Fast and sensitive method for the diagnosis and follow-up of anticoagulant rodenticides poisoning in animal whole blood.

Rodent control strategies are primarily based on the use of anticoagulant rodenticides (ARs), making them widely used worldwide. However, due to their high toxicity and availability, ARs are among the leading causes of animal poisoning in Europe. They are the primary agents involved in intoxication in cats and the second in dogs. Additionally, their long persistence in the body can lead to secondary exposure, particularly in wild predators. The laboratory findings and clinical signs of intoxication can range from increased clotting time (prolonged prothrombin time and activated partial thromboplastin time) to severe bleeding and death. Despite the prevalence and severity of this intoxication, only a few methods are available for the identification and quantification of ARs in animals, and most of them are suitable only for post-mortem diagnosis. In this study, we present the validation of a rapid and sensitive method for the identification and quantification of ARs in animal whole blood, using a small sample volume. The developed LC-MS/MS method demonstrated high accuracy and precision at the limit of quantification (LOQ), as well as at low, medium, and high concentrations. It exhibited higher sensitivity (LOQ 0.1 - 0.3 ng/mL) compared to previously published methods. After validation, the method was successfully applied to real cases of suspected poisoning events, resulting in the identification of several positive samples. The examples presented in this study highlight the utility of this method for diagnosis and follow-up, emphasizing the importance of method sensitivity in order to avoid misclassifying truly positive samples as negative.

Analysis of Metaldehyde in Animal Whole Blood and Serum by Gas Chromatography-Mass Spectrometry.

Metaldehyde, a widely used molluscicide, is the third cause of intoxication by pesticides in domestic animals in Europe. Most mammalian species are susceptible, and its exposure may lead to death within a few hours. While metaldehyde intoxication diagnosis is in most cases presumptive, based on the symptomatology or from "postmortem" analysis, few analytical methods are currently available for live animals. The aim of this work was to describe a fast analytical method for the specific and quantitative determination of metaldehyde in animal whole blood and serum at concentrations of toxicological significance. A liquid-liquid extraction with chloroform and gas chromatography-mass spectrometry quantification are proposed. The method limit of quantification (LOQ) was 0.04 µg/mL in serum and whole blood. The method was linear in the range from 0.04 to 200 µg/mL. The recovery was between 93% and 102% for LOQ, low, medium and high spike concentrations. Intra- and inter-assay relative standard deviation was <12% in all spike concentrations in both serum and whole blood, apart from one of the experiments at LOQ in whole blood, which accounted for 17.7%. The method was applied to real intoxication cases, and the concentration found in positive samples was between 29 and 69 µg/mL. The proposed method provides high sensitivity, accuracy and precision and can be used to assist in the diagnosis of metaldehyde poisoning.

Valorization of By-Products from Biofuel Biorefineries: Extraction and Purification of Bioactive Molecules from Post-Fermentation Corn Oil.

The aim of this work was to develop innovative and sustainable extraction, concentration, and purification technologies aimed to recover target substances from corn oil, obtained as side stream product of biomass refineries. Residues of bioactive compounds such as carotenoids, phytosterols, tocopherols, and polyphenols could be extracted from this matrix and applied as ingredients for food and feeds, nutraceuticals, pharmaceuticals, and cosmetic products. These molecules are well known for their antioxidant and antiradical capacity, besides other specific biological activities, generically involved in the prevention of chronic and degenerative diseases. The project involved the development of methods for the selective extraction of these minor components, using as suitable extraction technique solid phase extraction. All the extracted and purified fractions were evaluated by NMR spectroscopic analyses and UV-Vis spectrophotometric techniques and characterized by quali-quantitative HPLC analyses. TPC (total phenolic content) and TFC (total flavonoid content) were also determined. DPPH and ABTS radical were used to evaluate radical quenching abilities. Acetylcholinesterase (AChE), amylase, glucosidase, and tyrosinase were selected as enzymes in the enzyme inhibitory assays. The obtained results showed the presence of a complex group of interesting molecules with strong potential in market applications according to circular economy principles.

Valorization Potentials of Rapeseed Meal in a Biorefinery Perspective: Focus on Nutritional and Bioactive Components.

Rapeseed meal (RSM), a by-product of oilseed extraction connected to the agri-food and biofuel sectors, is currently used as animal feed and for other low-value purposes. With a biorefinery approach, RSM could be valorized as a source of bio-based molecules for high-value applications. This study provides a chemical characterization of RSM in the perspective of its valorization. A qualitative study of main functional groups by fourier transform infrared (FTIR) spectroscopy was integrated with a chemical characterization of macronutrients, minerals by inductively coupled plasma optical emission spectrometry (ICP-OES), phenolic acids and lipid components by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), HPLC-diode-array detector (HPLC-DAD) and gas chromatography-mass spectrometry/flame ionization detector (GC-MS/FID). The study, conducted on different lots of RSM collected over a one-year period from an oil pressing factory serving a biofuel biorefinery, highlighted a constant quality over time of RSM, characterized by high protein (31-34%), fiber (33-40%) and mineral (5.5-6.8%) contents. Polyphenol extracts showed a significant antioxidant activity and a prevalence of sinapic acid, accounting for more than 85% of total phenolic acids (395-437 mg kg-1 RSM). Results highlight the potentialities of RSM for further valorization strategies that may lead to the creation of new cross-sector interconnections and bio-based value chains with improvement of the economics and sustainability of the bioeconomy sectors involved.

Corn Bioethanol Side Streams: A Potential Sustainable Source of Fat-Soluble Bioactive Molecules for High-Value Applications.

This paper reports data from a characterization study conducted on the unsaponifiable lipid fraction of dry-grind corn bioethanol side streams. Phytosterols, squalene, tocopherols, tocotrienols, and carotenoids were quantified by High Performance Liquid Chromatography with Diode-Array Detector (HPLC-DAD) and Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) in different lots of post-fermentation corn oil and thin stillage collected from a bioethanol plant over a time-span of one year. Fat-soluble bioactives were present at high levels in corn oil, with a prevalence of plant sterols over tocols and squalene. Beta-sitosterol and sitostanol accounted altogether for more than 60% of total sterols. The carotenoid profile was that typical of corn, with lutein and zeaxanthin as the prevalent molecules. The unsaponifiable lipid fraction profile of thin stillage was qualitatively similar to that of post-fermentation corn oil but, in quantitative terms, the amounts of valuable biomolecules were much lower because of the very high dilution of this side stream. Results indicate that post-fermentation corn oil is a promising and sustainable source of health-promoting bioactive molecules. The concomitant presence of a variegate complex of bioactive molecules with high antioxidant potentialities and their potential multifaceted market applications as functional ingredients for food, nutraceutical, and cosmeceutical formulations, make the perspective of their recovery a promising strategy to create new bio-based value chains and maximize the sustainability of corn dry-grind bioethanol biorefineries.

Immune-spaying as an alternative to surgical spaying in Iberian x Duroc females: Effect on the sensory traits and volatile organic compound profile of dry-cured shoulders and dry-cured loins.

The aim of this study was to assess the effect of immune-spaying on sensory characteristics and the volatile organic compound (VOC) profile of dry-cured shoulders and loins by comparing Iberian × Duroc surgically spayed females, immune-spayed females and entire females. VOC profile of dry-cured shoulders was not significantly affected by the reproductive status, probably due to the large heterogeneity of dry-cured shoulders as a product. Correspondingly, dry-cured shoulders showed little differences among treatment groups, with better scores for marbling, hardness and chewiness attributes in the immune-spayed females. Dry-cured loin sensory traits such as brightness, marbling, chewiness and juiciness, presented better scores in immune-spayed females. Moreover, dry-cured loins showed a higher homogeneity that allowed the effects of spaying to be observed, thus the Principal Component Analysis performed on VOC profile data indicated a better separation of samples among treatment groups. Consequently, immune-spaying could be a viable alternative to surgical spaying from the point of view of meat quality.

Characterization of volatiles and identification of odor-active compounds of rocket leaves.

The volatile profile of crushed rocket leaves (Eruca sativa and Diplotaxis tenuifolia) was investigated by applying Headspace Solid-Phase MicroExtraction (HS-SPME), combined with GC-MS, to an aqueous extract obtained by homogenization of rocket leaves, and stabilized by addition of CaCl2. A detailed picture of volatile products of the lipoxygenase pathway (mainly C6-aldehydes) and of glucosinolate hydrolysis (mainly isothiocyanates), and their dynamics of formation after tissue disruption was given. Odor-active compounds of leaves were characterized by GC-Olfactometry (GC-O) and Aroma Extract Dilution Analysis (AEDA): volatile isolates obtained by HS-SPME from an aqueous extract and by Stir-Bar Sorptive Extraction (SBSE) from an ethanolic extract were analyzed. The most potent odor-active compounds fully or tentatively identified were (Z)- and (E)-3-hexenal, (Z)-1,5-octadien-3-one, responsible for green olfactory notes, along with 4-mercaptobutyl and 4-(methylthio)butyl isothiocyanate, associated with typical rocket and radish aroma. Relatively high odor potency was observed for 1-octen-3-one, (E)-2-octenal and 1-penten-3-one.

Classification of 7 monofloral honey varieties by PTR-ToF-MS direct headspace analysis and chemometrics.

Honey, in particular monofloral varieties, is a valuable commodity. Here, we present proton transfer reaction-time of flight-mass spectrometry, PTR-ToF-MS, coupled to chemometrics as a successful tool in the classification of monofloral honeys, which should serve in fraud protection against mispresentation of the floral origin of honey. We analyzed 7 different honey varieties from citrus, chestnut, sunflower, honeydew, robinia, rhododendron and linden tree, in total 70 different honey samples and a total of 206 measurements. Only subtle differences in the profiles of the volatile organic compounds (VOCs) in the headspace of the different honeys could be found. Nevertheless, it was possible to successfully apply 6 different classification methods with a total correct assignment of 81-99% in the internal validation sets. The most successful methods were stepwise linear discriminant analysis (LDA) and probabilistic neural network (PNN), giving total correct assignments in the external validation sets of 100 and 90%, respectively. Clearly, PTR-ToF-MS/chemometrics is a powerful tool in honey classification.

Immune-spaying as an alternative to surgical spaying in Iberian × Duroc females: Effect on the VOC profile of dry-cured shoulders and dry-cured loins as detected by PTR-ToF-MS.

Immunocastration in pigs has been proposed as a cruelty-free alternative to surgical castration. In this work the effect of immune-spaying of female pigs on the volatile compound profile of Iberian dry-cured products was evaluated. The head-space volatile compound of dry-cured shoulders and loins from surgically spayed, immune-spayed and entire Iberian × Duroc females was characterized by proton transfer reaction-time of flight-mass spectrometry. It was not possible to identify a significant effect of the castration modality on dry-cured shoulders, probably because of the heterogeneity of samples. Contrarily, Principal Component Analysis of dry-cured loins indicates a better homogeneity of samples and the separation of loins from surgically spayed and immune-spayed females. Some mass peaks tentatively identified as important flavor compounds in dry-cured products, 3-methylbutanal, 2,3-butanedione and 3-methylbutanoic acid, were significantly higher in the immune-spayed females. Therefore, immune-spaying seems to have a negligible effect on the volatile compound profile of dry-cured shoulders, whereas it could affect the VOC profile in the case of dry-cured loins.

SIFT-MS analysis of Iberian hams from pigs reared under different conditions.

The aim of this study was to investigate the usefulness of a Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) equipment to tentatively quantify relevant volatile compounds of dry-cured Iberian ham, and to differentiate Iberian hams from pigs reared at four different conditions yielding different commercial grades. The SIFT-MS analysis allowed the rapid quantification of 39 Iberian ham volatile compounds, 16 of them being significantly affected by the rearing conditions of pigs. The full spectra SIFT-MS data allowed the correct classification of 79.2% of hams according to diet, which is a smaller percentage than that obtained using intramuscular fatty acid data (95.8%) obtained by using a gas chromatograph-flame ionization detector after lipid extraction and transesterification. Therefore, the SIFT-MS analysis would be a rapid tool to tentatively quantify some relevant volatile compounds, and also would provide a rapid but rough classification of Iberian ham according to the rearing conditions of pigs.

Immune-spaying as an alternative to surgical spaying in Iberian×Duroc females: effect on quality characteristics and fatty acid profile in dry-cured shoulders and loins.

The aim of this study was to assess the effect of immune-spaying on meat quality characteristics and fatty acid profile of dry-cured shoulders and loins by comparing Iberian×Duroc surgically spayed females, immune-spayed females and entire females. There were no significant differences on the physicochemical composition of dry-cured shoulders; however the intramuscular fat content of dry-cured loins was higher in immune-spayed females than entire ones, but not significantly different from spayed females. Immune-spayed females showed the highest values, which could improve the sensory quality of Iberian dry-cured loins, since it is considered an index of high quality. On the other hand, the PUFA content of both dry-cured shoulders and loins proved to be lower in immune-spayed than spayed females, which could prevent excessive fat oxidation responsible for rancidity. Therefore, immune-spaying could be a viable alternative to surgical spaying from the meat quality point of view.

Immune-spaying as an alternative to surgical spaying in Iberian×Duroc females: effect on carcass traits and meat quality characteristics.

The aim of this study was to assess the effect of immune-spaying on production traits and meat quality characteristics of fresh loin (longissimus dorsi) by comparing 3 groups of Iberian×Duroc females (N=12 per batch): surgically spayed, immune-spayed and entire females. Carcass traits and physicochemical parameters, including fatty acid profile, were investigated. The only carcass trait significantly affected by castration was the ham fat thickness, where both immune-spayed and surgically spayed females showed higher values against entire females (57±9.5mm, 62±2.5mm and 51±10.1mm, respectively; p<0.05). Furthermore, there were no significant differences in the quality parameters of fresh meat. These results are important regarding animal welfare, since in Europe, there is a plan to voluntarily end the surgical castration of pigs by 2018. Therefore, with an adequate vaccination protocol, immune-spaying might represent a good alternative to surgical spaying. The reliability of immune-spaying over long periods should be evaluated.

PTR-ToF-MS characterisation of roasted coffees (C. arabica) from different geographic origins.

Characterisation of coffees according to their origins is of utmost importance for commercial qualification. In this study, the aroma profiles of different batches of three monoorigin roasted Coffea arabica coffees (Brazil, Ethiopia and Guatemala) were analysed by Proton-Transfer-Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS). The measurements were performed with the aid of a multipurpose autosampler. Unsupervised and supervised multivariate data analysis techniques were applied in order to visualise data and classify the coffees according to origin. Significant differences were found in volatile profiles of coffees. Principal component analysis allowed visualising a separation of the three coffees according to geographic origin and further partial least square regression-discriminant analysis classification showed completely correct predictions. Remarkably, the samples of one batch could be used as training set to predict geographic origin of the samples of the other batch, suggesting the possibility to predict further batches in coffee production by means of the same approach. Tentative identification of mass peaks aided characterisation of aroma fractions. Classification pinpointed some volatile compounds important for discrimination of coffees.

Amino acid and nucleotide contents and sensory traits of dry-cured products from pigs with different genotypes.

The free amino acid and nucleotide contents of dry-cured ham, shoulder and loin from two genetic lines selected from pigs according to the paternal allele (homozygous AA and heterozygous AG) of the insulin-like growth factor-II gene were studied by HPLC. Their influence on the flavor and taste characteristics was also studied. The increase of lean content caused by the IGF-II mutation could affect proteolysis during the ripening process and therefore the sensory characteristics. The lower intramuscular fat content in the AA ham batch had a positive effect on the free amino acid content. However, similar flavor traits between ham batches were found, but the AG loin batch showed greater value. The enhancing effect of the IMP on the overall flavor intensity was limited by the amino acid and the IMF contents in dry-cured ham and loin, while in dry-cured shoulder, the IMP could be the reason for the significant differences in after taste and cured flavor scores.

Quality of dry-cured ham compared with quality of dry-cured shoulder.

The physicochemical and sensory properties of 30 dry-cured hams and 30 dry-cured shoulders were analyzed to determine the relationships between them. The variables used to characterize both products were: compositional parameters, instrumental texture, amino acid and fatty acid composition, and sensory profile. Despite being products from the same animal and composed mainly of fat, lean, and bone, their morphological differences determine the conditions of the processing time, which produced differences between products in most of the parameters evaluated. Dry-cured shoulders showed lower moisture content and greater instrumental hardness due to their morphology and muscular structure. Besides, these samples showed lower amino acid content according to the shorter ripening time. For the same reason, the dry-cured hams showed higher moisture content, lower instrumental hardness, and higher amino acid content. However, the differences in the muscular structure did not affect the sensory characteristics, which were more related with some compositional parameters, such as chloride, moisture, and amino acid content and with the length of the curing process.

Effect of origin, breeding and processing conditions on the isotope ratios of bioelements in dry-cured ham.

The stable isotope ratios (SIR) of the bioelements ((2)H/(1)H, (13)C/(12)C, (15)N/(14)N, (18)O/(16)O, (34)S/(32)S) of the defatted dry matter and marbling and subcutaneous fat fractions, were assessed on 86 ham samples belonging to six different types, with the aim of ascertaining the effect of origin and production system on 11 isotopic ratios. The ham types were obtained from pigs reared in three regions, examining in every location one different production factor at two levels of expression: pig genotype (local breed vs. industrial hybrid) in Friuli (Italy), pig feeding regime (Bellota vs. Campo) in Extremadura (Spain) and ham seasoning time (mid vs. end) in Emilia (Italy). The isotopic composition of meteoric water and the dietary abundance of C(4) plants allowed to distinguish Italian PDO from Spanish hams. The contrasting treatments within the regional batches generated promising differences in SIR, potentially useful for tracing the whole ham production system, including the processing procedure.

Effect of IGF-II (insulin-like growth factor-II) genotype on the quality of dry-cured hams and shoulders.

The aim of this study was to investigate the effect of the paternal allele (homozygous AA and heterozygous AG) of the IGF-II gene on the fat content, fatty acid composition and sensory characteristics of dry-cured hams and shoulders. The effects were more evident in the subcutaneous fat thickness than in the intramuscular fat (IMF) content, and in the dry-cured hams rather than the dry-cured shoulders. Subcutaneous fat thickness was significantly higher in AG dry-cured hams and shoulders; however, IMF content was only significantly higher in AG dry-cured hams. These effects produce changes in fatty acid composition and sensory characteristics when comparing both batches of each product, but the behavior differed with the type of product. Sensory characteristics were similar in both batches of dry-cured hams in spite of the differences in IMF content. Nevertheless, AG dry-cured shoulders showed higher scores in most of the attributes evaluated, despite the IMF content being similar between batches.

Physico-chemical, textural and structural characteristics of sous-vide cooked pork cheeks as affected by vacuum, cooking temperature, and cooking time.

This paper describes the influence of different factors on sous-vide cooked pork. Pork cheeks were cooked at different combinations of temperature (60°C or 80°C), time (5 or 12h) and vacuum (vacuum or air packaged). Weight losses were lower and moisture content higher in samples cooked for a shorter time (P=0.054) and at a lower temperature (P<0.001). Samples cooked at 60°C showed more lightness (L*) and redness (a*) (P<0.001). Lipid oxidation showed an interaction between cooking time and temperature (P=0.007), with higher TBARs values for samples cooked for 12h at 60°C and lower for those cooked for 12h at 80°C. Samples cooked at 80°C for 12h showed lower (P<0.05) values for most textural parameters than all the other types of samples. Vacuum packaging showed no influence on any of the studied variables. For the treatments evaluated, cooking temperature×time combination seems to be more important than vacuum packaging in the textural and colour parameters of pork cheeks.