Search

Edible Insects (EIs) are an alternative source of bioactive compounds such as proteins or fatty acids and micronutrients as vitamins or minerals, thus showing potential to replace traditional foodstuffs in an economical and environmentally friendly way. Nonetheless, EIs can accumulate hazardous chemicals such as mycotoxins and heavy metals. The aim of the present study is to determine mycotoxins and heavy metal content in raw insect samples and those resulting products obtained after supercritical fluid extraction (SFE). Insect samples included Acheta domesticus (cricket) meal, Tenebrio molitor (mealworm) meal, Alphitobius diaperinus (buffalo worm), and Locusta migratoria (locust). For this purpose, a QuEChERS method followed by LC-MS/MS analysis was optimized with good results for the analysis of mycotoxins, principally Aflatoxins (AFs), Ochratoxin A (OTA), and Enniatins (ENNs). In contrast, heavy metals (As, Cd, Hg, Pb) were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The results obtained revealed that Locust was positive for AFG2 at a level of 115.5 μg/kg, and mealworm was only contaminated with OTA at 58.1 μg/kg. Emerging mycotoxins (ENNA, ENNA1, ENNB, and ENNB1) were detected at lower levels < 2.2 µg/Kg. Concerning heavy metals, limits exceeding regulation were detected for Cd in the insect species studied, with levels up to 219 μg/kg, and for Pb in crickets (100.3 μg/kg). Finally, the analysis of the post-extraction solids after SFE processing revealed that heavy metals remained in the resulting SFE cakes, while mycotoxins were detected at negligible levels (up to 1.3 µg/Kg).

Summary Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost‐effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum‐based expression system that consisted of the paf gene promoter, signal peptide (SP)‐pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (−OS) or under stress conditions (+OS) (5 mM of H2O2). LC-MS/MS identified 33 differentially expressed proteins (−OS: 14, +OS: 19) that were included By Gene Ontology analysis in biological processes: biosynthesis of amino acids, carbohydrate metabolism and reactive oxygen species metabolic process. In a gene-knockout strains study, eight proteins were identified as putative candidates for being involved in the protective mechanism of cocoa polyphenols against OS induced by H2O2. CPEX was able to exert its antioxidant activity in yeast mainly through the regulation of: (a) amino acids metabolism proteins by modulating the production of molecules with known antioxidant roles; (b) stress-responsive protein Yhb1, but we were unable to fully understand its down-regulation; (c) protein Prb1, which can act by clipping Histone H3 N-terminal tails that are related to cellular resistance to DNA damaging agents.

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum , P. italicum and P. expansum , PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

During the summer and autumn of 2001, symptoms of interveinal yellowing, bronzing, brittleness, and rolling of lower leaves were observed in greenhouse- and field-grown tomato (Lycopersicon esculentum) plants in Castellon Province in eastern Spain. Symptoms resembled those caused by the whitefly-transmitted criniviruses (1,2). Total RNA was extracted from 28 samples of symptomatic leaves collected in three greenhouses and one field and analyzed by reverse transcription-polymerase chain reaction using primers specific for Tomato chlorosis virus (ToCV) (1) and Tomato infectious chlorosis virus (TICV) (2). The 501-bp TICV-specific DNA fragment was amplified in four samples collected during the summer in three greenhouses and one field, and the 439-bp ToCV-specific DNA fragment was amplified in 15 samples collected during the autumn in the same three greenhouses; no mixed infections were found. The DNA fragments amplified from TICV were sequenced and showed 99 to 100% identity with the TICV isolates (GenBank Accession Nos. U67449 and AY048855) from the United States and Italy, respectively, confirming the diagnosis. One sequence was deposited as GenBank Accession No. AF479662. To our knowledge, this is the first report of TICV in Spain and the second in Europe. References: (1) D. Louro et al. Eur. J. Plant Pathol. 106:539, 2000. (2) A. M. Vaira et al. Phytoparasitica. In Press.

At the beginning of 2000, a damaging disease developed on protected tomato (Lycopersicon esculentum) crops grown in polyethylene greenhouses in different regions of Spain. Production losses were estimated at 15 to 80%. The tomato plants showed a variety of symptoms. The most common symptoms were leaf distortion, chlorosis, and mosaic. Some plants showed a dark green mosaic and bubbling of the leaf surface. Green striations were also observed on the stem and sepals. Most of the diseased plants had discolored fruits. Symptoms decreased as environmental temperature increased. The involvement of Pepino mosaic virus (PepMV) was suspected. To identify the etiological agent, ≈500 symptomatic tomato plants were collected from several locations in Alicante, Murcia, Almeria and the Canary Islands. Flexuous viral particles 510 nm long were observed by transmission electron microscopy, suggesting the presence of a potexvirus in the tissue extracts analyzed. All samples were tested by ELISA (enzyme-linked immunosorbent assay), using polyclonal antibodies to Narcissus mosaic virus (Adgen, Auchincriuve, Scotland), a virus serologically related to PepMV, and two antisera specific to PepMV (Adgen, Scotland and DMSZ, Braunschweig, Germany). PepMV was detected in 35% of the samples. Like PepMV, the virus infected (as confirmed by ELISA) greenhouse-grown Datura stramonium, Nicandra physalodes, Nicotiana benthamiana, N. clevelandii, Solanum tuberosum, and Vigna sinensis and did not infect Capsicum anuum, Cucumis sativus, Chenopodium amaranticolor, C. quinoa, Petunia × hybrida, Phaseolus vulgaris, Physalis floridana, N. glutinosa, N. rustica, or N. tabacum. The virus did infect Gomphrena globosa, which normally is not infected by PepMV. The first report of PepMV was on pepino (Solanum muricatum) in Peru in 1974 (1), but this virus has been recently reported in the Netherlands, England, Germany, and France on protected tomato crops (2). To our knowledge, this is the first report of PepMV in Spain, including the Canary Islands. References: (1) R. A. C. Jones et al. Ann. Appl. Biol. 94:61, 1980. (2) European and Mediterranean Plant Protection Organisation (EPPO). Alert List Viruses. On-line publication/2000/003.

In autumn of 1999 and winter-spring 2000, tomato (Lycopersicon) crops grown in the Regions of Las Palmas de Gran Canaria and Tenerife (Canary Islands) showed upward curling of leaves, yellowing of leaf margins, crumpling of new leaves, reduction of leaflet area, and stunting of shoots. These symptoms were similar to those described for tomato yellow leaf curl disease. Symptomatic samples were collected from Las Palmas de Gran Canaria (33 samples) and Tenerife (45 samples) for polymerase chain reaction (PCR) identification analysis. The degenerate primers pair of Begomovirus (AV494/AC1048) (3) was used to amplify the "core" region of the capsid protein gene. Two tomato plants experimentally infected with Tomato yellow leaf curl virus-Is (TYLCVIs) or TYLCV-Sar served as positive controls. Electrophoretic analysis of all samples showed a single fragment of the expected size (550 bp). To identify the type of TYLCV (TYLCV-Sar or TYLCV-Is), the PCR products were digested by endonucleases (AluI, HaeIII, HpaII, RsaI, Sau3A, TaqI, DdeI, and ScrFI). Twenty-six samples from Las Palmas de Gran Canaria showed the same restriction pattern of TYLCV-Sar, and seven samples from Las Palmas de Gran Canaria and all 45 samples from Tenerife showed the same restriction pattern of TYLCV-Is. These results confirm that TYLCV-Sar and TYLCV-Is are present in Las Palmas de Gran Canaria and TYLCV-Is is present in Tenerife. The presence of TYLCV-Is in Morocco (2) and TYLCV-Sar in the Canary Islands and Morocco has been recently described (1). However, this is the first report of TYLCV-Is in the Canary Islands. References: (1) F. Monci et al. Plant Dis. 84:490, 2000. (2) M. Peterschmitt et al. Plant Dis. 83:1074, 1999. (3) S. D. Wyatt et al. Phytopathology 86:1288, 1996.