Analysis of PCR-based methods for characterization of Listeria monocytogenes strains isolated from different sources

Listeria monocytogenes strains, isolated from various sources (food, environment, and animals), were used to test different PCR-based methods to investigate their capability to define the strain origin. RAPD-PCR with three primers and the SAU-PCR method, in which the DNA was first digested with the Sau3A restriction endonuclease and then amplified with a primer designed on the restriction site, were carried out, and the profiles obtained were used to perform cluster analysis. Based on the cluster analysis of Listeria spp. strains, obtained from international collections, the coefficient of similarity was selected. The results obtained showed that the methods tested in the study gave different levels of differentiation between the strains tested. The RAPD protocol using the P1254 primer and the SAU-PCR gave appreciable results only for strains isolated from animals and from a food processing plant in two different periods of the year 2003. Better differentiation was observed using the RAPD-PCR with primer D8635. As a matter of fact, it was able to distinguish L. monocytogenes obtained from different species of animals, different food samples and strains from the same production plant isolated in different periods of the year. Also primer M13 gave positive results, but the coefficient of similarity to use had to be increased to 80%. On the basis of the results observed, RAPD-PCR with primers D8635 and M13 should be considered reliable tools for epidemiological investigations focusing on L. monocytogenes.     

In vitro fermentation characteristics of diets with different forage/concentrate ratios: comparison of rumen and faecal inocula

BACKGROUND: The aim of this trial was to evaluate the replacement of rumen fluid with faeces as inoculum in studying the in vitro fermentation characteristics of diets for ruminants using the in vitro gas production technique. Six iso‐protein diets with different forage/concentrate ratios were incubated with rumen fluid (RI) or faeces (FI) collected from sheep. RESULTS: Most of the fermentation parameters were influenced by diet and inoculum (P < 0.01). With both inocula, organic matter degradability (dOM), cumulative gas production (OMCV) and maximum fermentation rate (R_max) increased as the amount of concentrate in the diet increased. R_max was lower with FI vs RI (P < 0.01); dOM was higher with FI vs RI and the diet × inoculum interaction was significant. As expected, with both inocula, R_max increased as the neutral detergent fibre content of the diet decreased. Significant correlations were obtained using both inocula between OMCV/dOM and gas/volatile fatty acid (VFA), while the correlation VFA/dOM was significant only with FI. The microbial biomass yield calculated by stoichiometric analysis for all diets was higher with FI vs RI. With FI the organic matter used for microbial growth showed an overall decreasing trend as the amount of concentrate in the diet increased. CONCLUSION: The results indicate that both faeces and rumen fluid from sheep have the potential to be used as inoculum for the in vitro gas production technique. Copyright © 2011 Society of Chemical Industry     

Content of phytoestrogens in soy-based dietary supplements

The isoflavone content of 14 soy-based dietary supplements intended to help alleviate perimenopausal and menopausal symptoms on sale in Italy were analysed using HPLC with UV detection. The aim was to quantify soy isoflavones after hydrolysis as aglycones, which are the bioactive part of isoflavone molecules. In the examined products, the amounts of isoflavones were frequently expressed ambiguously, and none of the products stated whether the isoflavone content of the product was expressed as aglycones or as conjugates. Each product revealed a different aglycone concentration profile. These supplements have different “fingerprints”, probably due to different sources of raw materials and to methods used in processing and preparation of extracts. In more than half the supplements tested, the actual values contained were below those stated and below those expected to help alleviate perimenopausal and menopausal symptoms.     

A new HLA antigen apparently not controlled by the known HLA loci

The human allogeneic serum RM3 recognizes a lymphocyte structure inherited with the HLA chromosome. Population studies show several positive associations with antigens of the HLA-A and HLA-B series. Negative association have been found with antigens of the HLA-C series. Moreover the distribution of the RM3 factor is in Hardy-Weinberg equilibrium with that of the HLA-C alleles. However, serological investigations (namely the 'lysostrip' technique) bring evidence that RM3 serum does not appear to contain antibodies directed against any of the antigens of the HLA-A, B and C series. The study of a segregant family, showing a crossing over between the HLA-A and HLA-B loci, shows that the RM3 factor segregates on the side of the HLA-A locus. In conclusion, within the limits of the serological approach used, the present study presents evidence of a new serologically defined HLA locus, different from those so far described.     

In Vitro and In Vivo Pharmaco-Toxicological Characterization of 1-Cyclohexyl-x-methoxybenzene Derivatives in Mice: Comparison with Tramadol and PCP

1-cyclohexyl-x-methoxybenzene is a novel psychoactive substance (NPS), first discovered in Europe in 2012 as unknown racemic mixture of its three stereoisomers: ortho, meta and para. Each of these has structural similarities with the analgesic tramadol and the dissociative anesthetic phencyclidine. In light of these structural analogies, and based on the fact that both tramadol and phencyclidine are substances that cause toxic effects in humans, the aim of this study was to investigate the in vitro and in vivo pharmacodynamic profile of these molecules, and to compare them with those caused by tramadol and phencyclidine. In vitro studies demonstrated that tramadol, ortho, meta and para were inactive at mu, kappa and delta opioid receptors. Systemic administration of the three stereoisomers impairs sensorimotor responses, modulates spontaneous motor activity, induces modest analgesia, and alters thermoregulation and cardiorespiratory responses in the mouse in some cases, with a similar profile to that of tramadol and phencyclidine. Naloxone partially prevents only the visual sensorimotor impairments caused by three stereoisomers, without preventing other effects. The present data show that 1-cyclohexyl-x-methoxybenzene derivatives cause pharmaco-toxicological effects by activating both opioid and non-opioid mechanisms and suggest that their use could potentially lead to abuse and bodily harm.     

Comparison of the compositional, microbiological, biochemical and volatile profile characteristics of three Italian PDO fermented sausages

Three Italian PDO fermented sausages, Varzi, Brianza and Piacentino, were compared for compositional, microbiological, biochemical and volatile profile characteristics. Mean values for the gross composition varied especially due to moisture, fat, total protein and nitrate concentration which reflected differences in the ingredients and some technological parameters. Cell numbers of the major microbial groups were almost similar among sausages. The major differences were found for Brochothrix thermosphacta, enterococci and moulds. Apart from their use as starters, Lactobacillus sakei and Lactobacillus curvatus were the dominant lactic acid bacteria and, as well as Staphylococcus xylosus, dominated the population of coagulase-negative staphylococci. Sausages differed for the hydrolysis of myofibrillar proteins and secondary proteolysis. Varzi, the sausage subjected to prolonged fermentation at 23–25 °C for 10 days before ripening, showed the highest degree of secondary proteolysis. Varzi and Brianza, the two fermented sausages manufactured by using microbial starters, showed the highest concentration and similar profiles of free amino acids. The peptidase activities contained in the aqueous extracts agreed with the above findings. A total of 52 volatile components, mainly alcohols, aldehydes and terpenes, were identified by solid-phase micro-extraction coupled with gas chromatography–mass spectrometry analysis. The volatile profiles of the three Italian PDO fermented sausages differed in part and, except for terpenes, the highest levels of the other chemical classes were found in Varzi and Brianza sausages. The composition of free fatty acids of the three Italian PDO sausages was rather similar. Monounsaturated fatty acids (MUFA) were found at the highest relative percentage followed by saturated (SFA) and polyunsaturated (PUFA) fatty acids. Oleic, palmitic, linoleic and stearic were the main free fatty acids found in all fermented sausages.