Effects of Phenolic Acids on the Growth and Production of T-2 and HT-2 Toxins by Fusarium langsethiae and F. sporotrichioides.

The effect of natural phenolic acids was tested on the growth and production of T-2 and HT-2 toxins by Fusarium langsethiae and F. sporotrichioides, on Mycotoxin Synthetic medium. Plates treated with 0.5 mM of each phenolic acid (caffeic, chlorogenic, ferulic and p-coumaric) and controls without phenolic acid were incubated for 14 days at 25 °C. Fungal biomass of F. langsethiae and F. sporotrichioides was not reduced by the phenolic acids. However, biosynthesis of T-2 toxin by F. langsethiae was significantly reduced by chlorogenic (23.1%) and ferulic (26.5%) acids. Production of T-2 by F. sporotrichioides also decreased with ferulic acid by 23% (p < 0.05). In contrast, p-coumaric acid significantly stimulated the production of T-2 and HT-2 toxins for both strains. A kinetic study of F. langsethiae with 1 mM ferulic acid showed a significant decrease in fungal biomass, whereas T-2 production increased after 10 days of incubation. The study of gene expression in ferulic supplemented cultures of F. langsethiae revealed a significant inhibition for Tri5, Tri6 and Tri12 genes, while for Tri16 the decrease in gene expression was not statistically significant. Overall, results indicated that phenolic acids had a variable effect on fungal growth and mycotoxin production, depending on the strain and the concentration and type of phenolic acid assayed.

The Effects of Aflatoxin B1 Intake in Assaf Dairy Ewes on Aflatoxin M1 Excretion, Milk Yield, Haematology and Biochemical Profile.

The aim of this study was to investigate the in vivo transfer of aflatoxin B1 (AFB1) to Assaf ewes' milk (aflatoxin M1, AFM1) and its effect on animal performance and health. Thirty Assaf ewes were allocated to three groups (C, L, H), and received a different individual daily dose of AFB1 (0, 40 and 80 µg) for 13 days. Milk (days 1, 2, 3, 4, 7, 14, 16 and 18) and blood (days 1, 7, 14 and 18) samples were collected. Milk yield, composition (except protein) and somatic cell counts (SCC) were not affected by AFB1 intake (p > 0.05). Haemoglobin concentration increased (p < 0.05) and haematocrit and alanine aminotransferase levels tended to increase (p < 0.10) in group H on day 14. AFM1 excretion was highly variable and detected in L and H animals from days 1 to 16 (3 days increase, 10 days steady-state, 3 days clearance). Carry-over rate (0.23%) was significantly higher in L (0.22-0.34%) than in H (0.16-0.19%) animals (p < 0.05). AFB1 daily doses of 40 to 80 µg do not impair milk yield; however, it may start affecting animals' health. Milk AFM1 depends mainly on the AFB1 intake whereas carryover rate is positively influenced by the level of milk production.

Inhibition of Aspergillus Parasiticus Growth and Aflatoxins Production by Natural Essential Oils and Phenolic Acids.

Aflatoxins represent a significant risk to food safety, and strategies are being implemented to reduce their entry into the food chain. The aim of this study was to evaluate the in vitro effect of four essential oils (EOs) (lavandins Grosso and Abrial, Origanum virens, and Rosmarinus officinalis) and four natural phenolic acids (PAs) (caffeic, chlorogenic, ferulic, and p-coumaric) on the growth and aflatoxins (B1, B2, G1, and G2) production by Aspergillus parasiticus. Minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC) were determined by the broth macrodilution method. Additionally, the mycelia weight was determined at concentration levels lower than MIC. The antiaflatoxigenic activity was evaluated in the two concentrations of the EOs right before MIC and at concentrations below the MIC value for the PAs. To this end, in-house validated methodology based on high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection (HPLC-PHRED-FLD) was used. EOs of O. virens and lavandins (Grosso and Abrial) completely inhibited mold growth. In addition, a significant reduction in mycelial mass (p < 0.05) was observed for all EOs and PAs at different concentrations. In all cases except for lavandin Abrial, EO concentrations just before the MIC value strongly reduced (p < 0.05) aflatoxins synthesis. Aflatoxins production was completely inhibited by all PAs at a concentration of 20 mM; although at low concentrations, mycotoxin production was stimulated in some cases. The present study provides a scientific basis for further study of the inhibiting mechanisms.

Chemical composition and antimicrobial activity of essential oils of Thymus algeriensis, Eucalyptus globulus and Rosmarinus officinalis from Morocco.

BACKGROUND: The present study reports on the antimicrobial activity and chemical composition of the essential oils (EOs) of Thymus algeriensis, Eucalyptus globulus and Rosmarinus officinalis from Morocco. RESULTS: The composition of these species was analysed by GC-MS, and 65 components were identified. Eucalyptus globulus EO showed a great similarity with EOs from other regions, with 1,8-cineole (79.85%) the major component. Also rich in this constituent was Rosmarinus officinalis (43.99%). However, the chemical profile of Thymus algeriensis was rather different, and for the first time such a high content of borneol (23.48%) has been described in this EO. The antimicrobial activity of these species has also been studied against seven pathogenic and spoiling bacteria of significant importance. According to the results, Thymus algeriensis showed the best bacteriostatic and bactericidal effect, followed by Eucalyptus globulus and Rosmarinus officinalis. CONCLUSIONS: As far as we know this is the first time that minimum inhibitory and bactericidal concentration values have been reported for Eucalyptus globulus EO. Our data support the possible use of this EO as well as Thymus algeriensis EO, as potential natural agents in preservatives for food and pharmaceutical products.

Field Monitoring of Aflatoxins in Feed and Milk of High-Yielding Dairy Cows under Two Feeding Systems.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) that can be excreted in milk of cows after consuming contaminated feed. The aim of this study consisted of a field monitoring to assess the contamination levels of AFB1 in 60 feed samples from two feeding systems for high-yielding dairy cows and of AFM1 in the corresponding raw milk samples. The aflatoxins were analyzed by in-house validated methods based on high-performance liquid chromatography (HPLC) with fluorescence detection. AFB1 was detected in 55% of feed samples (mean 0.61 µg/kg, with 2 samples exceeding the European Union (EU) maximum level set at 5 µg/kg), with greater incidence and concentration in compound feed than in unifeed rations (p < 0.05). AFM1 was detected in 38.3% milk samples (mean 12.6 ng/kg, with 5 samples exceeding the EU maximum level set at 50 ng/kg), with a higher occurrence in milk of cows fed compound feed, as well as in spring milk compared to that produced in winter. The overall transfer ratio of aflatoxins from feed to milk was 3.22%, being higher in cows fed with compound feed and in spring milkings. In a selection of positive matched samples (n = 22), the ratio AFM1/AFB1 exceeded the European Food Safety Authority (EFSA) estimated 6% threshold for high-yielding dairy cows.

Supercritical Carbon Dioxide Antisolvent Fractionation for the Sustainable Concentration of Lavandula luisieri (Rozeira) Riv.- Mart Antimicrobial and Antioxidant Compounds and Comparison with Its Conventional Extracts.

Lavandula stoechas subsp. luisieri is a Spanish subspecies from the Lamiaceae family. Its essential oil has been traditionally used for several medical applications though little is known about other extracts. Similar to many other studies aiming to obtain traditional plant extracts to be used in different applications, this work evaluated the antioxidant and antimicrobial activities of Lavandula luisieri extracts and the correlation with their composition. Traditional hydrodistillation and ethanolic maceration were used to obtain the essential oil and the maceration extract, respectively. A green and sustainable methodology was applied to the maceration extract that was under a Supercritical Antisolvent Fractionation process to obtain a fine solid enriched in rosmarinic acid and the terpenes oleanolic and ursolic acids. Antimicrobial activities of all extracts and pure identified compounds (rosmarinic and ursolic acids) were evaluated against five bacterial strains; Listeria monocytogenes, Enterococcus faecium, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli and were compared with the pure compounds identified, rosmarinic and ursolic acids. All strains were sensitive against L. luisieri essential oil. The solid product obtained from the supercritical process was concentrated in the identified actives compared to the maceration extract, which resulted in higher antimicrobial and DPPH scavenging activities. The supercritical sustainable process provided L. luisieri compounds, with retention of their antimicrobial and antioxidant activities, in a powder exemptof organic solvents with potential application in the clinical, food or cosmetic fields.

Occurrence and Exposure Assessment of Aflatoxins and Deoxynivalenol in Cereal-Based Baby Foods for Infants.

Aflatoxins are carcinogenic to humans and deoxynivalenol causes digestive disorders, and both mycotoxins occur frequently in cereal-based foods. The purpose of this study was to investigate the occurrence and levels of aflatoxins (B1, B2, G1 and G2) and deoxynivalenol (DON) in cereal-based baby foods as well as to calculate the estimated daily intakes (EDI) in different stages of infancy. Sixty samples of infant cereals (wheat-, corn-, rice-, oat-, and mixed grain-based) were collected during a 2-year period and analyzed by validated methods. Aflatoxins were detected in 12 samples (20%), six of which exceeded the EU maximum level for aflatoxin B1 set at 0.10 µg/kg. Deoxynivalenol appeared in 20% of baby food samples, with one sample exceeding the EU maximum level established at 200 µg/kg. There were no significant differences between gluten-free products for babies aged 4⁻6 months and multi-cereal products for infants aged 7⁻12 months, nor between whole-grain-based and refined ingredients. However, baby food products of organic origin showed significantly higher levels of deoxynivalenol than conventional ones (p < 0.05). It is proposed for the health protection of infants and young children, a vulnerable group, to establish the lowest maximum level for the sum of aflatoxins (B1, B2, G1 and G2) in baby food.

Evaluation of GC-ion trap-MS/MS methodology for monitoring PCDD/Fs in infant formulas.

The application of high resolution gas chromatography in combination with low resolution mass spectrometry with electron ionization and MS/MS detection (HRGC-MS/MS) is tested for its use in the analysis of PCDD/Fs in infant formulas. Development of the analytical method was based upon EPA directrices and international recommendations. Calibration linearity was tested and average relative response for any native and labelled compound over the five-point calibration range below 14% was found. The precision and accuracy of the proposed analytical procedure are also presented. Results obtained are in agreement with EPA criteria. The method is applied to the analysis of a number of initial and follow-on milk based infant formulas. In general, HRGC-MS/MS constitutes an interesting method for the analysis of dioxins in such matrices.

Inhibition of Fusarium Growth and Mycotoxin Production in Culture Medium and in Maize Kernels by Natural Phenolic Acids.

The possible role of natural phenolic compounds in inhibiting fungal growth and toxin production has been of recent interest as an alternative strategy to the use of chemical fungicides for the maintenance of food safety. Fusarium is a worldwide fungal genus mainly associated with cereal crops. The most important Fusarium mycotoxins are trichothecenes, zearalenone, and fumonisins. This study was conducted to evaluate the potential of four natural phenolic acids (caffeic, ferulic, p-coumaric, and chlorogenic) for the control of mycelial growth and mycotoxin production by six toxigenic species of Fusarium . The addition of phenolic acids to corn meal agar had a marked inhibitory effect on the radial growth of all Fusarium species at levels of 2.5 to 10 mM in a dose-response pattern, causing total inhibition (100%) in all species except F. sporotrichioides and F. langsethiae . However, the effects of phenolic acids on mycotoxin production in maize kernels were less evident than the effects on growth. The fungal species differed in their responses to the phenolic acid treatments, and significant reductions in toxin concentrations were observed only for T-2 and HT-2 (90% reduction) and zearalenone (48 to 77% reduction). These results provide data that could be used for developing pre- and postharvest strategies for controlling Fusarium infection and subsequent toxin production in cereal grains.

Occurrence and exposure assessment of Fusarium mycotoxins in maize germ, refined corn oil and margarine.

Analytical methods were validated for the analysis of fumonisins (FB1 and FB2), deoxynivalenol (DON) and zearalenone (ZEA) in maize germ, corn oil and margarine. A survey of 74 samples consisting of 12 wet-milled maize germ, 12 dry-milled maize germ, 25 refined corn oil, and 25 corn oil margarine was conducted. Results revealed that 100% and 87.5% of maize germ samples presented FB1 and FB2, respectively, attaining concentrations for the sum of both toxins of 1302±541 µg kg(-1) in wet-milled and 820±831 µg kg(-1) in dry-milled maize germ. The lower incidence of FB1, FB2 and DON in edible oil and margarine (4-8%) may be related with the industrial processes for their obtaining besides the high water-solubility of these mycotoxins. In contrast, 25% of maize germ samples were positive for ZEA as well as 32% of corn oil and 24% of margarine, which may be related with its lipophilic nature. A number of samples exceeded the maximum limits indicating that strict control is needed, though estimated dietary exposure was less than 0.2% tolerable daily intakes in all cases.

Relevance of carnosic acid, carnosol, and rosmarinic acid concentrations in the in vitro antioxidant and antimicrobial activities of Rosmarinus officinalis (L.) methanolic extracts.

The importance of the diterpenic and rosmarinic acid content in the biological activities of rosemary extracts has been studied previously, but how the relationship between the concentration of these components affects their antioxidant and antibacterial activities has received little attention. Accordingly, from a total of 150 plants, 27 methanolic extracts were selected, for their similar diterpene contents but different ratios between carnosic acid and carnosol concentrations. In extracts with similar rosmarinic acid contents but differing proportions between carnosic acid and carnosol, the two diterpenes were seen to equally affect the in vitro antioxidant activity; however, and related with the antibacterial efficiency, this biological activity improved when carnosol was the major diterpene component.

Evaluation of daily intake of PCDD/Fs and indicator PCBs in formula-fed Spanish children.

Human exposure to polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) occurs predominantly via food intake. In this study, the exposure assessment of these contaminants has been estimated for infant formula-fed children up to 1 year of age. PCDD/F concentrations in the infant formulae was low, ranging between 0.09 and 0.17 pg WHO-TEQ g(-1) fat and between 0.30 and 0.46 pg WHO-TEQ g(-1) fat when results were calculated with the lower and medium bound values, respectively. Indicator PCB contamination levels were below 1 ng g(-1) fat in all cases. Thus, the estimated daily intake of PCDD/Fs and indicator PCBs for infants has been assessed taking into account the above-mentioned contamination levels as well as different scenarios of body weight and food consumption data for babies aged 0-12 months. The results vary in the different scenarios considered but, on the whole, the daily estimated dioxin and indicator PCBs intake of the average infant population due to the consumption of infant formulae does not exceed the tolerable daily intake (TDI) of 2 pg WHO-TEQ kg(-1) bw day(-1) recommended by the Scientific Committee on Food (available at http://ec.europa.eu/food/fs/sc/scf/out90_en.pdf) nor the threshold value of 10 ng kg(-1) bw day(-1) proposed by the Dutch National Institute of Health and Environment (RIVM) (Baars et al. 2001. Report no. 711701025, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands).