Proteomics as a New-Generation Tool for Studying Moulds Related to Food Safety and Quality.

Mould development in foodstuffs is linked to both spoilage and the production of mycotoxins, provoking food quality and food safety concerns, respectively. The high-throughput technology proteomics applied to foodborne moulds is of great interest to address such issues. This review presents proteomics approaches useful for boosting strategies to minimise the mould spoilage and the hazard related to mycotoxins in food. Metaproteomics seems to be the most effective method for mould identification despite the current problems related to the bioinformatics tool. More interestingly, different high resolution mass spectrometry tools are suitable for evaluating the proteome of foodborne moulds able to unveil the mould's response under certain environmental conditions and the presence of biocontrol agents or antifungals, being sometimes combined with a method with limited ability to separate proteins, the two-dimensional gel electrophoresis. However, the matrix complexity, the high ranges of protein concentrations needed and the performing of multiple steps are some of the proteomics limitations for the application to foodborne moulds. To overcome some of these limitations, model systems have been developed and proteomics applied to other scientific fields, such as library-free data independent acquisition analyses, the implementation of ion mobility, and the evaluation of post-translational modifications, are expected to be gradually implemented in this field for avoiding undesirable moulds in foodstuffs.

Effect of selected agents for ochratoxin A biocontrol on the colour, texture and volatile profile of dry-cured fermented sausages.

BACKGROUND: Traditional dry-cured fermented sausages favour the growth of an autochthonous microbial population, which plays an important role in their sensory aspects. However, some moulds can produce mycotoxins such as ochratoxin A (OTA). The biocontrol agents (BCAs) Debaryomyces hansenii FHSCC 253H and Staphylococcus xylosus FHSCC Sx8 have been demonstrated to reduce OTA production in dry-cured meat products, but their influence in the sensory characteristics of sausages has to be tested. The aim of this study was to evaluate the effect of these BCAs on the colour, texture and volatile profile of dry-cured fermented sausages. RESULTS: D. hansenii caused few differences in the tested parameters with respect to the control batch. S. xylosus modified the texture and colour, although the values found were within the range expected for dry-cured fermented sausages 'salchichón'. Additionally, the volatile profile revealed the potential antioxidant effect of both BCAs and their ability to produce compounds associated with the ripened aroma that could increase product acceptability. CONCLUSION: The results indicate that there were no inconveniences in implementing both BCAs during the processing of dry-cured fermented sausages 'salchichón'. Moreover, D. hansenii FHSCC 253H could improve the volatile profile of this product. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of the activity of the antifungal PgAFP protein and its producer mould against Penicillium spp postharvest pathogens of citrus and pome fruits.

Postharvest fungal diseases are among the main causes of fresh fruit losses. Chemical control is against claims for "natural" or "chemical-free" products. Biocontrol agents, such as antifungal proteins or their producing moulds, may serve to combat unwanted pathogens. Since the effectiveness of these bioprotective agents depends on the food substrate, their effect must be tested on fruits. The objective of this work was to study the effect of the antifungal protein PgAFP and its producer, Penicillium chrysogenum, against Penicillium expansum and Penicillium digitatum growth on apple and oranges respectively, and the PgAFP effect on eleven P. expansum, Penicillium italicum, and P. digitatum strains in vitro, and on patulin production on apple substrate. The sensitivity upon PgAFP was P. digitatum > P. expansum > P. italicum. In oranges, broadly, no inhibitory effect was obtained. PgAFP and P. chrysogenum did not inhibit the P. expansum CMP-1 growth on Golden Delicious apples, however, a successful effect was achieved on Royal Gala apples. On apple substrate, patulin production by P. expansum CMP-1 rose in parallel to PgAFP concentrations, linked with high reactive oxygen species levels. PgAFP cannot be proposed as a bioprotective agent on apple. However, P. chrysogenum is a promising agent to be used on Royal Gala apples.

Biocontrol of aflatoxigenic Aspergillus parasiticus by native Debaryomyces hansenii in dry-cured meat products.

Dry-cured meat products, such as dry-cured ham or dry-fermented sausages, are characterized by their particular ripening process, where a mould population grows on their surface. Some of these moulds are hazardous to the consumers because of their ability to produce mycotoxins including aflatoxins (AFs). The use of native yeasts could be considered a potential strategy for controlling the presence of AFs in dry-cured meat products. The aim of this work was to evaluate the antagonistic activity of two native Debaryomyces hansenii strains on the relative growth rate and the AFs production in Aspergillus parasiticus. Both D. hansenii strains significantly reduced the growth rates of A. parasiticus when grown in a meat-model system at different water activity (aw) conditions. The presence of D. hansenii strains caused a stimulation of AFs production by A. parasiticus at 0.99 aw. However, at 0.92 aw the yeasts significantly reduced the AFs concentration in the meat-model system. The relative expression levels of the aflR and aflS genes involved in the AFs biosynthetic pathway were also repressed at 0.92 aw in the presence of both D. hansenii strains. These satisfactory results were confirmed in dry-cured ham and dry-fermented sausage slices inoculated with A. parasiticus, since both D. hansenii strains significantly reduced AFs amounts in these matrices. Therefore, both tested D. hansenii strains could be proposed as biocontrol agents within a HACCP framework to minimize the hazard associated with the presence of AFs in dry-cured meat products.

Increased prevalence of human herpesvirus type 8 (HHV-8) genome among blood donors from North-Western Argentina.

The prevalence of HHV-8 infection varies widely in South American populations, displaying geographical variations in its distribution. The heterogeneous genetic contributions provided by the transatlantic parental populations that modified the Native American genomes may explain this epidemiological observation. Aiming to determine the prevalence of HHV-8 genome among healthy South American blood donors and its potential association with genetic ancestry, 772 individuals were screened by a highly sensitive PCR protocol and ancestry was assessed in 414 samples. HHV-8 DNA was significantly more prevalent among North-western Argentines than among those from the metropolitan region (P = 0.001) and Bolivians (P = 0.0008), but no differences were found when compared with Peruvians and Paraguayans. Although significant differences were observed in the ancestry components of the studied populations, no association was found in the genetic admixture between HHV-8 [+] and HHV-8 [-] samples from the same place. These results support the hypothesis of the existence of geographical factors related to HHV-8 prevalence which could be explained by the presence of specific risk factors, cultural characteristics or behaviors, probably related to contaminated saliva and/or sexual transmission. The presence of HHV-8 in South American blood units available for transfusion and an increased risk of infection in some provinces of North-western Argentina represent a hazard for immunosuppressed recipients. J. Med. Virol. 89:518-527, 2017. © 2016 Wiley Periodicals, Inc.

Role of HLA-DP and HLA-DQ on the clearance of hepatitis B virus and the risk of chronic infection in a multiethnic population.

BACKGROUND & AIMS: HBV infection exhibits geographical variation in its distribution in South America. While HBV rates are low in central Argentina, the north-western region exhibits intermediate HBV rates. Unfortunately, the reasons that could explain this difference are still unknown. METHODS: A total of 1440 Argentines were recruited and grouped into HBV patients, HBV-resolved individuals and healthy controls. Genetic ancestry was assessed by analysis of biparental lineages and ancestry autosomal typing. SNPs of HLA-DPA1 (rs3077), HLA-DPB1 (rs9277542), HLA-DQB1 (rs2856718) and HLA-DQB2 (rs7453920) were determined, and HBV genotyping was performed by phylogenetic analysis in HBV patients. RESULTS: Native American ancestry prevailed in the north-western region when compared with central Argentina (P<.0001). However, no differences were observed among the three groups of each region. The distribution of HBV genotypes revealed significant differences (P<.0001). Three SNPs (rs3077, rs9277542 and rs7453920) showed a significant association with protection against chronic HBV and viral clearance in both regions. The remaining SNP showed a significant association with susceptibility to chronic HBV. The frequency rates of rs3077-T, related to protection against chronic HBV and viral clearance, were lower in north-western Argentina when compared with central Argentina. The same uneven frequency rates were observed for SNP rs9277542. CONCLUSIONS: This is the first study addressing the associations between the HLA-DP and HLA-DQ loci and the protection against chronic HBV and viral clearance in a multiethnic South American population. The uneven distribution of HLA-DP and HLA-DQ supports the HBV epidemiological differences observed in these two regions of Argentina with dissimilar ancestry genetic background.

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products.

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP.

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods.

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include 'omics' approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex ß subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products.

Influence of ethnicity on the distribution of genetic polymorphisms associated with risk of chronic liver disease in South American populations.

BACKGROUND: The global burden of chronic liver disease is rising. Besides environmental, behavioral, viral and metabolic factors, genetic polymorphisms in patatin-like phospholipase-3 (PNPLA3) and vitamin D receptor (VDR) genes have been related to the development of chronic liver disease and progression towards liver cancer. Although their prevalence differs remarkably among ethnic groups, the frequency of these polymorphisms in South American populations -whose genetic background is highly admixed- has been poorly studied. Hence, the aim of this study was to characterize polymorphisms related to chronic liver disease and their association with the genetic ancestry of South American populations. RESULTS: DNA samples from 258 healthy unrelated male volunteers were analyzed. The frequencies of G and C alleles of rs738409 polymorphism (PNPLA3 gene) were 74 % and 26 %, respectively; whereas the bAt (CCA) haplotype (VDR gene) was observed in 32.5 % of the samples. The GG genotype of PNPLA3 rs738409 and the bAt (CCA) haplotype -associated with an increased risk of chronic liver disease and progression towards liver cancer- were significantly more frequent among samples exhibiting maternal and paternal Native American haplogroups (63.7 % and 64.6 %), intermediate among admixed samples (45.1 % and 44.9 %; p = 0.03) and the lowest for Non-native American ancestry (30.1 % and 29.6 %; p = 0.001 and p = 0.0008). CONCLUSIONS: These results suggest that individuals with Native American ancestry might have a high risk of chronic liver disorders and cancer. Furthermore, these data not only support the molecular evaluation of ancestry in multi-ethnic population studies, but also suggest that the characterization of these variants in South American populations may be useful for establishing public health policies aimed at high risk ethnic communities.

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a ß subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit ß CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

Selection and evaluation of Debaryomyces hansenii isolates as potential bioprotective agents against toxigenic penicillia in dry-fermented sausages.

Biocontrol using autochthonous Debaryomyces hansenii isolates is a potentially suitable strategy for inhibiting toxigenic moulds in dry-cured meat products. The antifungal activity of 280 D. hansenii isolated from dry-cured meat products as well as the mode of action of the most active isolates against toxigenic penicillia were evaluated in this work. A 13.9% of the D. hansenii isolates showed inhibitory activity in a radial inhibition assay. The effects on penicillia growth of both the cell-free culture filtrate and volatile compounds from active yeast isolates were analysed. Penicillia growth inhibition by D. hansenii was probably based on additive or synergistic effects of several inhibiting factors such as competition for nutrient and space, and production of soluble or volatile compounds. When four D. hansenii isolates were tested on dry-fermented sausage, two of them produced a significantly growth reduction of the ochratoxigenic Penicillium verrucosum, keeping its counts under the level considered as hazardous for the mycotoxin presence. Therefore, the use of these two D. hansenii isolates during the processing of dry-fermented meat product could be a promising tool to control toxigenic moulds in the meat industry.

The diet of Myotis lucifugus across Canada: assessing foraging quality and diet variability.

Variation in prey resources influences the diet and behaviour of predators. When prey become limiting, predators may travel farther to find preferred food or adjust to existing local resources. When predators are habitat limited, local resource abundance impacts foraging success. We analysed the diet of Myotis lucifugus (little brown bats) from Nova Scotia (eastern Canada) to the Northwest Territories (north-western Canada). This distribution includes extremes of season length and temperature and encompasses colonies on rural monoculture farms, and in urban and unmodified areas. We recognized nearly 600 distinct species of prey, of which ≈30% could be identified using reference sequence libraries. We found a higher than expected use of lepidopterans, which comprised a range of dietary richness from ≈35% early in the summer to ≈55% by late summer. Diptera were the second largest prey group consumed, representing ≈45% of dietary diversity early in the summer. We observed extreme local dietary variability and variation among seasons and years. Based on the species of insects that were consumed, we observed that two locations support prey species with extremely low pollution and acidification tolerances, suggesting that these are areas without environmental contamination. We conclude that there is significant local population variability in little brown bat diet that is likely driven by seasonal and geographical changes in insect diversity, and that this prey may be a good indicator of environment quality.

Prevalence and trends of markers of hepatitis B virus, hepatitis C virus and human Immunodeficiency virus in Argentine blood donors.

BACKGROUND: Transfusion-transmitted infections are a major problem associated with blood transfusion. The aim of this study was to determine prevalence and trends of HBV, HCV and HIV in blood donors in Argentina. METHODS: A retrospective study was carried out in blood donors of 27 transfusion centers covering the whole country over a period of eight years (2004-2011). Serologic screening assays for HBsAg, anti-HBc, anti-HCV, and anti-HIV were performed in all centers and nucleic acid amplification testing (NAT) was performed in 2 out of the 27 centers. RESULTS: The 2,595,852 samples tested nationwide from 2004 to 2011 showed that the prevalence of HBsAg decreased from 0.336% to 0.198% (p < 0.0001), that of anti-HBc from 2.391% to 2.007% (p < 0.0001), that of anti-HCV from 0.721% to 0.460%, (p < 0.0001) and that of anti-HIV from 0.208% to 0.200 (p = 0.075). The prevalence of HBV, HCV and HIV was unevenly distributed among the different regions of the country. Two out of 74,838 screening- negative samples were positive in NAT assays (1 HIV-RNA and 1 HCV-RNA); moreover, HBV-DNA, HCV-RNA and HIV-RNA were detected in 60.29, 24.54 and 66.67% of screening-positive samples of the corresponding assays. As regards donors age, positive HBV-DNA and HCV-RNA donors were significantly older than healthy donors (46.6, 50.5 and 39.5 y respectively, p < 0.001). CONCLUSIONS: Argentina has a low prevalence of HBsAg, anti-HCV and anti-HIV in blood donors, with a decreasing trend for HBsAg, anti-HBc and anti-HCV but not for anti-HIV over the last 8 years. The uneven distribution of transfusion-transmitted infections prevalence among the different regions of the country highlights the need to implement regional awareness campaigns and prevention. The discrepancy between samples testing positive for screening assays and negative for NAT assays highlights the problem of blood donors who test repeatedly reactive in screening assays but are not confirmed as positive upon further testing. The uneven distribution of age between healthy donors and NAT-positive donors could be related to changes in risks of these pathogens in the general population and might be attributed to a longer exposure to transmission risk factors in elderly people.

Deciphering Staphylococcus xylosus and Staphylococcus equorum mode of action against Penicillium nordicum in a dry-cured ham model system.

Penicillium nordicum is one of the major producers of ochratoxin A (OTA) in dry-cured ham. Staphylococcus xylosus Sx8 and Staphylococcus equorum Se31 have been previously proposed as biocontrol agents (BCAs) to prevent the OTA contamination, although their antifungal mode of action has not been established yet. Thus, the aim of this work was to elucidate their mode of action against P. nordicum in a dry-cured ham model system. For this, the effect of live cells, dead cells, and cell-free broth; the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays, antifungal effect of volatile compounds, OTA detoxification, and effect on fungal proteome were determined. No fungal growth was observed after 14 days of co-culture with live cells of each staphylococcus at 15 or 20 °C. However, such inhibition was not observed with either dead cells or extracellular extracts. The number of carbon sources utilised by P. nordicum was higher than those used by both cocci at 20 °C, whilst the opposite occurred at 15 °C. According to NOI, nutritional dominance depends on temperature, at 20 °C P. nordicum dominated the niche, but at 15 °C the mould is dominated by the BCAs. The volatile pattern generated by each coccus did not show antifungal effect, and both staphylococci failed to degrade or adsorb OTA. However, in the interaction assay, S. xylosus and S. equorum were able to decrease the fungal growth and its OTA production. In addition, proteomic analyses showed changes in the abundance of proteins related to the cell wall integrity (CWI), carbohydrate metabolism and the biosynthesis of secondary metabolites such as OTA. In conclusion, overall, the antagonistic effects of the two studied cocci against P. nordicum are greater at 15 °C than at 20 °C, being linked to competition for space and nutrients, triggering alterations in CWI pathway, OTA biosynthesis, and carbohydrate metabolism.

Assessing the effect of prophylactic ankle taping on ankle and knee biomechanics during landing tasks in healthy individuals: A cross-sectional observational study.

BACKGROUND: Current research supports the fact that prophylactic ankle taping (AT) is effective in preventing ankle injuries in amateur and elite sports athletes. OBJECTIVE: This study aimed to investigate the effect of AT on balance, knee valgus during drop jump and single-leg countermovement jump (SL-CMJ) landings, and ankle range of motion (ROM) restriction in healthy participants. DESIGN AND SETTING: A cross-sectional observational study was conducted at the Universidad Europea de Madrid, Madrid, Spain. METHODS: Participants: Thirty-nine healthy individuals participated in this study and performed the movements under two conditions (with and without tape). Outcome measurements: ankle ROM, balance, SL-CMJ height, flight time, ground time, and knee valgus. Before any intervention, a random process was developed with a 1:1 allocation ratio, and the participants were assigned to groups A (tape-no tape) and B (no tape-tape). RESULTS: Significant differences between tape and no-tape moments were observed for drop jump knee valgus flexion (P = 0.007), with an increase in knee valgus in participants with ankle taping. Similarly, the Y-balance testshowed a significant decrease in all variables (P = 0.001 and), ankle dorsiflexion (P = 0.001) in participants with ankle taping. CONCLUSIONS: AT is effective for immediate ankle ROM restriction. However, an increase in knee valgus during drop jump task and a decrease in lower limb balance were observed during drop jump task. Based on these results, it can be concluded that AT application in healthy individuals should not be recommended as it results in increase in injury risk factors.

Biocontrol of ochratoxigenic Penicillium nordicum in dry-cured fermented sausages by Debaryomyces hansenii and Staphylococcus xylosus.

Penicillium nordicum is the main ochratoxin A (OTA) producing mould in dry-cured meat products. The use of autochthonous microorganisms as protective cultures is a promising strategy to control this hazard. The aim of this work was to evaluate the effect of Debaryomyces hansenii and Staphylococcus xylosus isolated from dry-cured meat products as biocontrol agents (BCAs) against P. nordicum during the ripening of dry-cured fermented sausages. The BCAs were added to the dough, and P. nordicum were inoculated on the surface after stuffing. Then, the sausages were ripened following a traditional processing. The growth of the microorganisms was determined by plate count at the beginning and at the end of ripening. To assess the implantation of BCAs in the sausages, the yeasts and staphylococci isolated from the sausages at the end of processing were identified by sequencing the 16S and 18S rRNA respectively, and they were characterized by pulsed field gel electrophoresis (PFGE) of the chromosomal DNA. OTA was quantified by UHPLC-MS/MS. BCAs were able to colonise and develop throughout the processing. Although none of the BCAs reduced the growth of P. nordicum, a OTA decrease was observed in the sausages inoculated with D. hansenii individually or combined with S. xylosus. The drop of OTA amount was particularly marked in the portions where the casing was damaged allowing the mould to grow inside the sausages. In these areas, OTA was not detected in the inoculated batches. In conclusion, D. hansenii could be proposed as BCA individually or in combination with S. xylosus for the biocontrol of OTA hazard in dry-cured fermented sausages.

Proteomic evaluation of the effect of antifungal agents on Aspergillus westerdijkiae ochratoxin A production in a dry-cured fermented sausage-based medium.

Aspergillus westerdijkiae may produce large amounts of ochratoxin A (OTA) in dry-cured meat products. Natural strategies to control ochratoxigenic moulds using biocontrol agents (BCAs) are currently in the spotlight. The aim of this study was to test the effects of Debaryomyces hansenii and its combination with rosemary derivatives and with a commercial antifungal preparation composed by natamycin and potassium sorbate (AP) against A. westerdijkiae in a dry-cured fermented sausage based-medium. The yeast and rosemary leaves were added to the medium, and rosemary essential oil and AP were added on the casings put on the medium surface to simulate the real product. The growth rate, OTA production and comparative proteomics were analysed. The mould growth in the presence of each treatment was not indicative of their efficiency on OTA repression. The treatment with AP did not affect to the OTA concentration, maybe as consequence of the stressful stimulation of the subinhibitory doses used. D. hansenii added alone or with rosemary showed the best results, decreasing the OTA production >80 %, suggesting that it can be useful as preservative agent during industrial processing. Attending to the proteomic results, its antifungal activity seems to be based on the reduction in abundance of proteins involved in OTA biosynthesis and in the cell wall integrity pathway.

Impact of the antifungal protein PgAFP on the proteome and patulin production of Penicillium expansum on apple-based medium.

Apples are prone to be contaminated with Penicillium expansum, which produces the mycotoxin patulin, posing a risk for human health. Antifungal treatments are required to control this fungal pathogen, although consumers demand products free of synthetic additives. Then, the use of antifungal proteins produced by moulds represents a novel and promising strategy. Although its inhibitory effect on P. expansum has been reported, the impact of these proteins on patulin production has been scarcely studied, pointing to a possible patulin overproduction. The aim of this work was to evaluate the effect of the antifungal protein PgAFP on the proteome and patulin biosynthesis of P. expansum grown in apple-based agar, intending to decipher these effects without the apple in vivo physiological response to the fungal infection. PgAFP increased the production of patulin on three of the five P. expansum strains evaluated. The proteome of the PgAFP-treated P. expansum showed five proteins involved in patulin biosynthesis in higher abundance (fold change 2.8-9.8), as well as proteins related to pathogenicity and virulence that suggest lower ability to infect fruits. Additionally, several proteins associated with oxidative stress, such as glutathione peroxidase, redoxin, or heat shock proteins were found in higher abundance, pointing to a response against oxidative stress elicited by PgAFP. These results provide evidence to be cautious in applying this antifungal protein in apples, being of utmost relevance to provide knowledge about the global response of P. expansum against an antifungal protein with many shared characteristics with others. These findings significantly contribute to future studies of assessment and suitability of not only these antifungal proteins but also new antifungal compounds.

Influence of an industrial dry-fermented sausage processing on ochratoxin A production by Penicillium nordicum.

Dry-fermented sausages are prone to be colonised by Penicillium nordicum, which is one of the main ochratoxin A (OTA)-producing species. Its ability to produce this mycotoxin on dry-fermented sausages has been reported. However, the influence of the conditions of a traditional processing of a Spanish dry-fermented sausage and the intrinsic physicochemical parameters of this product such as water activity (aw) and pH on OTA production has not been studied yet. Thus, the aim of this study was to evaluate the influence of traditional processing (interaction of relative humidity (RH) x temperature x ripening days) on the evolution of pH and aw during maturation of dry-fermented sausage "salchichón" and its relationship with OTA synthesis by P. nordicum. The expression of otapks and otanps genes, both involved in the biosynthesis of the mycotoxin, was also assessed. For this, 27 raw sausages were inoculated with P. nordicum and ripened for 26 days in a drying chamber (3 days at 5 °C and 84% RH, 17 days at 12 °C and 84% RH, and 6 days at 12 °C and 80% RH). From results, although it seems that the pH slightly influenced on OTA biosynthesis, the aw had a great impact on this mycotoxin production. In fact, the two highest OTA concentrations found coincided with a dramatic rise of the aw value (0.92 aw) by day 18 of incubation when the RH of the drying chamber was still 84% and at the end of the incubation time when the aw decreased noticeably (0.87 aw). The expression of the otapks and otanps genes correlated with the OTA produced by P. nordicum. Results from this work confirm that the traditional processing of Spanish dry-fermented sausages favours itself OTA synthesis by P. nordicum. Our findings may help in informed decision-making in relation to RH/temperature of drying chambers and shortening of the ripening process. This may be then effectively incorporated into the hygienic production system in the framework of HACCP together with other measures including the use of Penicillium nalgiovense as protective culture or the monitoring of otapks gene expression, and aw during the processing of dry-fermented sausages. All these strategies together may put ochratoxigenic Penicillia at a disadvantage and minimise OTA contamination risks in dry-fermented sausages.