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The combined effect of temperature, water activity (a w) and NaCl content, usually found in dry-cured ham, on the growth and expression of the virulence and stress-related genes of Listeria monocytogenes was evaluated in a dry-cured ham model system. The highest growth of this pathogen was observed at 15 °C and, at 0.98 and 0.96 a w values. At 0.94 and 0.92 a w values, moderate NaCl levels stimulated the L. monocytogenes growth and repressed the expression of the four tested genes. At 7 °C, the expression of the plcA gene was favored while at 15 °C the hly and iap genes were activated. Preventive actions based on temperature, a w and salt content should be taken to minimise risks associated with growth and gene expression of L. monocytogenes in dry-cured ham. [ABSTRACT FROM AUTHOR]

'La Vera' smoked paprika is a traditional Spanish product regulated under a protected designation of origin. Mycotoxins are possible contaminants in paprika, yet there is little information about mycotoxin production during the processing of smoked paprika. In this study, samples of dried peppers collected from six traditional dryers from four producers were evaluated for physicochemical parameters, mycotoxins, and mycotoxin-producing fungi. The moisture content and water activity of the peppers ranged from 11.0 to 16.3% and 0.513 to 0.611, respectively, with significant differences among the dryers (P ≤ 0.05). Culture methods revealed fungal counts of 2.6 to 5.7 log CFU/g, with significant differences among the dryers (P ≤ 0.05), and real-time PCR revealed aflatoxin-producing fungi (2.00 to 3.42 log CFU/g) in all dryers. However, mycotoxins were not detected in dried pepper samples. Sixty-seven mold species isolated from dried peppers were identified by sequencing of the ITS1–5.8S rRNA–ITS2 region and characterized by mycotoxigenic ability. Four isolates of Penicillium expansum, four isolates of Penicillium thomii, and one isolate of Aspergillus parasiticus were producers of patulin, penicillic acid, and aflatoxins, respectively. Toxigenic fungi were inoculated onto smoked dried peppers and stored at 84, 91, 94, and 97% relative humidity (RH) at 20°C for 30 days. Patulin was not detected under any of these conditions. Penicillic acid was detected in dried samples stored at 91 to 97% RH, although the optimum condition was isolate dependent. Aflatoxins G2, B1, and B2 were detected at 91 to 97% RH, with the highest concentrations at 94% RH. According to our results, hazard analysis critical control point systems should be used to control the drying and storage conditions of dried peppers until the milling step to avoid rehydration, which encourages fungal growth and mycotoxin production. [ABSTRACT FROM AUTHOR]

Penicillium nordicum is well known for its ability to produce high amounts of ochratoxin A (OTA) in cured meat-derived products. On the other hand, Aspergillus westerdijkiae , one of the most relevant OTA-producing species of the genus Aspergillus , is usually considered a major risk in carbon-rich food matrices of plant origin. The objective of this work was to evaluate, for the first time, the ecophysiological conditions governing growth, OTA production and sporulation of A. westerdijkiae (the type-strain and one ham-native strain), in comparison with P. nordicum , in dry-cured ham based medium. For that purpose, the interaction between temperature (15, 20, 25 and 30 °C) and water activity (a w ) (0.99, 0.97, 0.93, 0.90 and 0.85), achieved with a combination of ionic (NaCl) and non-ionic (glycerol) solutes, was studied by using dry-cured ham-based medium as a model system. Different OTA production profiles were found between the two genera, and also between the two strains of A. westerdijkiae , mostly in terms of amounts of OTA produced. The optimal OTA production conditions for A. westerdijkiae were at 0.94–0.97 a w and 20–25 °C, and for P. nordicum at 0.95–0.97 a w between 18 and 22 °C. Under these conditions, A. westerdijkiae produced 1934 ng/g agar, while P. nordicum produced 712 ng/g. None of the strains was able to produce detectable amounts of OTA at 0.85 a w , under all temperatures tested. Growth and sporulation were not good indicators of OTA production by A. westerdijkiae or P. nordicum . The results obtained show that A. westerdijkiae may represent a great potential risk of OTA contamination in dry-cured ham due to the high production under a wide range of conditions. Knowledge of the ecophysiology of important Aspergillus and Penicillium species and of their adaptability to the matrices can be determinant to adopt appropriate technological modifications during ham ripening process. [ABSTRACT FROM AUTHOR]

Linear growth retardation and anemia are the most prevalent nutritional problems in the world; effective interventions are urgently needed. We evaluated Ecuador's National Food Nutrition Program (PANN 2000) that included a micronutrient-fortified complementary food (FCF), Mi Papilla, in poor periurban and rural communities of Ecuador. The program is preventive and targeted to all infants and young children living in poor communities and receiving government health services. We compared dietary intake, micronutrient status, and growth over 11 mo in a cohort of children from the catchment areas of the PANN 2000 with same-age control children in nearby communities eligible to enter the program 1 y later. PANN 2000 children enrolled in the program when they were age 9-14 mo and were age 20-25 mo at the final survey. They consumed significantly more energy, protein, fat, iron, zinc, vitamin A, and calcium than control children because of their FCF consumption. Anemia, 76% in both groups at baseline, fell to 27% in PANN 2000 children but only to 44% in control children (P < 0.001). The odds of being anemic were 58% lower for PANN 2000 children (P = 0.003). The effects on linear growth and weight were limited to children who were older when the program began (12-14 mo) and were significant for weight (interaction with age, 0.38 kg; P = 0.029) and positive but not significant for length (0.66 cm; P = 0.08). An FCF, including ferrous sulfate, delivered through public health services, is highly effective in improving weight and hemoglobin and reducing anemia. [ABSTRACT FROM AUTHOR]

The objectives of this study were to compare the effect of different Stevia-based sugar substitutes (S1–S3), sucrose alone and a mixture of sucrose + S1 on: (a) humectant properties, (b) relative colonisation rates of sponge cake slices at 0.90 a w by strains of Aspergillus flavus , Eurotium amstelodami , Fusarium graminearum and Penicillium verrucosum at 20 and 25 °C and (c) shelf-life periods in days prior to visible growth. Results showed that sucrose, S1 commercial sugar substitute and the mixture of sucrose + S1 in water solutions were able to reach water activity levels similar to those of glycerol and glucose mixtures. The S2 and S3 commercial sugar substitutes were unable to reduce a w levels significantly. At 25 °C, colonisation of sponge cake slices by E. amstelodami , A. flavus and P. verrucosum occurred in all the treatments. Growth of F. graminearum only occurred on sponge cake slices containing S2 and S3 Stevia-based products at both temperatures. The best control of growth (30 days) was achieved in cake slices modified with sucrose or S1 Stevia treatments inoculated with A. flavus and in the sucrose treatment for E. amstelodami at 20 °C. F. graminearum growth was completely inhibited when sucrose alone, S1 or sucrose + S1 treatments were used at both temperatures. This study suggests that, as part of a hurdle technology approach, replacing sucrose with low calorie sugar substitutes based on Stevia glycosides needs to be done with care. This is because different products may have variable humectant properties and bulking agents which may shorten the potential shelf-life of intermediate moisture bakery products. [ABSTRACT FROM AUTHOR]

Moulds responsible for black spot spoilage of dry-cured fermented sausages were characterised. For this purpose, samples were taken from those dry-cured fermented sausages which showed black spot alteration. Most of the mould strains were first tentatively identified as Penicillium spp. due to their morphological characteristics in different culture conditions, with one strain as Cladosporium sp. The Cladosporium strain was the only one which provoked blackening in culture media. This strain was further characterised by sequencing of ITS1-5.8S-ITS2 rRNA and β-tubulin genes. This mould strain was able to reproduce black spot formation in dry-cured fermented sausage ‘salchichón’ throughout the ripening process. In addition, a specific and sensitive real-time PCR method was also developed to detect Cladosporium oxysporum responsible for the black spot formation in sausages. This method could be of great interest for the meat industry to detect samples contaminated with this mould before spoilage of product avoiding economic losses for this sector. [ABSTRACT FROM AUTHOR]

Abstract: Rapid and specific detection of viable Escherichia coli O157:H7 cells in ready-to-eat (RTE) meat products, by duplex quantitative PCR (qPCR) procedures with mRNA and SYBR Green and TaqMan methodologies were developed. Specific primers and probes were designed based on the serotype of E. coli O157:H7, fliCh7 and rfbE genes. No cross-reactivity with other microorganisms was observed. The detection limit of the assays was 101 or 102 CFU/g for artificially contaminated meat products, and after a 4h enrichment period at 37°C, the detection limit decreased to about 1CFU/g. Time-to completion of the assay was approximately 8h. Thus, these qPCR methods offer a useful, rapid and efficient tool for screening viable E. coli O157:H7 in RTE meat products. This tool could also be proposed for monitoring these foodborne pathogens in HACCP programs. [Copyright &y& Elsevier]

Abstract: A multiplex qPCR method to quantify the implantation of the antifungal-peptide-producing Penicillium chrysogenum RP42C and aflatoxin-producing moulds including a non-competitive IAC in dry-cured ham was developed. For this, the primer pairs F/R-Pc, F/R-omt and Tub-F1/R1 and the TaqMan probes, P-Pc, OMTprobe and Tubprobe targeting the pgafp, omt-1 and β-tubulin genes, respectively, were used. The ability of the optimized qPCR method to quantify simultaneously both non-toxigenic and toxigenic moulds in inoculated dry-cured ham was demonstrated since efficiency values ranged from 102.1 to 111.1%, and the limit of detection was between 2 and 3 log cfu/cm2 for both antifungal-peptide- and aflatoxin-producing moulds. In addition, suitability of multiplex qPCR to test implantation of protective P. chrysogenum as well as growth of aflatoxigenic strain in a controlled model system was carried out successfully. The multiplex qPCR allowed demonstrating that the protective strain of P. chrysogenum RP42C limits growth of the aflatoxin-producing Aspergillus flavus strain on dry-cured ham. These findings were confirmed by the results of aflatoxin analysis, since aflatoxin B1 was not detected when the protective P. chrysogenum RP42C was inoculated. Furthermore, the multiplex qPCR including an IAC quantified efficiently the implantation of protective culture P. chrysogenum RP42C in dry-cured ham after 6 months of incubation. Thus, the multiplex qPCR should be considered a sensitive and rapid tool to monitor the implantation of fungal protective cultures and to determine growth of aflatoxin-producing moulds in dry-cured ham as well. [Copyright &y& Elsevier]

• Firms from CEE transition economies take advantage of domestic and international external knowledge sources to innovate. • The effect of external knowledge –domestic and international- on innovation strengthens as governance imperfections rise. • International knowledge sources are more valuable for firms' innovation as the level of institutional development drops. In this study, we analyze how the acquisition of domestic and international external knowledge contributes to the innovation performance of firms in transition economies and how the institutional conditions of the home country may affect these relations. We test our hypotheses via the responses of 645 firms from 18 Central and Eastern European countries. Our findings show that both external knowledge sources—domestic and international—contribute positively to the number of new products in transition economies. Our results also indicate that a country's governance imperfections positively moderate the relations between both domestic and international external knowledge and the number of new products. Additionally, our findings highlight that the benefits of international external knowledge for product innovation are greater in contexts with weaker institutional conditions than in environments with stronger institutional conditions. In contrast, the benefits of domestic external knowledge for product innovation do not vary substantially between scenarios with stronger institutional conditions and those with weaker ones. These findings lead us to conclude that the institutional conditions of transition economies moderate the relation between domestic and international external knowledge and innovation performance differently, with international external knowledge proving particularly valuable for product innovation when these conditions are weak. [ABSTRACT FROM AUTHOR]

"Torta del Casar" is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti- Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a "Torta del Casar" cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in "Torta del Casar" cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures. • Some of the LAB showed antagonist potential against Listeria monocytogenes in a soft cheese-based model. • The ProH presence repressed L. monocytogenes growth but stimulated its virulence determinants. • The strain B2 alone and combined with B4 controlled L. monocytogenes growth. [ABSTRACT FROM AUTHOR]

Most strains of Aspergillus parasiticus are able to produce high concentrations of aflatoxin (AF) B1 and G1 which are among the most potent mutagenic, teratogenic, and carcinogenic mycotoxins. Molecular studies in relation to activity of secondary metabolite gene clusters are crucial to improving food safety. In the present work, reverse transcriptase quantitative PCR (RT-qPCR) was used to monitor the influence of temperature, substrates containing nitrogen and incubation time on the omt-1 gene expression in A. parasiticus. Phenotypic AFB1 and G1 production was also evaluated by high-performance liquid chromatography–mass spectrometry (HPLC–MS). The results demonstrated that temperature (25°C and 30°C) influenced relative expression of omt-1 gene throughout the time (maximum at 25°C) while substrate composition was not affected by it. However, when effect of temperature and substrate was analyzed at each incubation time, significant effects were found. Optimal conditions for biosynthesis of AFB1 and AFG1 were similar, and they were related to changes in temperature and sodium nitrate. The highest AFB1 and G1 production levels were found at 25°C. However, lower AFB1 and G1 values were obtained when A. parasiticus grew on the substrate containing sodium nitrate and there was no production of these AFs at 37°C in any of the conditions tested. In addition, omt-1 gene expression was correlated to AFB1 and G1 syntheses at the different conditions. Use of temperature conditions and sodium nitrate concentrations which limit production of AFs holds potential for preventing AF from entering the food chain. [ABSTRACT FROM AUTHOR]

Abstract: Accumulation of ochratoxin A (OTA) on the surface and to a 0.5cm 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 7.3logcfu/cm2. Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum. [Copyright &y& Elsevier]

Abstract: The growth of aflatoxin B1 (AFB1), ochratoxin A (OTA) and patulin producing strains and the hazard of their mycotoxins production in dry-cured ham were evaluated. In addition, effectiveness of real-time quantitative PCR (qPCR) for the detection and quantification of toxigenic moulds in this product before mycotoxins production was analyzed. For this, slices of dry-cured ham were inoculated in separate assays with AFB1, OTA and patulin producers and incubated at 97% and 84% relative humidity (RH) for 21 days at 25 °C. Higher counts were reached by AFB1 and patulin producing species at 97% RH, and by OTA producing species at 84% RH. The production of AFB1, OTA and patulin on dry-cured ham slices was demonstrated. The estimation of toxigenic moulds by qPCR was highly correlated with the counts obtained by plating in culture media. All the qPCR protocols assayed detected and quantified toxigenic moulds in the hams before mycotoxins were produced. Thus, qPCR should be considered for a reliable and rapid quantification of toxigenic moulds in dry-cured ham. [Copyright &y& Elsevier]

Abstract: This paper studies the synthesis of structured triacylglycerols (STAGs) by a four-step process: (i) obtaining 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil with several alcohols, catalyzed by lipases Novozym 435, from Candida antartica and DF, from Rhizopus oryzae, (ii) purification of 2-MAGs, (iii) formation of STAGs by esterification of 2-MAGs with caprylic acid catalyzed by lipase DF, from R. oryzae, and (iv) purification of these STAGs. For the alcoholysis of cod liver oil, absolute ethanol, ethanol 96% (v/v) and 1-butanol were compared; the conditions with ethanol 96% were then optimized and 2-MAG yields of around 54–57% were attained using Novozym 435. In these 2-MAGs, DHA accounted for 24–31% of total fatty acids. In the operational conditions this lipase maintained a stable level of activity over at least 11 uses. These results were compared with those obtained with lipase DF, which deactivated after only three uses. The alcoholysis of cod liver oil and ethanol 96% catalyzed by Novozym 435 was scaled up by multiplying the reactant amounts 100-fold and maintaining the intensity of treatment constant (IOT=3glipaseh/g oil). In these conditions, the 2-MAG yield attained was about 67%; these 2-MAGs contained 36.6% DHA. The synthesized 2-MAGs were separated and purified from the alcoholysis reaction products by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield and purity of the target product were approximately 96.4% and 83.9%, respectively. These 2-MAGs were transformed to STAGs using the optimal conditions obtained in a previous work. After synthesis and purification, 93% pure STAGs were obtained, containing 38% DHA at sn-2 position and 60% caprylic acid (CA) at sn-1,3 positions (of total fatty acids at these positions), i.e. the major TAG is the STAG with the structure CA-DHA-CA. [Copyright &y& Elsevier]

Abstract: Quantification of ochratoxin A (OTA)-producing molds in foods by real-time quantitative PCR (qPCR) may be affected by the DNA extraction method used. In the present work, 6 different methods for extraction of DNA from ochratoxigenic molds in foods were tested. Several combinations of mechanical and thermal lysis of conidia with commercialized DNA extraction kits and enzymatic treatments or resins were evaluated. DNA recovery and quality of extracted DNA was measured by testing the extracted DNA with a conventional PCR and an SYBR Green qPCR amplifying the β-tubulin gene and the non-ribosomal peptide synthetase gene, otanpsPN. Inhibition of conventional and qPCR was not observed when the DNA-extraction method includes an initial thermal disruption of conidia before use of commercialized extraction kit or resin, enzymatic treatment and/or lysis buffer. Of the six methods tested, the one combining thermal lysis of conidia followed by a short enzymatic treatment and incubation with Chelex-100 resin and final extraction with the EZNA kit was selected, since the extracted DNA showed good amplification by conventional PCR for β-tubulin gene and the highest DNA recoveries when tested by qPCR. The method was subsequently validated in different food products such as ripened foods, nuts, and grapes inoculated with Penicillium and Aspergillus species. With this Chelex100-enzymatic-EZNA method good DNA recoveries ranging from 69 to 99% were obtained for all food matrices and fungal species tested. This fast method is a promising tool to be used as routine analysis in HACCP systems in the food industry for quantifying OTA-producing molds by qPCR. [Copyright &y& Elsevier]

Abstract: A multiplex real-time PCR (qPCR) method to quantify aflatoxin, ochratoxin A (OTA) and patulin producing molds in foods was developed. For this, the primer pairs F/R-omt, F/R-npstr and F/R-idhtrb and the TaqMan probes, OMTprobe, NPSprobe and IDHprobe targeting the omt-1, otanpsPN and idh genes involved in aflatoxin, OTA and patulin biosynthesis, respectively, were used. The functionality of the developed qPCR method was demonstrated by the high linear relationship of the standard curves constructed with the omt-1, otanpsPN and idh gene copies and threshold cycle (Ct) values for the respective producing molds tested to quantify aflatoxin, OTA and patulin producing molds. The ability of the optimized qPCR protocol to quantify producing molds was evaluated in different artificially inoculated foods (fruits, nuts, cereals and dry-ripened meat and cheese products). Efficiency values ranged from 81 to 110% in all inoculated foods. The detection limit was between 3 and 1logcfu/g for aflatoxin, OTA and patulin producing molds. The developed multiplex qPCR was shown be an appropriate tool for sensitive quantification of growth of toxigenic fungi in foods throughout the incubation time. Thus, the multiplex qPCR is a useful, rapid and efficient method to quantify simultaneously aflatoxin, OTA and patulin producing molds in food products. [Copyright &y& Elsevier]

Abstract: Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography–mass spectrometry (HPLC–MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain. [Copyright &y& Elsevier]

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10² to 10³ CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods. [ABSTRACT FROM AUTHOR]

Abstract: Patulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 × 102 to 2.7 × 103 conidia g−1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food products. [Copyright &y& Elsevier]

Abstract: Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1x104 to 10conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods. [Copyright &y& Elsevier]

Abstract: Coho salmon (Oncorhynchus kisutch) supports an important farming production in parallel with capture delivery, giving rise to products of great economic importance in many countries. This review covers the research carried out during the last decades related to its employment as a food product. In the first part, studies carried out concerning the chemical constituent composition and nutritional value are reviewed; special attention is accorded to the wild/farmed fish comparison and to the effect of diet on lipid composition variations. In agreement to the great lability of chemical constituents of aquatic foods, the second part of the manuscript provides a revision of coho salmon research related to the chemical component changes produced during technological processing and their effects on nutritional and sensory losses; in this case, special attention is accorded to studies employing advanced technological strategies focused to partially inhibit the development of the different damage pathways. [ABSTRACT FROM AUTHOR]

Abstract: A relevant farmed fish species (coho salmon; Oncorhynchus kisutch) was studied as a raw material for the canning process. The effects of preliminary chilling storage and thermal treatment (cooking and sterilisation) on the chemical constituents (lipids and non-protein nitrogen compounds) of the canned fish were analysed. An increasing previous chilling time led to an important autolysis (K value) development, and to an increasing formation of free fatty acids, and interaction compounds (fluorescence and browning assessments) (p <0.05) in the canned product. The thermal treatment led to the formation of volatile amines (total and trimethylamine), free fatty acids, secondary lipid oxidation compounds (anisidine and thiobarbituric acid values) and interaction compounds in canned fish. Interaction compound assessment was found the most useful tool to study the lipid oxidation and non-enzymatic browning developments, while the K value showed to be an interesting index for assessing the freshness stage of the raw material employed. [Copyright &y& Elsevier]

• Relevance of geographic proximity and international diversity of R&D collaboration. • Nearer geographic partners contribute more to innovation success than failure. • More distant geographic partners contribute more to innovation failure than success. • Lower international diversity contributes more to innovation success than failure. • Higher international diversity contributes more to innovation failure than success. We aim to clarify the role of research partnerships on the success and failure of innovation projects by examining the geographic proximity and diversity of partners. First, we argue that collaboration with geographically near partners will contribute relatively more to innovation success than it does to innovation failure, while collaboration with geographically distant partners will contribute relatively more to innovation failure than it does to innovation success. Second, we postulate that lower levels of international diversity will contribute relatively more to innovation success than it does to innovation failure, while higher levels of international diversity will contribute relatively more to innovation failure than it does to innovation success. Using a large dataset of firms for the period 2008–2013, we perform a joint analysis of failed and successful innovations. Our empirical findings support our theoretical arguments. Our results highlight the relevance of studying both failed and successful innovations and the importance of knowing their determinants to manage the innovation process successfully. Moreover, our findings should alert managers to the importance of geographic location when choosing collaboration partners. It is noteworthy that beyond a certain threshold, international diversity begins to act as a brake on innovation success and to increase the likelihood of failure. [ABSTRACT FROM AUTHOR]

Water pollution due to microplastics (MPs) is recognized as a major anthropogenic impact. Once MPs reach the ecosystems, they are exposed to a variety of other pollutants, which can be sorbed on them, transported and eventually desorbed. In this work, we tested the hypothesis that MPs can behave as conveyors for delivering chemicals toxic to aquatic microorganisms by investigating the vector role of MPs of polyethylene terephthalate (PET), polylactic acid (PLA), polyoxymethylene (POM) and polystyrene (PS) to the macrolide antibiotics azithromycin (AZI) and clarithromycin (CLA). AZI and CLA were chosen, as they are included in the Watch List for EU monitoring concerning water policy by Decision (EU) 2018/840. MPs were loaded in contact with 500 μg/L of AZI or 1000 μg/L of CLA. Results showed that both antibiotics were sorbed on all tested MPs. The more hydrophobic AZI was sorbed in higher proportion than CLA. Both antibiotics were desorbed from MPs upon contact with water with percentages between 14.6 ± 2.6% for AZI and 1.9 ± 1.4% for CLA of the concentrations to which the MPs were initially exposed. Virgin MPs were not toxic to the cyanobacterium Anabaena sp. PCC7120. However, antibiotic-loaded MPs significantly inhibited the growth and chlorophyll content of the cyanobacterium. Most of the sorbed antibiotics became released upon contact with cyanobacterial cultures, which was the cause for the observed toxicity. Therefore, MPs can play a role as vectors of antibiotics in freshwaters systems affecting the basic trophic level of photosynthetic microorganisms. Image 1 • Macrolide antibiotics were sorbed on all tested microplastics. • The antibiotics desorbed from loaded microplastics once in contact with water. • Desorption of antibiotics from loaded MPs negatively affected primary producers. • Microplastics can play a role as vectors of antibiotics in freshwaters systems. [ABSTRACT FROM AUTHOR]

Penicillium nordicum , an ochratoxin A (OTA) producer, widely contaminates the surface of dry-cured fermented sausages. Although the meat industry uses antifungal synthetic compounds during ripening, consumers' preferences currently stand up for natural preservatives. The aim of this study was to evaluate the antifungal effect of spices commonly added to dry-cured fermented sausages during their processing on P. nordicum for establishing their value as an alternative strategy to synthetic compounds. The mould strain was grown on a dry-cured fermented sausage-based medium containing oregano, rosemary or thyme. Moreover, an antifungal commercial preparation containing potassium sorbate and natamycin was tested alone and in combination with the spices. The growth rate, OTA accumulation and relative expression of genes involved in OTA biosynthesis (otapks and otanps) and some stress pathways (Hog1 and Rho1) were evaluated. In the growth assessment, the oregano alone or in combination with the antifungal preparation significantly reduced the growth rate. Different patterns were observed at both sampling times for the OTA production and gene expression analyses, and even differences between treatments at the same incubation period were observed. Regarding OTA accumulation, significant reductions were found when adding oregano or rosemary in the presence and absence of the antifungal compounds. While a stimulation of the expression of most of the tested genes was obtained at the initial stages, a repression was generally found at the end of the incubation. This makes sense since the mycotoxin accumulation was usually higher at the initial stages than at the end of the incubation. Accordingly, such spices would allow satisfying the current consumers' demand for natural preservatives minimising the hazard associated with the OTA presence in cured meats at the same time. • Oregano significantly reduced the growth rate of P. nordicum. • Oregano and rosemary significantly reduced the OTA accumulation. • The reduction of OTA amount correlated with a repression of stress-related genes. • Spices provide safer dry-cured fermented sausages regarding the OTA occurrence. [ABSTRACT FROM AUTHOR]

• Potential benefits of alternative schools on child development are reviewed. • Montessori and Waldorf are the main schools studied. • Most studies focus on executive function, creativity, and academics achievement. • Limited focus on emotional and social development. • Alternative schools show benefits or no difference with conventional schools. Alternative schools such as Montessori, Reggio Emilia or Waldorf emerged on the educational scene over a century ago but have proliferated internationally in the last 15–20 years. In addition to being considered as educational alternatives to conventional approaches, these schools are often associated with enhanced benefits in cognitive, social, emotional, and personal development of attending children. This assumption stems from the fact that these approaches are aligned with the basic principles of child development, especially because in these schools, daily practices are organized according to children's developmental strengths and considering individual learning rhythms. However, empirical research on this assumption is scarce and little is known about the type of schools studied and the aspects of development analyzed. Thus, this systematic review aims to address two objectives: to identify which types of alternative schools have captured the interest of researchers and to explore the most studied areas of cognitive and socioemotional development during childhood, along with the main findings. The review includes studies conducted in the last decade that compare the effects of attending alternative schools versus conventional preschools, elementary schools, or high schools. Twenty-four articles were included, most of them focused on Montessori and, to a lesser extent, Waldorf schools. Other types of alternative schools (democratic, Freinet) had limited representation. Executive function, creativity and academic achievement have received more attention in research compared to well-being, social competence, or independence. Overall, the results show a better performance in children from alternative schools or no differences with their counterparts in conventional schools. However, this study provides a critical perspective on these findings, highlighting limitations that should be considered when interpreting them and guiding future research endeavors. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • P. griseofulvum is able to produce CPA during dry-fermented sausage ripening. • Neither D. hansenii nor P. acidilactici affected CPA synthesis on sausage medium. • P. chrysogenum lowered CPA on dry-fermented sausage-based medium and on sausages. • The PgAFP gene expression increase is related to inhibition of CPA production. [ABSTRACT FROM AUTHOR]

The environmental conditions reached during the ripening of dry-cured meat products favour the proliferation of moulds on their surface. Some of these moulds are hazardous to consumers because of their ability to produce ochratoxin A (OTA). Biocontrol using Debaryomyces hansenii could be a suitable strategy to prevent the growth of ochratoxigenic moulds and OTA accumulation in dry-cured meat products. The aim of this work was to evaluate the ability of two strains of D. hansenii to control the growth and OTA production of Penicillium verrucosum in a meat model under water activities (a w ) values commonly reached during the dry-cured meat product ripening. The presence of D. hansenii strains triggered a lengthening of the lag phase and a decrease of the growth rate of P. verrucosum in meat-based media at 0.97 and 0.92 a w . Both D. hansenii strains significantly reduced OTA production (between 85.16 and 92.63%) by P. verrucosum in the meat-based medium at 0.92 a w . Neither absorption nor detoxification of OTA by D. hansenii strains seems to be involved. However, a repression of the expression of the non-ribosomal peptide synthetase ( otanps PN) gene linked to the OTA biosynthetic pathway was observed in the presence of D. hansenii. To confirm the protective role of D. hansenii strains, they were inoculated together with P. verrucosum Pv45 in dry-fermented sausage and dry-cured ham slices. Although P. verrucosum Pv45 counts were not affected by the presence of D. hansenii in both meat matrices, a reduction of OTA amount was observed. Therefore, the effect of D. hansenii strains on OTA accumulation should be attributed to a reduction at transcriptional level. Consequently, native D. hansenii can be useful as biocontrol agent in dry-cured meat products for preventing the hazard associated with the presence of OTA. [ABSTRACT FROM AUTHOR]

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA , of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA -based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments. [ABSTRACT FROM AUTHOR]

Anaerobic digestion is a growing technology to manage organic waste and produce bioenergy. To promote this technology, it is essential to know, at the molecular level, the dynamics of microbial communities, specifically the methanogenic community. In the present study, three primer pairs were selected from seven primer pairs which were designed and tested with different concentrations and conditions to detect Methanosarcina , Methanoculleus and Methanobacterium by real-time PCR based on the SYBR Green System. The functionality of the developed methods was demonstrated by the high linear relationship of the standard curves, and the specificity of each primer was empirically verified by testing DNA isolated from methane-producing and non-producing strains. These assays also exhibited good repeatability and reproducibility, which indicates the robustness of the methods. The described primers were successfully used to investigate the methanogenic communities of 10 samples from an anaerobic co-digestion. The genus Methanosarcina was the dominant methanogenic group. • Development of SYBR Green-based qPCR methods to quantify methane-producing archaea. • The qPCR methods showed high sensitivity, specificity and robustness. • Methanosarcina was the dominant methanogenic archaea in the digestate studied. • Reliable quantification of three genera of methane-producing archaea was obtained. [ABSTRACT FROM AUTHOR]

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii , Pichia kudriavzevii , Kluyveromyces lactis , and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics. [ABSTRACT FROM AUTHOR]

Abstract: The ability of the osmotolerant yeast Debaryomyces hansenii to inhibit Penicillium nordicum, the most common ochratoxigenic mould encountered in dry-cured meat products, was evaluated. The antagonistic effect of ten D. hansenii strains isolated from dry-cured ham was screened in vitro using malt extract media and meat extract peptone media with the water activity (a w) adjusted to 0.97 and 0.90. A significant inhibition of the two tested P. nordicum strains by D. hansenii cells and cell-free supernatants was observed. At 0.97 a w, increasing D. hansenii inoculum concentrations significantly improved the inhibition of mould growth on solid medium, whereas at 0.90 a w this was not always the case. As observed by bright field microscopy, most D. hansenii strains were able to delay P. nordicum spore germination when co-cultured in malt extract broth. D. hansenii FHSCC 253H showed the highest overall in vitro inhibition of ochratoxigenic mould growth, and was therefore chosen for co-cultivation assays in dry-cured ham slices incubated at 0.94 and 0.84 a w simulating ham ripening. Regardless of the experimental conditions tested, lower levels of the inoculated P. nordicum strain were detected in co-cultivation batches compared with batches without D. hansenii. The highest level of mould growth inhibition was observed in batches at 0.94 a w. Ochratoxin A (OTA) production in ham samples was detected by HPLC-MS. Co-culturing of P. nordicum with D. hansenii FHSCC 253H resulted in lower OTA levels compared with control samples without D. hansenii. The decrease of the mycotoxin presence due to D. hansenii FHSCC 253H was more efficient at 0.94 a w (OTA was below the detection limit). In conclusion, D. hansenii is potentially suitable as a biopreservative agent for preventing ochratoxigenic mould growth and OTA accumulation in dry-cured meat products. The inoculation of D. hansenii should be made at the beginning of processing (at the end of post salting) when the a w of the product is still high (near 0.94). This action in addition to application of appropriate hygienic actions and control of temperature and relative humidity throughout ripening is required to reduce health risks due to OTA exposure. [Copyright &y& Elsevier]

The processing of dried figs in the industry involves a number of stages that present a significant risk of filamentous fungal infection of the fruit and subsequent mycotoxin contamination, due to the changes in temperature and water activity (a w) to which dried figs are exposed. In this study, the environmental conditions and the physicochemical parameters of dried figs at different processing stages were evaluated in 3 different industries, and were associated with fungal counts and the presence of toxigenic moulds and their mycotoxins. For this, dried figs at 5 relevant stages of industrial processing (curing, sizing, blanching, storage, and final product) in 3 industries located in Extremadura (Spain) were sampled. Changes in moisture content and a w of dried figs during processing were observed and they influenced the mycological quality of figs. Among the fungal genera, Aspergillus spp. predominated in most stages except blanching, where Penicillium spp. prevailed. About 10% of the dried fig samples were contaminated with aflatoxins (AFs) and 6% with ochratoxin A (OTA). Based on findings, longer drying times are necessary after blanching to reduce a w and to avoid the development of toxigenic moulds. In addition, all stages covering industry processing, final storage, and retailing of dried figs are advisable to be conducted at refrigeration conditions and controlled relative humidity to avoid mycotoxin production. The enumeration of AFs- and OTA- producing moulds by real-time PCR seems to be a good indicator for integration into prevention strategies to control filamentous fungal hazards and subsequent mycotoxin synthesis during the processing of dried figs. • Evaluation of 5 high risk stages of dried fig processing on their mycological quality • Aspergillus spp. prevailed in most stages except in blanching, where Penicillium did. • ≤ 10% of aflatoxin- or ochratoxin A- contaminated dried-fig samples were found. • Toxigenic mould enumeration by qPCR may be used into prevention strategies to control fungal hazards in figs • Longer drying times after blanching and post-harvest at ≤4 °C control toxigenic moulds [ABSTRACT FROM AUTHOR]

Abstract: Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 102–104 cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs. [Copyright &y& Elsevier]

Abstract: We report a case of chronic myeloid leukemia in chronic stage with 48 chromosomes and four BCR/ABL1 fusion signals on two out of three chromosomes 9 and two signals on the two Philadelphia chromosomes. These abnormalities were detected by both conventional cytogenetic analysis and metaphase and interphase fluorescence in situ hybridization studies in ∼90% of the cells at diagnosis. Real-time–polymerase chain reaction studies on peripheral blood showed b3a2(p210) and e1a2(p190) BCR/ABL1 fusion transcripts. During treatment with imatinib, the patient was asymptomatic with hematological remission. Cytogenetic and fluorescence in situ hybridization analysis revealed that only 6.6% of cells had the initial majority line karyotype, with disappearance of the p210 but increased p190 transcript, which led to the treatment being changed. We discuss the implication of cytogenetic and molecular alterations in the patient''s evolution and treatment. [Copyright &y& Elsevier]

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 (a w) 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 a w 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. • Penicillium chrysogenum and Debaryomyces hansenii reduced the growth of P. nordicum. • D. hansenii showed dominance at distance over P. nordicum. • The action of P. chrysogenum over Penicillium nordicum is based on nutritional competition. • P. chrysogenum and D. hansenii limited OTA production by P. nordicum. • P. chrysogenum and D. hansenii can be proposed as biocontrol agents in dry-cured meats. [ABSTRACT FROM AUTHOR]

Dry-cured ham may be contaminated with aflatoxin B 1 (AFB 1) produced by Aspergillus spp. Temperature and water activity (a w) are two key parameters that affect both ham ripening and AFB 1 production. The objective of this study was to predict AFB 1 production by Aspergillus parasiticus and Aspergillus flavus strains in conditions related to dry-cured ham ripening using data mining techniques. J48 decision tree, isotonic regression (IR), and multiple linear regression (MLR) were tested to (a) classify and predict AFB 1 concentration as a function of different days, temperatures and a w values and (b) predict the beginning of AFB 1 production as a function of different temperatures and a w values. For this, a model system based on a dry-cured ham-based medium was used. The percentage of correct classification was higher than 75%. R values to predict the concentration of AFB 1 when applying MLR were 0.81, being higher than those obtained after using IR. The models developed were validated with experimental data obtained after inoculating samples of dry-cured ham with two aflatoxigenic strains. The predicted AFB 1 concentration showed correlation coefficients ≥0.74 and prediction errors ≤0.38, confirming the feasibility of the prediction equations obtained. This information may help to make informed decisions to minimise the hazard posed by AFB 1 in dry-cured ham. • Data mining was applied to predict AFB 1 production by Aspergillus in dry cured ham. • A MLR model to predict aflatoxin risk in dry-cured ham was developed. • AFB 1 production was classified applying J48 decision tree. • The predictions were validated in dry cured ham with high correlation coefficients. • Data mining techniques could be used as predictive tools for AFs risk assessment. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to analyse the pattern and diffusion capacity of secondary metabolites produced by ochratoxin A (OTA)- and cyclopiazonic acid (CPA)-producing mould species commonly growing in dry-cured meat products. Dry-fermented sausage and dry-cured ham pieces were inoculated with Penicillium nordicum , Penicillium verrucosum and Penillium griseofulvum , and incubated at 20 °C for 15 days at 94% relative humidity. After incubation, the samples were divided into 3 subsamples: A (0–1 cm; including the fungal colony), B (1–2 cm) and C (2–3 cm). The subsamples were analysed with a UHPLC-Q trap-MS method capable of detecting and quantifying 33 mycotoxins at values around 1 μg/kg. Mould strains produced from 5 to 12 secondary metabolites on the surface of the meat products. Besides, a higher number of metabolites was encountered in dry-fermented sausage than in dry-cured ham. Some of the fungal secondary metabolites diffused from the surface (layer A) into the inner core of meat products (layers B and C) contaminating the products up to 3 cm depth. In general, the concentration of fungal metabolites able to diffuse generally decreased as they diffused inward; while other compounds remained on the surface. This supposes a problem for the industry given that the removal of mouldy surface of dry-cured meats seems not to protect consumers' health. Highlights • Penicillium produces various mycotoxins and secondary metabolites in cured meats. • Some metabolites may diffuse into the inner core of cured meats up to 3 cm depth. • More metabolites in dry-fermented sausage than in dry-cured ham were found. • In general, metabolite concentration decreased as it diffused inward in cured meats. • Removal of cured meat surface seems not to be enough to protect consumers' health. [ABSTRACT FROM AUTHOR]

Understanding of the mechanisms implicated in the protective role of probiotic bacteria is of the utmost scientific interest. This study provides original insight into the genetic and molecular basis of the responses of Lactobacillus reuteri PL503 against hydrogen peroxide (H 2 O 2)-induced oxidative stress. Six experimental groups were considered depending on the addition and concentration of H 2 O 2 and resveratrol: 1. CONTROL (L. reuteri in MRS broth); 2. H 2 O 2 (L. reuteri in MRS broth + 0.5 mM H 2 O 2); 3. LRES (L. reuteri in MRS broth + 20 μM resveratrol); 4. HRES (L. reuteri in MRS broth + 100 μM resveratrol); 5. H 2 O 2 -LRES (L. reuteri in MRS broth + 0.5 mM H 2 O 2 + 20 μM resveratrol); 6. H 2 O 2 -HRES (L. reuteri in MRS broth + 0.5 mM H 2 O 2 + 100 μM resveratrol). Three replicates were incubated at 37 °C for 24 h in microaerophilic conditions sampled at 12, 16, 20 and 24 h. The NADH-dependent-oxidoreductase encoded by the dhaT gene is a plausible candidate to be strongly implicated in the antioxidant response of L. reuteri. Resveratrol (100 μM) is found to protect L. reuteri against protein carbonylation plausibly through various mechanisms including direct scavenging of reactive oxygen species (ROS), upregulation of the dhaT gene and promoting the synthesis of sulfur containing compounds. The hypothesis formulated on the ability of L. reuteri to detoxify H 2 O 2 and its underlying mechanism needs to be clarified. Furthermore, the consequences of protein carbonylation as a reflection of oxidative damage to bacteria and its role in the responses of bacteria to oxidative stress need to be further investigated. Image 1 • Hydrogen peroxide induced thiol depletion and protein carbonylation in L. reuteri. • The NADH-dependent-oxidoreductase (dhaT gene) is activated against the oxidative threat. • Resveratrol protects L. reuteri against hydrogen peroxide-induced protein oxidation. • Protein carbonyls and resveratrol derivatives may act as signaling molecules. [ABSTRACT FROM AUTHOR]

Abstract 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 (a w ; 0.95 and 0.98 a w) 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. Highlights • The antifungal protein PgAFP reduced Alternaria growth on a wheat-based medium. • PgAFP caused a reduction in mycotoxin accumulation at both 0.95 and 0.98 a w. • A commercial fungicide applied at fungistatic level triggered mycotoxin production. • The stress-related CWI pathway and alternariols accumulation may be associated. • PgAFP is a promising natural strategy for the control of Alternaria spp. in wheat. [ABSTRACT FROM AUTHOR]

Abstract Ochratoxigenic penicillia can grow on the cured meat products surface during their ripening. Ingredients added to cured meat products throughout their processing, and specifically of dry-fermented sausages, consist of a potential strategy to prevent or minimise the hazard associated with ochratoxin A (OTA) in these products. The aim of this work was to evaluate the effect of different concentrations of NaCl, KCl and sucrose in a conducive medium on the growth and OTA production by two Penicillium verrucosum strains. In general, there were no statistical differences between their growth rates. Although OTA production was not completely avoided in the presence of KCl and sucrose, it was reduced by comparison with NaCl. The concentration of the ingredients also affected the OTA production by P. verrucosum. These results would mean that the replacement of NaCl with KCl would imply the production of potentially safer cured meat products in terms of the OTA presence, which is of great importance for the current trends to reduce the NaCl levels in this kind of products. Furthermore, the addition of sucrose should be considered one of the approaches of the hurdle technology for minimising OTA accumulation in cured meat products. Highlights • The role of cured meat ingredients on mould growth and OTA production was evaluated. • Two ochratoxigenic Penicillium verrucosum strains were investigated. • Type and amount of cured meat ingredients influenced growth and OTA accumulation. • Large differences between the tested two P. verrucosum strains were not found. • Formulation changes might provide safer cured meats in terms of the OTA presence. [ABSTRACT FROM AUTHOR]

An extraction method and an UHPLC–MS/MS method for the quantification of CPA in dry-cured ham were developed and validated. To optimise detection and quantification of CPA, the composition of mobile phase, flow rates, gradient–related factors and solvents used for resuspension of dry extracts were evaluated. Besides, four extraction methods were tested. The best peak shape and resolution were obtained by eluting the mobile phase consisting in acetic acid-ammonium acetate buffer pH 5.75/methanol in gradient mode at a flow rate of 0.2 mL/min. The method 4 relied on the QuEChERS methodology was the most effective one. Almost half of the 61 dry-cured ham samples examined were contaminated with CPA, with values ranging from 36.1 to 540.1 ng/g. The combination of a QuEChERS-based extraction method and analysis by UHPLC–MS/MS allows highly sensitive, fast, reliable and cheap detection and quantification of CPA for routine analysis in ham. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]