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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.