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The environmental conditions during the ripening of dry-cured meats and their nutritional composition promote the colonisation of their surface by Penicillium spp., including P. nordicum producer of ochratoxin A (OTA). The objective of this work was to study the competitiveness of three potential biocontrol candidates (Debaryomyces hansenii FHSCC 253H, Enterococcus faecium SE920 and Penicillium chrysogenum CECT, 20922) against the ochratoxigenic P. nordicum FHSCC4 under environmental and nutritional conditions simulating the ripening of dry-cured meat products. For this, the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays and OTA production were determined. The number of carbon sources (CSs) metabolised depended on the microorganism and the interacting water activity (aw) x temperature conditions. The number of CSs utilised by both filamentous fungi was quite similar and higher than those utilised by D. hansenii and E. faecium. The yeast isolate metabolised a number of CSs much larger than the bacterium. The NOI values showed that, in general, P. nordicum nutritionally dominated E. faecium and D. hansenii regardless of the environmental conditions evaluated. The relationship between the toxigenic and non-toxigenic fungal isolates depended on the aw x temperature combinations, although in none of the conditions a dominance of P. nordicum was observed. According to the interaction assays, both D. hansenii and P. chrysogenum decreased the growth of P. nordicum. The effect of D. hansenii could be attributed to the production of some extra-cellular compounds, while the action of P. chrysogenum is likely related to nutritional competition. In addition, both P. chrysogenum and D. hansenii reduced the OTA levels produced by P. nordicum. The effect of the yeast was more pronounced decreasing the concentration of OTA at quantities lower than the limit established by the Italian legislation. Therefore, P. chrysogenum and D. hansenii can be suggested as biocontrol candidates in the manufacture of 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.

BackgroundTomato fruits are susceptible to Alternaria spp. spoilage. A correct postharvest management is necessary to prevent mould growth and mycotoxin accumulation, being the temperature one of the main factors. The effect of different postharvest temperatures (5, 12, 25 and 35 °C) on growth, mycotoxin production and a stress-related gene expression by two Alternaria spp. was assessed. ResultsGrowth rates decreased rapidly when temperature was higher than the optimum (25 °C), while a gradual reduction was detected at lower temperatures. Tenuazonic acid (TeA) was strongly synthesised at all temperatures evaluated, with a maximum between 12 and 25 °C. Alternariol monomethyl ether (AME) was produced only at the two lowest temperatures; with a peak at 12 °C. Regarding the expression of the stress-related RHO1 gene, during active fungal growth both Alternaria spp. showed more copies of the gene as temperature increased. At the stationary phase, the RHO1 gene expression was significantly higher at 12 °C, coinciding with AME highest accumulation. ConclusionChanges on temperatures related to different postharvest stages of tomato fruits markedly affect toxigenic Alternaria spp. The highest levels of both mycotoxins were recorded at 12 °C, a common storage temperature for tomato fruit. Additionally, an association between alternariols biosynthesis and the cell wall integrity pathway was noticed in relation to temperature, suggesting that temperature may act as stressor stimulating the RHO1 gene expression, which in turn triggers this mycotoxin synthesis. These results will be useful in developing new strategies to efficiently control Alternaria spoilage in tomato fruit and by-products. Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Universidad de Extremadura; España Fil: Rodríguez, Alicia. Universidad de Extremadura; España Fil: Delgado, Josué. Universidad de Extremadura; España Fil: Patriarca, Andrea Rosana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 μg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 μg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Background This study aimed to investigate the molecular mechanisms involved in the onset of the white striping (WS) myopathy with particular attention to the role of oxidative stress and protein oxidation in the loss of meat quality. Results It was found that WS-M (moderate degree; white stripes 1 mm thickness) breast presented higher pH, hardness, redness, lipid, and collagen content, and lower lightness than normal breast. Compared with the latter, WS-S had a more severe loss of protein thiols (70.7% less thiols than in N), reduced activity of antioxidant enzymes such as catalase (23 versus 40 U g-1 ), glutathione peroxidase (0.21 versus 0.54 U g-1 ), and superoxide dismutase (56 versus 73 U g-1 ), and consequently, had greater accretion of thiobarbituric acid reactive substances (0.64 versus 0.22 mg MDAkg-1 muscle), allysine (3.1 versus 1.9 nmol mg-1 protein) and Schiff base structures (645 versus 258 fluorescent units). The analysis of sarcoplasmic proteins revealed that muscles severely affected by the myopathy suffered a chronic impairment of physiological (upregulation of sarcoplasmic reticulum Ca2+ ATPase, sarcalumenin and calsequestrin-2) and metabolic processes (downregulation of pyruvate kinase, creatine kinase, and l-lactate dehydrogenase). Conclusion The overexpression of ribonuclease / angiogenin inhibitor 1 and Kelch-like proteins in WS chicken breasts indicates altered protein turnover plausibly mediated by oxidative stress and accumulation of oxidized proteins. © 2020 Society of Chemical Industry.

Toxigenic moulds can develop on the surface of dry-cured meat products during ripening due to their ecological conditions, which constitutes a risk for consumers. A promising strategy to control this hazard is the use of antifungal microorganisms usually found in these foods. However, to date, the effectiveness of gram-positive catalase-positive cocci (GCC+) has not been explored. The aim of this work was to select GCC+ isolates with antifungal activity to study its effectiveness in a dry-cured ham model system at the environmental conditions reached during the ripening. Forty-five strains of GCC+ were evaluated and the isolate Staphylococcus xylosus Sx8 was selected to assess its efficacy at two different concentrations (106 and 104 cfu/mL) against Penicillium nordicum, Aspergillus flavus, Aspergillus parasiticus, and Penicillium griseofulvum at 15, 20, and 25 &deg, C. The results showed that the inoculation of 106 cfu/mL of S. xylosus completely inhibited the growth of most fungi. In addition, in the presence of this strain at 104 cfu/mL, a significant reduction in fungal growth and mycotoxins production was observed at the three temperatures studied. In conclusion, S. xylosus Sx8 possesses great potential as a biological agent to control toxigenic moulds in dry-cured meat products.

Maize, peanuts and wheat are exposed to colonisation by Aspergillus flavus and Aspergillus parasiticus, which are mould species able to produce aflatoxins B 1 (AFB 1 ) and G 1 (AFG 1 ). New tools based on molecular data can be used to obtain critical information to prevent AFs accumulation in these crops. The objective of this study was to analyse the time course of the relative expression of the aflR , aflS and aflP genes by reverse transcription real-time PCR (RT-qPCR) in relation to AFs production by A. flavus and A. parasiticus on maize, peanuts and wheat under controlled conditions during nine days of incubation. Different gene expression profiles for the three target genes were observed in maize, peanut and wheat. In general, AFB 1 amounts produced by both species were increased over incubation time, although A. parasiticus produced higher AFB 1 quantities than A. flavus in peanuts and wheat. Regarding AFG 1 production, quantities were higher at the end of the incubation time but lower than AFB 1 amounts. High relationships were found between gene expression by day 4 and AFs production by days 7 and 9. Thus, gene expression data allow predicting early AFs contamination of maize and wheat. In the case of peanuts further studies are needed.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to its high toxicity and carcinogenicity. New strategies to avoid the risk associated with OTA are therefore necessary.; Results: A comparison was made between the effects of the antifungal protein PgAFP and potassium sorbate (E-202) on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels, 0.95 and 0.93 aw . The results showed that PgAFP successfully controlled OTA production, whereas E-202, despite being able to reduce Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus.; Conclusion: PgAFP protein, a biological compound with an antifungal activity, is safer to use than E-202 and may be proposed as a food preservative and a useful strategy to control ochratoxigenic A. carbonarius in raisins. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Iberian dry-cured ham is an important meat product with high consumption worldwide. The special ecological conditions occurring throughout its ripening favour surface colonisation of filamentous fungi. Normally, moulds contribute to the development of the sensory qualities of the ham; however, some toxigenic species, such as Penicillium nordicum, are able to successfully adapt to the NaCl-rich environment found in dry-cured ham and produce ochratoxin A (OTA) in this product. Moreover, it was suggested that the biosynthesis of OTA by P. nordicum itself may support the adaptation to this food environment. However, this mechanism has not been completely elucidated yet. The objective of this work was to evaluate the influence of different concentrations of commercial OTA (cOTA, at 0, 0.2, 1 and 5 ppb) on growth rate, biosynthetic- and stress-related gene expression and OTA production by two P. nordicum strains (Pn15 and Pn69) on dry-cured ham based-media. Two NaCl conditions (0% and 10%) were evaluated for each cOTA level. In general, no intra-strain and inter-strain differences in growth rates were found among the conditions tested. The stress-related Hog1 gene expression of the strain Pn15 was affected by cOTA and NaCl concentration whilst the strain Pn69 was not affected by these variables. The expression of OTA-related otapks and otanps genes of the strain Pn15 was affected by several NaCl and cOTA combinations. However, the strain Pn69 showed no differences in relative gene expression. Regarding to OTA production, different behaviours were displayed by the two strains. The strain Pn15, which produced high OTA amounts by itself, produced OTA without the necessity of the presence of NaCl or cOTA as stressors. However, the presence of cOTA triggers OTA production by the weak OTA producing Pn69 in the absence of NaCl. In addition, although a moderate correlation was found between the expression of the OTA-related genes and mycotoxin produced by P. nordicum in the absence of NaCl, none was obtained between Hog1 gene expression and mycotoxin production. This study is a step forward for a better understanding of the ability of P. nordicum producers of OTA to colonise NaCl-rich habitats such as Iberian ham for proposing actions to minimise OTA contamination in this meat product. Fil: Delgado, Josué. Universidad de Extremadura; España Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Extremadura; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina Fil: Rodríguez, Mar. Universidad de Extremadura; España Fil: Rodríguez, Alicia. Universidad de Extremadura; España

Dry-cured meat products are frequently colonised by toxigenic Penicillium nordicum and Penicillium verrucosum throughout their ripening that can produce ochratoxin A (OTA). The predictive nature of the molecular analyses can be used to determine the risk of toxigenic species. For this reason, the objective of the present work was to analyse the temporal changes in the expression of the otapks and otanps genes by P. nordicum and P. verrucosum in relation to OTA production on slices of dry-fermented sausage salchichon and dry-cured ham. Gene expression was higher in P. nordicum than in P. verrucosum in both meat matrices. The otapks gene was overexpressed by both Penicillium species, especially in salchichon. OTA was only detected in inoculated salchichon regardless of the ochratoxigenic species used. The high significant correlation found between the early relative expression of the otapks gene and OTA production in salchichon leads to propose the relative gene expression of the otapks gene as a good indicator to predict OTA accumulation throughout the ripening of this product.

Penicillium nordicum is among the main ochratoxin A (OTA) producers in dry-cured fermented sausages. Recent strategies to prevent its development on the surface of these products include the substitution of synthetic antifungals, such as potassium sorbate, by natural compounds. However, the mechanisms of action of the latter are still unclear. The Proteomic analysis is a useful tool to elucidate the basis of the antifungal compound effects on moulds. Therefore, the aim of this study was to evaluate the efficiency of biocontrol agents on the OTA repression in P. nordicum and its relation to proteome changes. Seven different combinations of rosemary leaves, rosemary essential oil, acorn shell extract and the yeast Debaryomyces hansenii have been tested as biocontrol agents against two ochratoxigenic P. nordicum strains on a dry-cured fermented sausage-based medium. The rosemary essential oil and the combination of rosemary leaves with D. hansenii provoked significant inhibition of OTA production in both moulds (p ≤ 0.05). The acorn shell extract and D. hansenii individually decreased OTA only in one of the mould strains. The Proteomic analyses revealed that rosemary essential oil reduced the abundance of proteins involved in the polyketide synthase enoylreductase domain in both moulds, which would explain the OTA reduction. However, the combination of rosemary leaves with D. hansenii showed inter-strain differences, reducing the abundance of proteins related to the cell wall integrity and to the purine pathway, depending on the tested mould. In conclusion, rosemary essential oil and the combination of rosemary leaves with D. hansenii can be proposed as efficient biocontrol agents against ochratoxigenic P. nordicum on dry-cured fermented sausages, although some inter-strain differences at proteome level were found regarding the mechanism of action involved in the reported OTA repression.

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.

Dry-cured hams may be colonised by aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus during the ripening process. The objective of this study was to evaluate the interaction between non-ionic water stress and temperatures may have on lag phases prior to growth, growth rates and aflatoxin production by two strains of each A. parasiticus and A. flavus on meat matrices over a period of 12days. Results showed that A. flavus CBS 573.65 had shorter lag phases than A. parasiticus CECT 2688, however the growth rates were quite similar. For both species, no growth occurred at 10°C and all aw tested and optimum growth happened at 25°C and 0.95 aw. Similar aflatoxin B1 production profiles between both species were found, however A. flavus produced much higher concentration of such toxin than A. parasiticus. Both species produced aflatoxins when the temperature and the aw were ≥15°C and ≥0.90.

Traditional dry-ripened foods require the contribution of molds to achieve their particular flavor, but ripening conditions favor growth of spoilage and toxigenic molds. To control unwanted molds, physical and chemical methods are not appropriate for these foods. Lactic acid bacteria are not adequate for the low water activity at the food surface where molds grow. Yeasts are not very effective by themselves to prevent mycotoxins in dry-ripened foods. Molds producing antifungal proteins offer a quite promising means to combat unwanted molds. However, their performance on dry-ripening foods is yet to be tested. The microbial ecology of the ripened food has to be carefully considered, paying special attention to the effect on mycotoxin metabolism.

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 β 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.

The effect of high pressure assisted thermal processing (HPTP) to preserve the pumpkin puree was evaluated. Pumpkin cv. Butternut puree was submitted to 9 different combinations of pressure and temperature of HPTP (300, 600, 900 MPa/60, 70, 80 °C/1 min). Native microorganisms’, instrumental color, polyphenol oxidase (PPO) enzyme activity, carotenoids content, total antioxidant activity and individual and total polyphenols content were examined after processing and compared to non-processed puree. In treated purees, the inactivation of native microorganisms increased proportionally with pressure and temperature; CIE L* increased while CIE a* decreased respect to control puree. Moreover, most of the treatments applied reduced markedly the PPO activity, although the treatment at 600 MPa/70 °C was the most effective to reduce its activity. All treatments applied were effective to maintain or increase the content of carotenoids and phenolic compounds in the puree. The combination of 600 MPa/70 °C would be the treatment of choice since it increased the contents of bioactive compounds and the antioxidant activity of the purees, while caused the largest reduction of PPO activity.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Alternaria spp. are major contaminants of wheat crops, causing both economic losses for producers and health risk for consumers due to the accumulation of toxic metabolites. The application of synthetic fungicides in the field may trigger mycotoxin accumulation, since fungistatic levels of those compounds might cause fungal responses to stress. Hence, new alternatives are needed for its control. The aim of this work was to compare the effects of a natural antifungal compound, the antifungal protein PgAFP, and a synthetic commercial one, on Alternaria tenuissima sp.-grp. growth, mycotoxin biosynthesis (tenuazonic acid and alternariols) and the expression of a stress-related gene associated with cell wall integrity (CWI) pathway, in a wheat-based medium at two water activities (aw; 0.95 and 0.98 aw) conditions associated with the ripening of this grain. The application of both antifungals produced comparable fungistatic effects on Alternaria spp. growth. However, the presence of PgAFP produced a significant reduction in mycotoxins accumulation, whereas this effect was not observed with the commercial antifungal. To our knowledge, this is the first study on the influence of fungicides on the expression of a key gene involved in CWI stress-related pathway in relation to Alternaria mycotoxins accumulation. This information is useful when developing new antifungal methods for foods. The application of PgAFP would be a promising natural strategy for its application in wheat for the control of Alternaria spp.

Dry-fermented sausages are very appreciated by consumers. The environmental conditions during its ripening favor colonization of their surface by toxigenic molds. These molds contribute to the development of sensory characteristics; however, some of them could produce mycotoxins such as cyclopiazonic acid (CPA). CPA is mainly produced by Penicillium commune and Penicillium griseofulvum which have been found in dry-cured meat products. Thus, strategies to prevent the CPA contamination in dry-fermented sausages are needed. The objective of this work was to evaluate the ability of P. griseofulvum to produce CPA in dry-fermented sausage during its ripening as well as to test different strategies to prevent CPA production. The ability of PgAFP antifungal protein-producing Penicillium chrysogenum, Debaryomyces hansenii and Pediococcus acidilactici for inhibiting CPA production by P. griseofulvum was tested on dry-fermented sausage-based medium. Only P. chrysogenum inhibited the CPA production, so this mold was co-inoculated with P. griseofulvum on sausages whose ripening was performed at low temperature. CPA reached around 800 ng/g in the control batch, being reduced to 20 ng/g by the presence of P. chrysogenum. This work demonstrates the risk posed by CPA on dry-fermented sausages, and provides a successful strategy to prevent this hazard.

Aflatoxigenic molds can grow and produce aflatoxins on dry-fermented meat and cheese. The small, basic, cysteine-rich antifungal protein PgAFP displays a time-limited inhibitory ability against unwanted molds by increasing reactive oxygen species (ROS), which can lead to increased aflatoxin production. However, calcium abolishes the inhibitory effect of PgAFP on certain Aspergillus spp. To maximize the antifungal effect, this protein may be combined with protective cultures. Yeasts and lactic acid bacteria may counteract the impact of calcium on PgAFP fungal inhibition. The objective of this work was to study the effect of PgAFP and different combined treatments with Debaryomyces hansenii and/or Pediococcus acidilactici against growth of and aflatoxin production by an aflatoxigenic strain of Aspergillus parasiticus in both culture media and dry-fermented foods with low or high calcium levels. Aflatoxins production was increased by PgAFP but dramatically reduced by P. acidilactici in low calcium culture medium, whereas in the Ca-enriched culture medium, all treatments tested led to low aflatoxins levels. To study whether PgAFP and the protective microorganisms interfere with ROS and aflatoxin production, the relative expression of genes foxA, which is involved in peroxisomal &beta, oxidation, and aflP, which is required for aflatoxin biosynthesis, were evaluated. The aflatoxin overproduction induced by PgAFP seems not to be linked to peroxisomal &beta, oxidation. The combination of PgAFP and D. hansenii provided a successful inhibitory effect on A. parasiticus growth as well as on aflatoxin production on sliced dry-fermented sausage and cheese ripened up to 15 days, whereas P. acidilactici did not further enhance the protective effect of the two former agents. Therefore, the combined treatment of PgAFP and D. hansenii seems to provide a promising protective mean against aflatoxin-producing A. parasiticus on dry-fermented foods.

The cell wall integrity (CWI) pathway is activated in response to cell wall stresses due to different food-related environments. Rho1 is one of the main regulators within such pathway. The objective of this work was to design an easy-to-use RT-qPCR technique for the evaluation of the Rho1 gene expression useful to measure responses to the presence of cell wall stressors such as the antifungal protein PgAFP. Two primer pairs were designed from published conserved regions. Their specificity initially was determined by in silico analysis for several fungal species. After optimising the qPCR, the primer pair Rho1-F1/R2 was selected due to the lowest Cq values obtained and its specificity. The qPCR method showed efficiencies between 97.5% and 100.5%. Applicability of the designed qPCR method was evaluated in the presence of the stressor PgAFP. The PgAFP-resistant Penicillium polonicum and the PgAFP-sensitive Aspergillus flavus showed Rho1 gene over- and under- expression, respectively, indicating that the CWI pathway is activated in the former species but not activated in the latter one in response to the stress caused by PgAFP. This novel qPCR methodology able to detect changes in CWI-related gene expression in filamentous fungi will be useful in future studies to evaluate physiological mould responses to different food environmental challenges. Fil: Da Cruz Cabral, Lucía Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina. Universidad de Extremadura; España Fil: Delgado, Josué. Universidad de Extremadura; España Fil: Andrade, María J.. Universidad de Extremadura; España Fil: Rodríguez, Mar. Universidad de Extremadura; España Fil: Rodríguez, Alicia. Universidad de Extremadura; España

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.

Dry-ripened foods favor the development of a superficial fungal population that may include toxigenic molds. To combat unwanted molds, an antifungal protein from Penicillium chrysogenum (PgAFP) can be useful. The aim of the present work was to study the antimicrobial activity of PgAFP against microorganisms common in dry-ripened foods, and to evaluate its sensitivity to proteolytic enzymes and heat treatments that may be applied to foods, as well as to different pH values. The inhibitory effect of the purified protein on 38 microbial strains grown in culture medium was determined. PgAFP sensitivity to various proteases, heat treatments, and preincubation at different pH values was tested by means of the residual activity on selected reference strains. Inhibitory activity of PgAFP against unwanted molds was tested in a dry-fermented sausage. This protein exhibited potent inhibitory activity against unwanted molds, including the main mycotoxin-producing species of Aspergillus and Penicillium of concern for dry-ripened foods. PgAFP withstood most proteases, intense heat and a wide range of pH values. PgAFP efficiently reduced counts of A. flavus and P. restrictum inoculated on a dry-fermented sausage. This protein can be of interest to control hazardous molds in dry-ripened foods.

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., Work in LGC’s lab has been partially co-funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (AGL2014-55802-R and AGL2017-88120-R) and the Generalitat Valenciana (PROMETEOII/2014/027). Work in the University of Extremadura lab was supported by the Spanish Ministry of Economy and Competitiveness, Government of Extremadura and FEDER (AGL2013-45729-P, AGL2016-80209-P, GR15108).

Aspergillus flavus and Aspergillus parasiticus are mould species producers of aflatoxins (AFs) and may grow on dry-cured ham during the ripening process. In this study, the influence of different water activity (aw) and temperatures on the temporal relative expression of three genes involved in AFs biosynthesis and their relationship with AFs production on dry-cured ham-based medium were evaluated. In general, the regulatory aflR and aflS genes showed similar expression patterns, and the expression of the structural aflP gene was much higher than that obtained for aflR and aflS genes. Regarding A. flavus, a decrease of aw regardless of temperature caused an increase of the expression of the regulatory aflR and aflS genes. Concerning A. parasiticus, the highest and lowest expression values of the regulatory aflR and aflS genes were found at 0.95 aw and 0.85 aw, respectively. The expression of the structural aflP gene of both species was stimulated at low temperature and aw. The PCA analysis indicated that both toxigenic species showed a strong correlation between the relative expression of the aflR and aflS genes and the concentration of AFs under conditions which simulate dry-cured ham ripening. This suggests that an early detection of the expression of regulatory genes can be a good indicator of possible AFs contamination of dry-cured ham through ripening.

Accumulation of ochratoxin A (OTA) on the surface and to a 0.5 cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3 log cfu/cm². Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum.

Summary The decline in melatonin secretion with age seems to be one of the major reasons for increased sleep disruption in older animals. Previously, we showed that the administration with melatonin or its precursor, tryptophan, improved activity/rest rhythms in aged individuals. Here, it was evaluated the effect of a 10-day consumption of a Jerte Valley cherry-based nutraceutical product (patent no. ES2342141B1), which contains high levels of tryptophan, serotonin and melatonin, on the activity/rest rhythms of young and old rats (Rattus norvegicus) and ringdoves (Streptopelia risoria) as representatives of animals with nocturnal and diurnal habits, respectively, and its possible relationship with the serum levels of melatonin and glucose. Total diurnal and nocturnal activity pulses were logged at control, during, and up to 3 days after the treatment. Melatonin and glucose were measured with ELISA and testing kits respectively. In both young and old rats, the intake of the cherry nutraceutical decreased diurnal activity, whereas nocturnal activity increased. The opposite effect was observed for ringdoves. The treatment increased the circulating levels of melatonin in both species and restored the amplitude of the activity rhythm in the old animals to that of the non-treated young groups. The consumption of a Jerte Valley cherry-based nutraceutical product may help to counteract the impaired activity/rest rhythm found in aged animals.

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 β 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 β 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.

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (aw), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267–272 kelvins (–6 to –1°C), and growth of P. agglomerans did not change at varying pH levels (4.5–8.6). The minimum awfor growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3–42°C) or pH (5–8.6) regimes. This study has defined the range of environmental conditions (aw, pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.Key words: biocontrol, ecophysiology response, stress.

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.

Melatonin is involved in the regulation of both cellular and humoral immunity. In the present study we have evaluated the effect of the oral administration of melatonin and its precursor, the amino acid tryptophan, on humoral immune response in ringdoves (Streptopelia risoria) from different age groups. Male and female ringdoves of 4–5 years of age (young) and 12–14 years of age (old) were used in this study. The animals received a single capsule of 300 mg/kg b.w. of tryptophan (old animals) for 3 consecutive days 1 h after lights on or a single oral dose (0.25 or 2.5 mg/kg body weight/0.1 ml per animal/day, young and old animals, respectively) of melatonin, for 3 consecutive days 1 h before lights off. Blood samples were taken before beginning the treatment (basal values) and at the end of the treatment. Immunoglobulins, bactericidal and haemolytic activity were measured. Our results show that in old animals the humoral immune response was reduced with respect to the young. Both melatonin and tryptophan treatment increased the immunoglobulin concentration, with the nocturnal values being significantly higher than diurnal values and with a major effect in old animals. The bactericidal activity of the S. risoria serum against Staphylococcus aureus, after the treatment with melatonin or tryptophan, was increased at night with a greater effect in old animals. No significant differences were observed in the haemolytic activity of the serum in young animals, but there was an increase in old animals, with higher values at night after treatment with melatonin. In general, the oral administration of melatonin or tryptophan produced a stimulation of humoral immune response with greater effects in old ringdoves.

Spray drying could be a suitable method for preserving microorganisms, as it allows large quantities of cultures to be dried at low cost. The aims of this paper were to evaluate the effects of spray-drying conditions on survival of the biocontrol agent Pantoea agglomerans CPA-2, which has shown antifungal activity against Penicillium expansum and Penicillium digitatum on citrus fruits. Various compounds cited in the bibliography as carriers were tested in our spray drying, and some salts (MgSO4, K2SO4. and Na2CO3) and dairy products (lactoserum or nonfat skimmed milk [NFSM]) showed the best results in terms of recovered powder. Outlet temperature had more influence on the death of bacteria than inlet temperature. P. agglomerans was heat sensitive, and the activation energy was around 6 kcal/mol K when MgSO4 (10%) or NFSM (10%) were used as carriers and only 3 kcal/mol K when the combination of MgSO4 (10%) and NFSM (10%) was used. The highest powder recovery was obtained when NFSM was used as the rehydration medium. Although the percentage of powder recovery was not high (around 50%) and viability was low, the results suggest that with bigger spray dryers, we could expect a lower outlet temperature and probably an increased viability. Further research into spray-dryer design is needed in order to demonstrate this.