher than the PMTDI. As for inoculated macadamia nuts spiked with C. incanus extract (maximum: 46.1 ng AFB1 g−1), the exposure was 6.6 fold higher than the PMTDI. It is obvious that C. incanus extract inhibited the AFB1 production in macadamia nuts leading to a reduction of the daily exposure. Even though the daily intake was reduced, it was still higher than the PMTDI. However, this reduction is important because in cases of natural contamination, the AFB1 amounts will be probably lower than these produced in artificially inoculated macadamia (spore inoculum: 100 conidia). On the other hand, even negligible AFB1 quantities are undesirable, since it may contribute to the total daily intake of AFB1. To our knowledge in the literature, there are no studies concerning the occurrence of AFB1 in macadamia nuts. Due to this lack of the scientific research, it is obvious that macadamia nuts must be studied more, since they are used widely in pastry products, cereal breakfast, vegetarian dishes, etc. This type of nut is also used as source for edible oil and for cosmetics products as well. Therefore, there is a need for occurrence studies that will indicate the presence and levels of AFB1 in macadamia nuts while these data could be used for purposes of risk assessment and exposure.
day of observation where it was the maximum AFB1 production. T-test (p < 0.05) analysis showed that there is significant difference between YES medium and macadamia paste substrates for the whole period of observation (df = 10, Texp = 1.812, Ttheor = 3.118). The amounts of AFB1 produced in inoculated macadamia were from 4 μg kg−1 to 327 μg kg−1 (0.0040 μg g−1–0.3270 μg g−1). These levels were above the limit of 2 μg kg−1 as established by EU legislation (Commission Regulation (EC) No. 1881/2006). This fact reveals that macadamia is a substrate that can be easily contaminated with AFB1, even though it is not a favorable substrate for AFB1 production if compared with YES medium, as it was shown in the present study. The production of AFB1 was investigated previously in several substrates. In olives of two different black varieties and in caper the production was not significantly higher against control (YES medium). Although in green olives was stimulated (Ghitakou et al., 2006; Leontopoulos et al., 2003; Meimaroglou et al., 2009). The AFB1 production in YES medium was found to be approximately higher compared with the production in currants originated from Crete and Corinth (Kostarellou et al., 2014). In addition, the bee pollen and sesame seeds’ paste are favorable substrates for AFB1 production when invasion by an aflatoxigenic fungi such as A. parasiticus occurs (Kollia et al., 2016a,b; Pitta and Markaki 2010). Aflatoxin production and fungi’s growth are greatly affected by the identity and concentration of available nutritional factors. Macadamia nuts contain many nutritional components such as trace metals, vitamins, sugars and lipids. The total sugar content of macadamia was estimated to 8.2% while lipids are representing the 70% of their weight (Acquino-Bolanos et al., 2017; Jamieson, 1943; Rengel et al., 2015). Sucrose was identified as the main sugar constituent in macadamia nuts whereas traces of glucose were also detected (Fourie and Basson, 1990). Buchanan and Stahl, (1984) showed that among other sugars, glucose and sucrose supported high levels of aflatoxin production. Moreover, many studies report that the existence of lipids or their derivatives are essential for the fungus to produce aflatoxin (Fabbri et al., 1983; Fanelli and Fabbri, 1989; Meimaroglou et al., 2009). Consequently, macadamia nuts contain several major constituents that are potential fungal nutrient sources and possible stimulators of aflatoxin biosynthesis. Furthemore, the pre-harvest conditions, combined with delayed harvest during rainy seasons, increase the natural microflora of the raw macadamia kernels. So contamination with fungi may result to the mycotoxin occurrence in the final product (Wall, 2013). Another important factor is the moisture content of the nuts during storage (Beuchat, 1978). Meanwhile the nuts are often mixed with other foods without receiving any sterilization treatments, therefore the occurrence of aflatoxin-producing molds on them are of serious concern (Weinert, 1993).
4. Conclusions The present work comprises the first report on antiaflatoxigenic mode of action of C. incanus. It was shown that C. incanus extract displayed a significant inhibition of AFB1 production both in inoculated YES medium (87.1–90.1%) and macadamia nuts (72.5–85.9%). The C. incanus extract was found to inhibit aflatoxin B1 production effectively and it may be able to provide protection of food commodities, enhancing their shelf life. The anti-aflatoxigenic C. incanus extract can be possibly used for reducing or preventing pre/post-harvest AF production and combined with good agricultural, manufacturing and storage practices could lead to a product of guaranteed quality and safety. Aknowledgement This work was supported by the National and Kapodistrian University of Athens, Special Account for Research Grants (11400). References Acquino-Bolanos, E.N., Mapel-Velazco, L., Martin-del-Campo, S.T., Martinez, A.J., Verdalet-Guzman, I., 2017. Fatty acids profile of oil from nine varieties of macadamia nut. Int. J. Food Prop. 20 (6), 1262–1269. Aly, S., Sabry Shaheen, B.M., Hathout, A., 2016. Assessment of antimycotoxigenic and antioxidant activity of star anise (Illicium verum) in vitro. J. Saudi Soc. Agric. Sci. 15 (1), 20–27. Attaguille, G., Campisi, A., Savoca, F., Acquaviva, R., Ragusa, N., Vanella, A., 2000. Antioxidant activity and protective efect on DNA cleavage of extracts from Cistus incanus L. And Cistus monspeliensis L. Cell Biol. Toxicol. 16 (2), 83–90. Beuchat, L.R., 1978. Relationship of water activity to moisture content in tree nuts. J. Food Sci. 43 (3), 754–755. Buchanan, R.L., Stahl, H.G., 1984. Ability of various carbon sources to induce and support aflatoxin synthesis by Aspergillus parasiticus. J. Food Saf. 6, 59–67. Chun, H.S., Kim, H.J., Ok, H.E., Hwang, J.B., Chung, D.H., 2007. Determination of aflatoxin levels in nuts and their products consumed in South Korea. Food Chem. 102, 385–391. Daradimos, E., Markaki, P., Koupparis, M., 2000. Evaluation validation of two fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil. Food Addit. Contam. 17 (4), 65–73. Davis, N.D., Diener, U.L., Eldridge, D.W., 1966. Production of aflatoxin B1 and G1 by Aspergillus flavus in a semisynthetic medium. Appl. Microbiol. 14, 378–380. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed – Council statement. EFSA Panel on Contaminants in the Food Chain, 2007. Opinion of the scientific panel on contaminants in the food chain [CONTAM] related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and derived products. EFSA J. 5 (3), 127–446. European Commission, 2006. Commission Regulation 2006/1881/EC of 19 December 2006 replacing Regulation (EC) 466/2001 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. Union 49 L364/5-24. Fabbri, A.A., Fanelli, C., Panfili, G., Passi, S., Fasella, P., 1983. Lipoperoxidation and
3.4. Risk assessment The most significant aspects in risk analysis of chemical substances are to estimate the degree of human exposure (World Health Organization, 2002). As already stated, AFB1 is a genotoxic carcinogen and a strong acute toxin and for this reason is the only mycotoxin with maximum permitted levels (MPLs) set under Directive 2002/32/EC. Therefore the MPLs estimated for AFB1 have been set as low as reasonably achievable (ALARA) in order to protect public health (EFSA, 2007). Taking into account AFB1 toxicity, the authorities have not established a tolerable daily intake (TDI). Nevertheless, Kuiper-Goodman (1998) assessed a provisional maximum tolerable daily intake (PMTDI) of 1.0 ng AFB1 kg−1 body weight−1 for adults and children without hepatitis B. The established PMTDI has been used in this study for the assessment of aflatoxin ingestion. For the risk analysis, only the maximum amounts of AFB1 produced in each sample were used, while the risk assessment was estimated for an adult (70 kg) consuming approximately 10 g of macadamia nuts, daily. The daily exposure of an adult to AFB1 from inoculated macadamia 67
Industrial Crops & Products 111 (2018) 63–68
V. Kalli et al. aflatoxin biosynthesis by Aspergillus parasiticus and A. flavus. J. Gen. Microbiol. 129, 3447–3453. Fanelli, C., Fabbri, A.A., 1989. Relationship between lipids and aflatoxin biosynthesis. Mycopathologia 107, 115–120. Fourie, P.C., Basson, D.S., 1990. Sugar content of almond, pecan, and macadamia nut. J. Agric. Food Chem. 38 (1), 101–104. Garcia, D., Ramos, A.J., Sanchis, V., Marín, S., 2012. Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity. Int. J. Food Microbiol. 153, 21–27. Ghitakou, S., Koutras, K., Kanellou, E., Markaki, P., 2006. Study of aflatoxin B1 and ochratoxin A production by natural microflora and Aspergillus parasiticus in black and green olives of Greek origin. Food Microbiol. 23, 612–621. Gori, A., Ferrini, F., Marzano, M.C., Tattini, M., Centritto, M.C., Pogni, R., Brunetti, C., 2016. Characterisation and antioxidant activity of crude extract and polyphenolic rich fractions from C. incanus leaves. Int. J. Mol. Sci. 17 (8), 1344. International Agency for Research on Cancer (IARC), 2002. Aflatoxins, IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, vol. 82. IARC World Health Organization, Lyon, France, pp. 171–300. Jamieson, G.S., 1943. Vegetable Fats and Oils: Their Chemistry, Production and Utilization for Edible, Medicinal and Technical Purposes, second edition. Reinhold, New York. Kabak, B., 2014. Quantitation of Aflatoxins in walnut kernels by high-performance Liquid Chromatography with Fluorescence detection. Food Addit. Contam. Part B Surveill. 7 (4), 288–294. Kedia, A., Bhanu, P., Prashant, K.M., Dubey, N.K., 2014. Antifungal and antiaflatoxigenic properties of Cuminum cyminum (L.) seed essential oil and its efficacy as a preservative in stored commodities. Int. J. Food Microbiol. 1–7, 168–169. Kollia, E., Kanapitsas, A., Markaki, P., 2014. Occurrence of aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market. Food Addit. Contam.: Part B Surveill. 7 (1), 11–16. Kollia, E., Tsourouflis, K., Markaki, P., 2016a. Aflatoxin B1 in sesame seeds and sesame products from the Greek market. Food Addit Contam.: Part B 9 (3), 1–6. Kollia, E., Proestos, C., Zoumpoulakis, P., Markaki, P., 2016b. Inhibitory effect of Cynara cardunculus L. extract on aflatoxin B1 production by Aspergillus parasiticus in sesame (Sesamum indicum L.). Int. J. Food Prop. 20 (6), 1270–1279. Kollia, E., Proestos, C., Zoumpoulakis, P., Markaki, P., 2017. Comparison of different extraction methods for the determination of the antioxidant and antifungal activity of Cynara scolymus and C. cardunculus extracts and infusions. Nat. Prod. Commun. 12 (3), 423–426. Kostarellou, P., Kanapitsas, A., Pyrri, I., Kapsanaki-Gotsi, E., Markaki, P., 2014. Aflatoxin B1 production by Aspergillus parasiticus strains of Aspergillus section Nigri in currants of greek origin. Food control 43, 121–128. Krishna Reddy, V., Srinivas, M., Rajender Reddy, A., Srujana, G., Surekha, M., Reddy, S.M., 2011. Plant extracts in the management of aflatoxin production by aspergillus flavus. Int. J. Pharm. Bio Sci. 2 (2), B492–B498. Kuiper-Goodman, T., 1998. Food safety, mycotoxins and phycotoxins in perspective. In: Miraglia, M., van Egmond, H.P., Brera, C., Gilbert, J. (Eds.), Mycotoxins and Phycotoxins/developments in Chemistry. Toxicol. food Saf. Fort Collins (CO),
Alakens, pp. 25–48. Leontopoulos, D., Siafaka, A., Markaki, P., 2003. Black olives as substrate for Aspergillus parasiticus growth and aflatoxin B1 production. Food Microbiol. 20, 119–126. Loizzo, M.R., Ben Jemia, M., Senatore, F., Bruno, M., Menichini, F., Tundis, R., 2013. Chemistry and functional properties in prevention of neurodegenerative disorders of five Cistus species essential oils. Food Chem. Toxicol. 59, 586–594. Luchese, R.H., Harrigan, W.F., 1993. Biosynthesis of aflatoxin—the role of nutritional factors. J. Appl. Bacteriol. 74, 5–14. Maggio-Hall, L.A., Wilson, R.A., Keller, N.P., 2005. Fun-damental contribution of β oxidation to polyketide my-cotoxin production in planta. Mol. Plant-Microb. Int. 18 (8), 783–793. Masood, M., Iqbal, S.Z., Asi, M.R., Malik, N., 2015. Natural occurrence of aflatoxins in dry fruits and edible nuts. Food Control 55, 62–65. Meimaroglou, D.M., Galanopoulou, D., Markaki, P., 2009. Study of the effect of methyl jasmonate concentration on aflatoxin B1 biosynthesis by Aspergillus parasiticus in yeast extract sucrose medium. Int. J. Microbiol. 1–7. Pitta, M., Markaki, P., 2010. Study of Aflatoxin B1 production by Aspergillus parasiticus in bee pollen of Greek origin. Mycotox Res. 26 (4), 229–234. Rajasekaran, G., Ford Sethumadhavan, K., Carter-Wienties, C., Bland, J., Cao, H., 2017. Aspergillus flavus growth and aflatoxin production as influenced by total lipid content during growth and development of cottonseed. J. Crop Improv. 31 (1), 91–99. Rengel, A., Pérez, E., Piombo, G., Ricci, J., Servent, R., Tapia, M.S., Gibert, O., Montet, D., 2015. Lipid profile and antioxidant activity of macadamia nuts (Macadamia integrifolia) cultivated in Venezuela. Nat. Sci. 7, 535–547. Riehle, P., Volmer, M., Rohn, S., 2013. Phenolic compounds in Cistus incanus herbal infusions-antioxidant capacity and thermal stability during the brewing process. Food Res. Int. 53 (2), 891–899. Roidaki, A., Kollia, E., Chiou, A., Varzakas, T., Markaki, P., Proestos, C., 2016. Superfoods and superherbs: antioxidant and antifungal activity. Nutr. Food Sci. 4 (2), 138–145. Siciliano, I., Dal Bello, B., Zeppa, G., Spadaro, D., Gullino, M.L., 2017. Static hot air and infrared roasting are efficient methods for aflatoxin decontamination on hazelnuts. Toxins 9 (2), 72. Tzanidi, C., Proestos, C., Markaki, P., 2012. Saffron (Crocus sativus L.) inhibits aflatoxin B1 production by aspergillus parasiticus. Adv. Microbiol. 2, 310–316. Vergopoulou, S., Galanopoulou, D., Markaki, P., 2001. Methyl jasmonate stimulates aflatoxin B1 biosynthesis by Aspergillus parasiticus. J. Agric. Food Chem. 7, 3494–3498. Villa, P., Markaki, P., 2009. Aflatoxin B1 and ochratoxin A in breakfast cereals from Athens market: occurrence and risk assessment. Food Control 20 (5), 455–461. Wall, M.M., 2013. Improving the quality and safety of macadamia nuts. In: Harris, L.J. (Ed.), Improving the Safety and Quality of Nuts. Woodhead Publishing, pp. 274–296. Weinert, A.G., 1993. Macadamia Nut Processing. The Southern African Macadamia Growers Association Yearbook, pp. 54–75. World Health Organization, 2002. Evaluation of Certain Mycotoxins in Food. World Health Organization (WHO technical report series, 906), Geneva. Yazdani, D., Mior Ahmad, Z.A., Yee How, T., Jaganath, I.B., Shahnazi, S., 2013. Inhibition of aflatoxin biosynthesis in Aspergillus flavus by phenolic compounds extracted of Piper betle L. Iran J. Microbiol. 5 (4), 428–433.
68