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The increased risk to health by diverse pathologies, such as cancer, liver diseases, and endocrine alterations, caused by chemical residues in food, has led to the search for sustainable agricultural management alternatives, such as the use of essential oils for the development of natural and eco-friendly fungicides. The aim of this study was to evaluate the antifungal and antiaflatoxigenic activity of Rosmarinus officinalis L. essential oil (REO) against Aspergillus flavus Link. REO was obtained by hydrodistillation and its major components were identified as 1,8-cineole (eucalyptol, 52.2%), camphor (15.2%) and α-pinene (12.4%) by GC/MS and NMR. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were both 500 µg/mL. REO reduced the mycelial growth of A. flavus at a concentration of 250 µg/mL (15.3%). The results obtained from scanning electron microscopy (SEM) demonstrated a reduction in the size of conidiophores and in the thickness of hyphae in A. flavus caused by treatment with REO (250 µg/mL). The production of ergosterol and the biomass of mycelium were both reduced as the REO treatment concentration increased. The production of aflatoxins B 1 and B 2 was inhibited after treatment with 250 µg/mL REO, a concentration below the MIC/MFC, indicating that the antiaflatoxigenic effect of REO is independent of its antifungal effect and is likely due to its direct action upon toxin biosynthesis. The data demonstrated that REO may be used as an alternative to synthetic fungicides.

Members of the Fusarium genus are capable of contaminating agricultural commodities, compromising the quality of maize and other grains, which leads to severe quality and yield losses. Contamination with mycotoxins is also a concern. Essential oils are possible alternatives to the use of synthetic pesticides for control of fungal contamination, as many have antifungal and anti-mycotoxigenic properties and are innocuous to human health. They also do not cause any sort of microbial resistance and do not promote environmental pollution. The aim of this study was to evaluate the antifungal and anti-mycotoxigenic effects of Zingiber officinale Roscoe essential oil (GEO) upon Fusarium graminearum Schwabe in vitro. The essential oil was extracted by hydrodistillation and analysed by GC/MS. Antifungal and anti-mycotoxigenic activities were assessed by HPLC/UV by quantifying ergosterol and deoxynivalenol (DON), respectively. Results indicated that GEO inhibited ergosterol production at a concentration of 1000 µg/mL and DON production at a concentration of 500 µg/mL, evidencing that the anti-mycotoxigenic effect is independent of the antifungal effect due to its probable direct action upon toxin biosynthesis.

Deoxynivalenol (DON) is the most important of the trichothecenes in terms of amounts and occurrence in wheat. This compound was shown to be associated with a glomerulonephropathy involving an increase of immunoglobulin A in humans. This study assessed the occurrence of DON in wheat flour and the exposure of Brazilian teenagers, adults and elderly to this mycotoxin due to intake of wheat flour-based products. DON extraction in wheat flour was carried out by solid phase extraction and the quantification was performed by ultra-high proficiency liquid chromatography with diode-array detection. A total of 77.9% of all samples were positive for DON, with concentrations ranging from 73.50 to 2794.63 µg kg -1 . The intake was calculated for the average and 90th percentile of the contamination levels of DON in foods based-wheat for teenagers, adults and elderly in Brazil, and compared with the provisional maximum tolerable daily intakes (PMTDI). Females of all age groups were exposed to DON at higher levels when compared to males in regard of consumption of breads and pastas. Teenagers were the main consumers of foods derived from wheat flour, with maximum probable daily intakes of 1.28 and 1.20 µg kg -1 b.w. day -1 for females and males, respectively. This population is at an increased risk of exposure to DON due to consumption of wheat flour-based foods in Brazil.

Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 μg/mL and the MBC was 3.91 μg/mL, while theMIC of bromelain was 62.5 μg/mL and the MBCwas 250 μg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.

The antifungal and antiaflatoxigenic properties of Thymus vulgaris essential oil (TEO) were evaluated upon Aspergillus flavus "in vitro". Suspension containing 10(6) of A. flavus were cultivated with TEO in concentrations ranging from 50 to 500 μg/mL. TEO reached minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) at 250 μg/mL. Inhibition of ergosterol biosynthesis was detected at a concentration of 100 μg/mL of TEO. Morphological evaluation performed by both light microscopy and scanning electron microscopy showed that antifungal activity of TEO could be detected starting at a concentration of 50 μg/mL and the fungicide effect at a concentration of 250 μg/mL. TEO completely inhibited production of both B1 and B2 aflatoxins (AFB1 and AFB2) at a concentration of 150 μg/mL. This way, fungal biomass development and aflatoxin production were dependent on TEO concentration. Therefore, TEO was capable of controlling the growth of A. flavus and its production of aflatoxins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the presence of tetracycline residues in pasteurised cow's milk using high-performance liquid chromatography coupled with UV/VIS detection to determine the exposure of Brazilian's population to antibiotic residues. One hundred samples collected from the State of Paraná, Brazil, were analysed. Three of these samples were contaminated at the following concentrations: 121.8 µg·kg(-1) for oxytetracycline, 93.5 µg·kg(-1) for tetracycline and 134.6 µg·kg(-1) for chlortetracycline (61.6 µg·kg(-1)) and doxycycline (73.0 µg·kg(-1)). The median tetracycline residue concentration found in the samples was 42.3 µg·kg(-1), and the estimated daily intake (EDI) was 0.05 µg Kg(-1) bw day(-1) in Brazil. These results demonstrate that the occurrence of tetracycline in Brazilian milk was low (3%) and only for 2% above the maximum residue limit, so the risk to the population from the presence of these residues in milk was low (<1% of the acceptable daily intake).

The chemical composition of Rosmarinus officinalis L. essential oil (REO) was analysed by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The main compounds of the REO were 1.8 cineole (52.2%), camphor (15.2%) and α-pinene (12.4%). The mycelial growth of Fusarium verticillioides (Sacc.) Nirenberg was reduced significantly by 150 μg/mL of REO. Significant microscopic morphological changes were visualised, such as the rupture of the cell wall and the leakage of cytoplasm at 300 μg/mL of REO. At lower concentrations of REO, the effects on the production of ergosterol and the biomass of mycelium varied, as did the effects on the production of fumonisins, but at ≥300 μg/mL of REO, these processes were significantly inhibited, showing the effectiveness of the REO as an antifungal agent. The results suggested that the REO acts against F. verticillioides by disrupting the cell wall and causing the loss of cellular components, subsequently inhibiting the production of fumonisins and ergosterol., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The essential oil from Curcuma longa L. was analysed by GC/MS. The major components of the oil were ar-turmerone (33.2%), α -turmerone (23.5%) and β -turmerone (22.7%). The antifungal activities of the oil were studied with regard to Aspergillus flavus growth inhibition and altered morphology, as preliminary studies indicated that the essential oil from C. longa inhibited Aspergillus flavus Link aflatoxin production. The concentration of essential oil in the culture media ranged from 0.01% to 5.0% v/v, and the concentration of curcumin was 0.01-0.5% v/v. The effects on sporulation, spore viability, and fungal morphology were determined. The essential oil exhibited stronger antifungal activity than curcumin on A. flavus. The essential oil reduced the fungal growth in a concentration-dependent manner. A. flavus growth rate was reduced by C. longa essential oil at 0.10%, and this inhibition effect was more efficient in concentrations above 0.50%. Germination and sporulation were 100% inhibited in 0.5% oil. Scanning electron microscopy (SEM) of A. flavus exposed to oil showed damage to hyphae membranes and conidiophores. Because the fungus is a plant pathogen and aflatoxin producer, C. longa essential oil may be used in the management of host plants.

The antifungal activity of ginger essential oil (GEO; Zingiber officinale Roscoe) was evaluated against Fusarium verticillioides (Saccardo) Nirenberg. The minimum inhibitory concentration (MIC) of GEO was determined by micro-broth dilution. The effects of GEO on fumonisin and ergosterol production were evaluated at concentrations of 500-5000 μg/mL in liquid medium with a 5mm diameter mycelial disc of F. verticillioides. Gas chromatography-mass spectrometry showed that the predominant components of GEO were α-zingiberene (23.9%) and citral (21.7%). GEO exhibited inhibitory activity, with a MIC of 2500 μg/mL, and 4000 and 5000 μg/mL reduced ergosterol biosynthesis by 57% and 100%, respectively. The inhibitory effect on fumonisin B1 (FB1) and fumonisin B2 (FB2) production was significant at GEO concentrations of 4000 and 2000 μg/mL, respectively. Thus, the inhibition of fungal biomass and fumonisin production was dependent on the concentration of GEO. These results suggest that GEO was able to control the growth of F. verticillioides and subsequent fumonisin production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The productivity of wheat and corn crops depends on climatic conditions and resistance against phytopathogenic fungi such as those of the genus Fusarium. Some species of this genus produce zearalenone (ZEA), a mycotoxin with hyperestrogenic effects. The objective of this study was to investigate the presence of ZEA in samples of cracked wheat (n = 109), popcorn (n = 51) and corn grits (n = 50) commercialized in the State of Paraná, Brazil. Commercial samples of each crop were collected between September 2007 and June 2008 and analyzed by thin-layer chromatography. The method used for detection of the mycotoxin in wheat and corn derivatives presented a recovery rate of 94.5% and 99.5%, respectively, detection limit of 40 μg.kg(-1) and quantification limit of 55 μg.kg(-1). No contamination with ZEA was detected in cracked wheat samples. Among the corn derivatives, only one cracked corn sample was contaminated with ZEA (64 μg.kg(-1)). Despite the low contamination observed, monitoring the occurrence of mycotoxins in foods is important to ensure safety.

Ear rots caused by Fusarium spp. are among the main fungal diseases that contribute to poor quality and the contamination of maize grains with mycotoxins. This study aimed to determine the visual incidence of fungal-damaged kernels (FDKs), the incidence of two main Gibberella (a teleomorph of Fusarium) complexes (G. fujikuroi and G. zeae) associated with maize using a seed health blotter test, and the fumonisin levels, using high performance liquid chromatography, in samples of maize grains grown across 23 municipalities during the 2008/09 and 2009/10 growing seasons. Additionally, 104 strains that were representative of all of the analysed samples were identified to species using PCR assays. The mean FDK was seven per cent, and only six of the samples had levels greater than six per cent. Fusarium spp. of the G. fujikuroi complex were present in 96% of the samples, and G. zeae was present in 18% of the samples (5/27). The mean incidence of G. fujikuroi was 58%, and the incidence of G. zeae varied from 2 to 6%. FB1 was found in 58.6%, FB2 in 37.9%, and both toxins in 37.9% of the samples. The FB1 and FB2 levels were below the quantification limits for 41.3% of the samples, and the mean FB1 levels (0.66 μg/g) were higher than the mean FB2 levels (0.42 μg/g). The PCR identification separated the 104 isolates into three of the G. fujikuroi complex: F. verticillioides (76%), F. subglutinans (4%) and F. proliferatum (2%); and G. zeae (anamorph = F. graminearum) (18%). Our results confirmed the dominance of F. verticillioides, similar to other regions of Brazil, but they differed due to the relatively higher incidence of F. graminearum. Total fumonisin levels were below the maximum limit determined by current Brazilian regulations.

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic mycotoxins. Consumption of aflatoxin-contaminated food and commodities poses serious hazards to the health of humans and animals. Turmeric, Curcuma longa L., is a native plant of Southeast Asia and has antimicrobial, antioxidant and antifungal properties. This paper reports the antiaflatoxigenic activities of the essential oil of C. longa and curcumin. The medium tests were prepared with the oil of C. longa, and the curcumin standard at concentrations varied from 0.01% to 5.0%. All doses of the essential oil of the plant and the curcumin standard interfered with mycotoxin production. Both the essential oil and curcumin significantly inhibited the production of aflatoxins; the 0.5% level had a greater than 96% inhibitory effect. The levels of aflatoxin B(1) (AFB(1)) production were 1.0 and 42.7 μg/mL, respectively, for the samples treated with the essential oil of C. longa L. and curcumin at a concentration of 0.5%., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To estimate daily intake of aflatoxins from peanut products consumed by the population of Paraná State (Brazil), 100 samples of peanut products were collected between July 2006 and April 2007. Aflatoxins were determined by an HPLC method with fluorescence detection. There was a 50% occurrence of aflatoxins (B1, B2, G1 and G2) in concentrations ranging from 0.5 to 113 ng g(-1), with 13 samples with levels above 20 ng g(-1). Intake was calculated for average and high adult consumers of peanut products and it was compared with provisional maximum tolerable daily intake (PMTDI). The estimated probable daily intake (PDI) for aflatoxin B1 (AFB1) varied from 0.6 to 10.4 ng kg(-1) bw day(-1), exceeding the PMTDI of 0.4 ng kg(-1) bw day(-1) for carriers of hepatitis B virus.

In this study we examined the hygienic and sanitary quality of pasteurized cow's milk in the state of Paraná, Brazil, by determining the presence of coliforms and occurrence of antimicrobial residues. A total of 260 milk samples were collected from commercial establishments in different regions of the state. Coliform populations were estimated by the multiple-tube test, and antimicrobial residues were detected by enzyme-linked immunosorbent assay. Overall, 105 samples (40.4%) were unsuitable for consumption according to Brazilian legal standards. Among the coliforms, Escherichia coli and Klebsiella pneumoniae were respectively identified in 77.05 and 36.07% of the samples. The highest rates of resistance to antimicrobial agents were observed for ampicillin (19.2%), cephalothin (18.9%), and tetracycline (17.1%). Antimicrobial residues were detected in 80 samples (30.8%). Forty-eight samples (18.5%) were positive for tetracycline, 29 (17.4%) for neomycin, 9 (3.5%) for beta-lactams, 6 (2.3%) for gentamicin, 4 (1.5%) for chloramphenicol, and 1 (0.4%) for streptomycin-dihydrostreptomycin. The results demonstrate a high prevalence of coliforms and also a high occurrence of antimicrobial residues in pasteurized cow's milk from Paraná, Brazil.

Milk containing antimicrobial residues presents a health risk to the human population. The objective of this study was to use an enzyme-immunoassay technique to determine the occurrence of antimicrobial residues in 151 samples of pasteurized milk sold in cities of the State of Paraná, Brazil, from March 2005 to April 2006. Fifty-nine (41.3%) of the 151 samples contained antimicrobial residues. Residues of neomycin, streptomycin, and/or dihydrostreptomycin and chloramphenicol were found in three, two, and four, respectively. None of the samples with neomycin residues had levels above the maximum residue limit (MRL) permitted in this country, which is 500 microg/kg. Only one sample had a higher level of streptomycin-dihydrostreptomycin (260 microg/kg) than the MRL (200 microg/kg). The four samples positive for chloramphenicol had levels above the zero tolerance level. In the qualitative analysis, 41 of 151 samples contained tetracyclines (tetracycline, chlortetracycline, and/or oxytetracycline), 4 of 82 samples contained gentamicin, and 5 of 151 samples contained beta-lactams (amoxicillin, ampicillin, ceftiofur, cephapirin, and/or penicillin G). It was not possible to determine whether the levels of the antimicrobials found in the qualitative analyses (tetracyclines, gentamicin, and beta-lactams) were above the MRLs because the detection limits were below the MRLs in Brazil. In nine samples, two or more antimicrobial residues were found. The results demonstrate the need for monitoring various antimicrobial residues in pasteurized milk to ensure safety, quality, and integrity and to protect the health of the Brazilian population.

Purpose: We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade., Methods: Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated., Results: The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium., Conclusion: The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.