Monitoring of Pig Slaughter Stages and Correlation in the Prevalence of Pathogens and Levels of Microorganisms That Indicate Microbiological Quality and Hygiene Using a Predictive Model.

Pig production is relevant to the Brazilian economy. Different stages of the raising and slaughtering process influence the microbiological quality of pig products and by-products. Microbiological analysis and hazard analysis and critical control points (HACCPs) are tools for monitoring microbiological quality indicator microorganisms. The construction of predictive models can assist the process of monitoring the microbiological quality of pig products. This study aimed to map the slaughter stages and develop a model to predict the absence or presence of Salmonella based on the process variables (distance from the farm to the slaughterhouse and aerobic mesophilic) and analyze their influence on contamination indicator microorganisms. A total of 810 samples were collected at nine stages of the slaughter process (bleeding, scalding, dehairing, singeing, washing, evisceration, inspection, final washing, and chilling). The binary class predictive model was used as a microbiological quality predictor at the slaughter stages. Salmonella was identified at all process stages, with lower contamination levels at the scalding and chilling stages, whereas the highest levels were found at the dehairing and bleeding stages. The predictive model revealed an accuracy of about 85% for Salmonella being a tool to monitor the microbiological quality of pig slaughter.

Efeito combinado de carvacrol e tiabendazol contra Colletotrichum gloesporioides, Fusarium solani e Alternaria alternata / Combined effect of carvacrol and thiabendazole against Colletotrichum gloesporioides, Fusarium solani and Alternaria alternata

O objetivo deste trabalho foi determinar a atividade antimicrobiana do carvacrol e sua combinação com tiabendazol no controle de fungos patogênicos deteriorantes de frutas (Colletotrichum gloesporioides, Fusarium solani e Alternaria alternata). O carvacrol apresentou uma concentração inibitória mínima (CIM) de 282 a 563 μg mL-1 para os fungos testados. Quando avaliado em conjunto com o tiabendazol apresentou efeito aditivo contra C. gloesporioides e F. solani (FICI 0,5 e 1,0, respectivamente) e sinérgico contra a A.alternata (FICI 0,1). Houve redução da CIM do carvacrol de 50 a 88%. Este estudo mostra o potencial do uso.

ß-Glucanase Addition in Brewing Malt Produced by Reduced Time of Germination

Abstract The β-Glucans content has straight influence on the quality of malt and beer, mainly during the filtration step. Barley presenting high β-Glucan content demands longer germination time at malting. The application of commercial β-Glucanase is an alternative to accelerate the process and preserve the quality of malt. This work aimed to evaluate the effect of commercial β-Glucanase addition in malt produced within reduced germination time (64 h). Micro-malting was conducted with BRS-Caue and Elis barley cultivars at germination time 64 h and 96 h. The β-Glucanase concentration applied were 0, 25, 50 and 100 mg.kg-1. Barley, malt and wort samples were analyzed to check their physical-chemical features. Beers were produced with BRS-Caue malt and the physical-chemical and sensory attributes were analyzed. The commercial enzyme addition in BRS-Caue and Elis (64 h), at concentration 25 and 50 mg.kg-1, resulted in wort presenting proper β-Glucan content (≤ 178 mg.L-1). The beer produced with malt germinated for 64 h and added with 50 mg.kg-1 of β-glucanase was the one showing the largest number of physical-chemical and sensory parameters similar to the beer made with malt germinated for 96 h (conventional process). Commercial β-glucanase application in malt allowed accelerating the malting process without affecting the quality of the malt for beer production.

Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9.

Strain B-9, which has a 99% similarity to Sphingosinicella microcystinivorans strain Y2, is a Gram-negative bacterium with potential for use in the degradation of microcystin-related compounds and nodularin. We attempted to extend the application area of strain B-9 and applied it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and l-phenylalanine, and levels of fumonisin B1 gradually decreased after 96 h. However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds, such as 3,4-dimethyl hippuric acid and 4-benzyl aspartate, were readily hydrolyzed by strain B-9, although the sulfonamides remained unchanged. The ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9. Furthermore, the degradation of amide and ester compounds with amino acids was not inhibited by the addition of ethylenediaminetetraacetic acid (EDTA), indicating that the responsible enzyme was not MlrC. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE, as well as an esterase.

Natural occurrence of deoxynivalenol in wheat from Paraná State, Brazil and estimated daily intake by wheat products.

The occurrence of deoxynivalenol (DON) was evaluated in 113 wheat samples from the northern and central/southwestern regions of Paraná State, Brazil during the 2008 and 2009 growing seasons, and this rate of occurrence was used to estimate the DON dietary exposure. The DON determination was carried out by an indirect competitive enzyme-linked immunosorbent assay. DON was detected in 66.4% samples at levels ranging from 206.3 to 4732.3 µg/kg (mean 1894.9 µg/kg). The estimated daily intake (EDI) of DON through bread and pasta was evaluated in the inhabitants of Londrina City in northern Paraná State, Brazil. The average intake of these inhabitants was 0.79 µg/kg body weight (b.w.) for bread and 0.35 µg/kg b.w. for pasta. The total EDI was 1.13 µg/kg, which is above the Provisional Tolerable Daily Maximum Intake (PTDMI) of 1 µg/kg b.w. To our knowledge, this is the first report on natural DON occurrence in wheat and DON dietary exposure estimation from Paraná, Brazil.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Application of log D for the prediction of hydrophobicity in the advanced Marfey's method.

In the advanced Marfey's method, the resolution between the diastereomers derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) and 1-fluoro-2,4-dinitrophenyl-5-D-leucinamide (D-FDLA) is reflected by the difference of hydrophobicity of the two functional groups at the asymmetric carbon. However, no effective method has been developed for the estimation of hydrophobicity so far. For this purpose, we introduced log D from the ACD Labs LogD and applied it to relatively simple primary amines, amino acids and secondary alcohols in the present study. It was found that the difference of the retention times (Delta t(R)) correlated with that of log D (Delta log D) for both diastereomers based on the obtained experimental results. Based on these results, the following procedure was proposed for the non-empirical determination of the absolute configuration of primary amines including amino acids and secondary alcohols: (1) estimate the hydrophobicity by the calculation of log D for the two substituent groups at the asymmetric carbon, (2) locate the trans-type arrangement of the two more hydrophobic substituents in the L-DLA derivative and judge the asymmetric carbon to be R or S in the trans-type that is eluted first, (3) derivatize the desired compound with L- or D-FDLA and analyze by LC/MS, and (4) compare the elution order with the prospective one and determine the absolute configuration at the asymmetric carbon. Furthermore, log D could also be used to predict the retention times of unavailable amino acids and small peptides, indicating that the combination of the advanced Marfey's method with log D would provide more reliable structural information on a mixture composed of amino acids and small peptides. The developed method is being applied to more complicated compounds.

Further investigation of microbial degradation of microcystin using the advanced Marfey method.

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log D would be a standard technique for the structural characterization of a mixture of amino acids and peptides.

Mycotoxicological quality evaluation of corn samples used by processing industries in the Northern region of Paraná State, Brazil.

Based on fungal and fumonisin contamination of 870 freshly harvested samples, the quality of corn used by processing industries in the Northern region of Parana State, Brazil (2003 and 2004 crop-year) was evaluated. Sampling was carried out for each crop at two points in the production chain, i.e. at reception by the processors and at the pre-drying step. Corn samples were more frequently contaminated with Fusarium sp. (100%) and Penicillium sp. (84.1-95.3%) than Aspergillus sp. (5.6-19.8%). Fumonisin B(1) (FB(1)) was detected in all samples from the two points in both crop-years. FB(1) levels ranged 0.02-11.83 microg g(-1) in the reception and 0.02-10.98 microg g(-1) in the pre-drying samples of the 2003 crop. Samples from the 2004 crop showed FB(1) levels ranging 0.03-12.04 microg g(-1) in the reception and 0.06-7.74 microg g(-1) in the pre-drying samples. FB(2) levels ranged 0.02-5.25 microg g(-1) in the reception and 0.01-7.89 microg g(-1) in the pre-drying samples (2003 crop-year). In samples from the 2004 crop, FB(2) levels ranged 0.02-6.12 microg g(-1) in the reception and 0.05-3.47 microg g(-1) in the pre-drying samples. Low fumonisin levels were detected in most corn samples used by processors in the Northern region of Parana State, showing a decreasing trend in fumonisin contamination over the years.

Imunoistoquímica: detecção de microcistina em tilápia exposta ao extrato de Microcystis aeruginosa (Cyanobacteria) / Imunohistochemistry: detection of microcystin in tilápia exposed to Microcystis aeruginosa (Cyanobacteria) extract

A deterioração da qualidade de água pela piscicultura associa-se à eutrofização, com florescimento de cianobactérias. Microcystis aeruginosa destaca-se como principal produtora de microcistinas (MCs), grupo de hepatotoxinas com potencial promotor de tumor. No presente trabalho desenvolveu-se método imunoistoquímico para a detecção de MC em tilápias (Oreochromis niloticus) submetidas à injeção intraperitoneal (i.p.) ou imersão em extrato de M. aeruginosa BCCBUSP 262, empregando anticorpo monoclonal anti-MC (M8H5) e sistema polímero-peroxidase. As tilápias (N=42) foram submetidas a sete tratamentos, sendo três grupos inoculados i.p. com 2,0x105, 4,0x105 e 1,0x106 cels.Kg-1 de M. aeruginosa BCCBUSP 262 e quatro submetidos à imersão em diferentes concentrações do extrato da cianobactéria (variando de 1,0x104 a 1,0x105cel.mL-1). Analisando fígado e tecido muscular pelo ensaio imunoistoquímico, não se detectou marcação em tecido muscular. Todos os animais inoculados i.p. apresentaram marcação positiva para MC no fígado, mas em teste de imersão, apenas os expostos a maior dose (1,0x105 cels.mL- 1) apresentaram marcação positiva. Embora MC não seja detectada em tecido muscular, assim como no fígado de animais imersos em extrato de M. aeruginosa CCBUSP 262 em concentrações menores que 1,0x105 cels.mL-1, os resultados constituíram-se base para o desenvolvimento metodológico objetivando a aplicação da imunoistoquímica no diagnóstico rápido no controle de qualidade de pescados.

The deterioration of the water quality due to aquaculture is associated with eutrophication, with bloom of cyanobacteria. Microcystis aeruginosa is distinguished as main producer of microcystins (MCs), group of hepatotoxins with tumor promoter potential. In the present work immunohistochemical method for detection of MC in tilápia (Oreochromis niloticus), fish submitted to intraperitoneal injection (i.p.) or immersion in extract of M. aeruginosa BCCBUSP 262 was developed, using monoclonal antibody anti- MC (M8H5) and polymer peroxidase system. The tilápias (N=42) had been submitted to the seven treatments, three groups inoculated i.p. with 2.0x105, 4.0x105 and 1.0x106 cells. Kg-1 of M. aeruginosa BCCBUSP 262 and four groups exposed to the immersion in different extract concentrations of cyanobacterium. Analyzing liver and muscular tissue for immunohistochemical assay, muscular tissue was not stained. All the animals inoculated i.p. presented positive marking for MC in the liver, but in immersion test, only the ones exposed in the highest dose (1,0x105 cels.mL-1) presented positive marking. Although MC was not detected in muscular tissue, as well as in the liver of animals immersed in extract of M. aeruginosa BCCBUSP 262 in concentrations less than 1.0x105 cels.mL-1, the results would constitute in the base for the methodological development aiming the application of the immunohistochemistry in the rapid diagnosis in quality control of fish.