Antifungal and antiaflatoxigenic assessment of new Cu(II)-pq complexes against Aspergillus parasiticus, in dark conditions and under visible irradiation.

The issue of food contamination by fungi and aflatoxins; constitutes a serious concern not only for human/animal health but also for agriculture and the economy. Aflatoxins are secondary metabolites produced by certain filamentous fungi and contaminate a variety of foodstuffs. In this context, control of fungal growth and aflatoxin contamination appears to be important. The present study aimed to investigate new Cu(I) and Cu(II)-quinoxaline complexes, namely [Cu(2,2´-pq)(NO3)](NO3) (1), [Cu(2,2´-pq)2(NO3)](NO3)·6H2O (2) and [Cu(2,2΄-pq)2](BF4) (3), where 2,2´-pq is 2-(2'-pyridyl quinoxaline), as antifungal agents against Aspergillus parasiticus. All complexes, the ligand and the starting material Cu(NO3)2-3H2O, regardless of the concentration used, caused inhibition of A. parasiticus growth ranged from 8.52 to 33.33%. The fungal growth inhibition was triggered when irradiation in visible (λ > 400 nm) was continuously applied (range 18.36-57.20%). The highest inhibitory activity was exhibited by the complex [Cu(2,2´-pq)2(NO3)](NO3)·6H2O and for this reason, it was selected to be studied for its ability to suppress aflatoxin B1 produced by A. parasiticus. AFB1 production after the irradiation process was found to be suppressed by 25% compared to AFB1 produced in dark conditions.

Capsaicin, an inhibitor of Ochratoxin A production by Aspergillus section Nigri strains in grapes (Vitis vinifera L.).

Food decay by spoilage fungi leads to significant economic losses and hazards to consumers' health due to the potential of mycotoxin occurrence. Ochratoxin A (OTA) is a mycotoxin known as nephrotoxic and carcinogenic to humans. Natural capsaicin was evaluated for its effectiveness against the growth of five Aspergillus section Nigri strains and accumulation of OTA in inoculated black grapes. Results showed that capsaicin was effective in inhibiting fungal growth and OTA production by new four Aspergillus section Nigri strains (ATHUM 6997, 6998, 6999, 7000) and by Aspergillus carbonarius as well. Moreover, capsaicin addition exhibited maximum inhibition of OTA produced by ATHUM 6997, 6998, 6999, and 7000 in black grapes at 28.9%, 8.6%, 68.4%, and 78.1%, respectively. Inhibition percentage of OTA production by A. carbonarius in grapes treated with capsaicin was estimated at 61.5%. These results suggest that capsaicin influences the OTA biosynthesis pathway of all Aspergillus section Nigri strains and therefore could be used as an effective natural preservative against OTA contamination of vineyards. Risk assessment revealed that when grapes are treated with capsaicin, consumers are less exposed to OTA.

Comparison of Different Extraction Methods for the Determination of the Antioxidant and Antifungal Activity of Cynara scolymus and C. cardunculus Extracts and Infusions.

Extracts and infusions of wild artichoke (Cynara cardunculus L.) and globe artichoke (C. scolymus L.) (heads, bracts and stems) were examined for their total phenolic content (TPC) and their antioxidant activity after performing Classical Extraction (CE) and Ultrasound-Assisted Extraction (UAE). UAE proved to be more effective, since extracts exhibited higher antioxidant activity and TPC values than CE extracts and infusions. Moreover C. cardunculus heads extract using UAE, displayed the maximum TPC values (1.57 mg gallic acid equivalents (GAE) g⁻¹ fresh weight (fw)), the highest DPPH· scavenging activity (IC50; 0.91mg mL⁻¹) and the highest ABTS·+ radical scavenging capacity (2.08 mg Trolox Equivalents (TE) g⁻¹ fw). Moreover, the effect of different concentrations of C. cardunculus head extracts (showing the highest TPC and antioxidant activity) on Aspergillus parasiticus growth was estimated in AFPA medium. The maximum inhibition was found to be 42.1% in comparison with the control.

Αntioxidant activity of Cynara scolymus L. and Cynara cardunculus L. extracts obtained by different extraction techniques.

Extracts of different parts (heads, bracts and stems) of Cynara cardunculus L. (cardoon) and Cynara scolymus L. (globe artichoke), obtained by two different extraction techniques (Ultrasound-Assisted Extraction (UAE) and classical extraction (CE)) were examined and compared for their total phenolic content (TPC) and their antioxidant activity. Moreover, infusions of the plant's parts were also analysed and compared to aforementioned samples. Results showed that cardoon's heads extract (obtained by Ultrasound-Assisted Extraction) displayed the highest TPC values (1.57 mg Gallic Acid Equivalents (GAE) g-1 fresh weight (fw)), the highest DPPH• scavenging activity (IC50; 0.91 mg ml-1) and the highest ABTS•+ radical scavenging capacity (2.08 mg Trolox Equivalents (TE) g-1 fw) compared to infusions and other extracts studied. Moreover, Ultrasound-Assisted Extraction technique proved to be more appropriate and effective for the extraction of antiradical and phenolic compounds.

Aflatoxin B1 in sesame seeds and sesame products from the Greek market.

Aflatoxin B1 (AFB1) is considered as the most potent liver carcinogen for humans. A method for determination in sesame seeds was developed. AFB1 was extracted by methanol-water, cleaned by immunoaffinity columns and determined by high-performance liquid chromatography with fluorescence detection. The recovery factor and the limit of detection (LOD) of AFB1 in sesame seeds were 111.5% and 0.02 ng g(-1), respectively. Thirty samples of sesame products were examined for the presence of AFB1. After analysis, 77.6% of samples were found to be contaminated. Eight samples exceeded the European Union (EU) limit (2 µg AFB1 kg(-1)). In 15 samples, AFB1 was below the EU limit. Seven samples remained below the LOD. The most contaminated (14.49 ng AFB1 g(-1)) sample was unpeeled packaged sesame seeds. In all samples, aflatoxigenic Aspergilli fungi as well as the risk for AFB1 presence in sesame seed was investigated.

Occurrence of aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market.

Twenty-six samples of dried vine fruits from Athens and Thebes (Central Greece) market were simultaneously extracted and cleaned up by immunoaffinity columns and analysed for aflatoxin B1 (AFB1) and ochratoxin A (OTA). A combination of ELISA and HPLC methods was applied for the determination of AFB1. Recovery was 97.6%, RSD 6.46%, while the limit of detection (LOD) and limit of quantification (LOQ) were 0.05 µg kg(-1) and 0.09 µg kg(-1), respectively. OTA concentrations were only estimated by ELISA. Results revealed the presence of AFB1 in 23% of the samples (mean 1.4 µg AFB1 kg(-1)), but none exceeded the EU limit (2 µg AFB1 kg(-1)). However, OTA was detected in 100% of the samples (mean 47.2 µg OTA kg(-1)). Six samples were found to be contaminated at high levels (median 120.6 µg OTA kg(-1)) and 18 exceeded the EU limit (10 µg OTA kg(-1)).

Study of aflatoxin B1 production by Aspergillus parasiticus in bee pollen of Greek origin.

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species such as A. parasiticus and A. flavus. The beneficial properties of bee pollen have transformed this commodity into an increasingly frequent component of the human diet. As bee pollen is a substrate on which aflatoxigenic fungi can grow, AFB1 production is likely. In the present study, we describe a method for aflatoxin B1 determination in bee pollen utilising high pressure liquid chromatography (HPLC) with a fluorescence detector (FD). The recovery factor of the method was found to be 111% (RSD% 1.61), while the detection limit (LOD) was 0.08 ng AFB1/g. An additional aim of this study was to investigate the growth of A. parasiticus and AFB1 production in bee pollen. Results indicated that no mycelial growth was observed and no AFB1 was detected in bee pollen samples containing natural microbiota throughout the entire observation period (20 days). In contrast, AFB1 production in treated bee pollen samples (15 g pollen/flask) inoculated with A. parasiticus was significantly higher (p ≤ 0.05) compared to control samples (treated but not inoculated) throughout the entire incubation period, while no mycelial growth was apparent. Maximum production was observed on the 12th day (79.29 ng AFB1/flask and 32.44 ng AFB1/flask for inoculated and non-inoculated bee pollen, respectively). As a result, AFB1 production in bee pollen is likely even following a minor contamination, which could occur randomly.

Study of the Effect of Methyl Jasmonate Concentration on Aflatoxin B(1) Biosynthesis by Aspergillus parasiticus in Yeast Extract Sucrose Medium.

Aflatoxin B(1) (AFB(1)) is a carcinogenic metabolite produced by certain Aspergillus species on agricultural commodities. AFB(1) biosynthesis is affected by jasmonic acid and also by its methylester (MeJA), a plant growth regulator derived from linoleic acid. This study reports the effect of MeJA on the growth of A. parasiticus and AFB(1) output in yeast extract sucrose (YES) medium when added at three different concentrations; namely, 10(-2) M, 10(-4) M, and 10(-6) M. AFB(1) determination was performed by immunoaffinity and HPLC. MeJA at 10(-4) and 10(-6) M concentrations had no significant effect on mycelial growth but did affect AFB(1) production after the 7th day of incubation; on the 12th day, AFB(1) production was increased by 212.7% and 141.6% compared to the control samples (addition of 10(-6) M and 10(-4) M MeJA, resp.). Treatment of A. parasiticus cultures with 10(-2) M MeJA inhibited mycelial growth and AFB(1) production as well. These results suggest that the effect of MeJA on AFB(1) biosynthesis by A. parasiticus depends on the MeJA concentration used.

Determination of patulin in fruit juices using HPLC-DAD and GC-MSD techniques.

A high performance liquid chromatography with a diode-array detector (HPLC-DAD) and a gas chromatography with a mass spectrometer (GC-MSD) are described for the determination of patulin (PAT) in apple juice. The limits of detection (DL) and quantification (QL) for the HPLC-DAD and GC-MSD method were found to be (DL=0.23µgkg(-1) QL=1.2µgkg(-1)) and (DL=5.8µgkg(-1) and QL=13.8µgkg(-1)), respectively. The recovery factors for HPLC-DAD and GC-MSD were found to be 99.5% (RSD%=0.73) and 41% (RSD%=10.03), respectively. The HPLC-DAD method was used to determine the occurrence of PAT in 90 samples of fruit juices. Results revealed the presence of PAT in 100% of the samples examined. The mean values of PAT in concentrated fruit juices and in the commercial fruit juices collected from the Greek market were found to be 10.54µg PAT kg(-1) and 5.57µg PAT kg(-1) juice, respectively. The most contaminated samples were four concentrated juices ranging from 18.10µg PAT kg(-1) to 36.8µg PAT kg(-1) juice. The daily exposure to patulin for the consumers of all ages in Greece, is ranging from 0.008µg PAT kg(-1) bw to 0.1µg PAT kg(-1) bw if the daily intake of fruit juices is from 0.1 to 0.5kg. With the exception to the most contaminated sample, the daily exposure due to the samples examined, is below the provisional maximum tolerable daily intake for PAT (0.4µg PAT kg(-1) bw).

Determination of ochratoxin A in grapes of Greek origin by immunoaffinity and high-performance liquid chromatography.

A simple analytical method for ochratoxin A (OTA) determination in grapes is described, using aqueous methanolic extraction, an immunoaffinity column clean-up step and high-performance liquid chromatography with fluorescence detection. Mean recovery was 94% (RSD = 4.0%) with a detection limit of 0.4 ng g(-1) and quantification limit of 1.20 ng g(-1). Repeatability (r) and reproducibility (R) were 1.17 and 1.34, respectively. OTA determinations were applied to 50 grape samples (23 different varieties) originating from representative regions of Greece. Results showed the presence of OTA in 86% of samples tested (n = 50). Traces were found in 56% of samples but OTA was not detectable in 14% of samples. Traces were also found in 4% of red, organically grown samples. The most contaminated were three samples of red grapes, two from Central Greece (2.69 and 1.41 ng g(-1)), both table and wine-making grapes. The third sample (1.46 ng g(-1)), originating from the island of Samos, was used only in wine-making. Mean (1.06 ng g(-1)) and median (0.76 ng g(-1)) OTA concentrations in red grapes were slightly higher compared to the mean (0.82 ng g(-1)) and median (0.65 ng g(-1)) concentrations in white grape samples. The study shows that the potential risk for a person of 60 kg ranged from 0.9 to 9 ng kg(-1) bw day(-1) and is dependent on the quantity of grapes consumed daily.

Study of aflatoxin B1 and ochratoxin A production by natural microflora and Aspergillus parasiticus in black and green olives of Greek origin.

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is classified as "possible carcinogen" and it is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. Fungi contaminate natural and processed olives which support AFB1 and OTA production. The aim of this study was to compare and investigate AFB1 and OTA production in three different varieties of damaged olives. For each variety two different treatments were applied: (1) olives with natural microflora and (2) olives inoculated with A. parasiticus after natural microflora elimination. AFB1 and OTA have been extracted simultaneously from olives, purified with immunoaffinity columns and quantitated by HPLC using fluorescence detector. The recoveries and detection limits of AFB1 and OTA were 94% and 0.15 ng AFB1 g(-1) and 102.7%, 0.41 ng OTA g(-1) respectively. Results showed that, meanwhile OTA was not found in any olive sample, AFB(1) production within the three varieties of olives with natural microflora was significantly (P< or =0.05) different regarding their substrate and time of incubation (18 days). AFB1 production in two different varieties of black olives after inoculation by A. parasiticus was not significantly higher compared with control samples. On the contrary, AFB1 production in green olives was stimulated after the 12th day. Additionally, investigation on the occurrence of AFB1 and OTA in 30 samples of olives and olive pasta from Athens market showed OTA's presence in two samples of olives contaminated at the levels of 1.18 and 1.86 ng OTA g(-1). Aflatoxin B1 was found at levels 0.15-1.13 ng AFB1 g(-1) in all samples tested.