Simultaneous determination of organophosphorus pesticides and phthalates in baby food samples by ultrasound-vortex-assisted liquid-liquid microextraction and GC-IT/MS.

Baby foods are either a soft, liquid paste or an easily chewed food since babies lack developed muscles and teeth to chew effectively. Babies typically move to consuming baby food once nursing or formula is not sufficient for the child's appetite. Some commercial baby foods have been criticized for their contents. This article focuses on the simultaneous determination of organophosphorus pesticides and phthalates by means of a method based on ultrasound-vortex-assisted liquid-liquid microextraction coupled with gas chromatography-ion trap mass spectrometry (GC-IT/MS). The protocol developed allowed the determination of six phthalates [dimethyl phthalate, diethyl phthalate, dibutyl phthalate, isobutyl cyclohexyl phthalate, benzyl butyl phthalate, bis(2-ethylhexyl) phthalate] and 19 organophosphorus pesticides. Freeze-dried product samples (0.1-0.2 g) were dissolved in 10 mL of warm distilled water along with 5 µL of an internal standard (anthracene at 10 mg mL-1 in acetone): the choice of extraction solvent was studied, with the most suitable being n-heptane, which is used for phthalate determination in similar matrices. The solution, held for 5 min in a vortex mixer and for 6 min in a 100-W ultrasonic bath to favor solvent dispersion and consequently analyte extraction, was centrifuged at 4000 rpm for 30 min. Then 1 µL was injected into the GC-IT/MS system (SE-54 capillary column; length 30 m, inner diameter 250 µm, film thickness 0.25 µm). All analytical parameters investigated are discussed in depth. The method was applied to real commercial freeze-dried samples: significant contaminant concentrations were not found. Graphical abstract Simultaneous and sensitive determination of organophosphorus pesticides and phthalates in baby foods by the ultrasound-vortex-assisted liquid-liquid microextraction Öªgas chromatography-ion trap mass spectrometry procedure. 1 methacrifos, 2 pirofos, 3 phorate, 4 seraphos, 5 diazinon, 6 etrimphos, 7 dichlofenthion, 8 chlorpyrifos-methyl, 9 pirimiphos-methyl, 10 malathion, 11 chlorpyrifos, 12 parathion-ethyl, 13 pirimiphos-ethyl, 14 bromophos, 15 chlorfenvinphos, 16 bromophos-ethyl, 17 stirophos, 18 diethion, 19 coumaphos, A dimethyl phthalate, B diethyl phthalate, C dibutyl phthalate, D butyl cyclohexyl phthalate, E benzyl butyl phthalate, F bis(2-ethylhexyl) phthalate, IS internal standard.

Comparative Study of Carcass Characteristics and Meat Quality of Local Mediterranean Donkey Breeds.

This study aimed to evaluate carcass and meat quality traits in Masri (n = 14) and North African (n = 14) male donkeys, raised in a semi-intensive breeding system, grazing on mountainous areas, with supplementation of 1 kg of barley per day per animal, slaughtered at 5 (±0.48) years old. Compared to Masri, the North African population exhibited higher (p < 0.05) final body weights (181.7 ± 2.1 and 212.5 ± 7.6 kg) and cold carcass weights (101.7 ± 1.3 and 116.2 ± 4.5 kg), but lower (p < 0.05) cold dressing percentages (56.0 ± 0.4 and 54.6 ± 0.4%). Meat quality analyses showed higher (p < 0.05) cooking loss values in Masri meat (43.9 ± 0.8 vs. 39.9 ± 1.2%). Among the meat color parameters, the hue value was higher (p < 0.05) in North African samples (0.42 ± 0.01 vs. 0.39 ± 0.1). Meat from North African donkeys had higher (p < 0.05) dry matter, fat, and protein contents. Meat amino acid analysis revealed abundant levels of lysine, leucine, and methionine, in both populations. Donkey meat from both populations presented a high polyunsaturated fatty acids content, resulting in polyunsaturated fatty acids/saturated fatty acids and omega 6/omega 3 ratios for all breeds close to the recommended values for human health. Atherogenic and thrombogenic indices were also suggested to have positive effects on consumers' artery health. The characteristics of donkey meat present intriguing nutritional aspects compared to ruminant meat, and its production should be encouraged in the rural development of inner-Mediterranean areas.

Thyroid hormones in milk and blood of lactating donkeys as affected by stage of lactation and dietary supplementation with trace elements.

The traditional utilization of donkeys (Equus asinus) as dairy animals has recently attracted substantial scientific interest with regard to human nutrition. Donkey milk is well tolerated by infants with cows' milk allergy, useful in the treatment of human immune-related diseases, in the prevention of atherosclerosis, and in-vitro studies showed an anti-proliferative effect. Active 3-3'-5-triiodothyronine (T3) in colostrum and milk could play different physiological roles, systemic and paracrine, for both the mother and the suckling offspring. The aim was to evaluate whether thyroid hormones (TH) concentrations in milk and blood of lactating donkeys change with the advancing lactation and whether they can be affected by dietary supplementation with several trace elements, some of them directly involved with TH synthesis (I), metabolism (Se) and action (Zn). Sixteen lactating jennies were divided into two groups (CTL and TE). Mixed feed for TE was added with Zn, Fe, Cu, Mn, I, Se and Co. Every 2 weeks milk and blood samples were collected at 11·00. Total concentrations of T3 in milk (T3M) and T3 and T4 in plasma (T3P and T4P) were assayed using ELISA kits, validated for the donkey species. T3M was not correlated with TH concentrations in blood, did not change with the stage of lactation, and was significantly higher in TE (4·09 ± 0·07 ng/ml, mean ± SE) than in CTL group (3·89 ± 0·08 ng/ml). T4P (81·8 ± 5·2 ng/ml) and T3P (15·2 ± 1 ng/ml) significantly changed with time, but were not significantly affected by dietary treatment. T3P/T4P ratio was significantly lower in TE group. This study indicates that in donkey milk the concentration of T3, a human-like bioactive compound, can be affected by trace elements intake.

Soybean vs. Pea Bean in the Diet of Medium-Growing Broiler Chickens Raised under Semi-Intensive Conditions of Inner Mediterranean Areas: Growth Performance and Environmental Impact.

The effects of Glycine max L. (SOY) vs. Pisum sativum L. (PEA) in the diet were investigated on in vivo performance of two medium-growing broiler genotypes and on environmental impact. Sixty Kabir Red Plus and sixty New Red chickens were randomly allocated in 20 pens (n = 6 birds per pen). Each pen, i.e., experimental unit, received 1.18 kg dry matter (DM) including soybean (3.39%) in SOY, or pea bean (6.78%) in PEA groups. DM intake, DM refusals and bodyweight (BW) were recorded on pen basis, and average daily gain (ADG) and feed conversion rate (FCR) were calculated. Data on in vivo performance were processed by ANOVA General Linear Model followed by Student−Newman−Keuls post hoc test. Greenhouse gas (GHG) emissions were evaluated on a year basis by GLEAM-i software. The diet did not affect (p > 0.05) DM intake and DM refusals, BW, ADG and FCR. Diet x genotypes affected (p < 0.05) DM intake and DM refusals. PEA diet, cheaper than SOY diet, allowed the reduction in total GHG and CO2, and of meat emission intensity. However, an increase in total CH4 and N2O emissions was observed. The replacement of soybean with pea bean can represent a possible management strategy to reach trade-off between good farming practices and environmental protection on small-scale poultry farms of inner Mediterranean areas.

Carcass and Meat Quality Traits of Medium-Growing Broiler Chickens Fed Soybean or Pea Bean and Raised under Semi-Intensive Conditions.

A study was carried out to evaluate the effect of the total replacement of flaked soybean (Glycine max L., SOY) with raw pea (Pisum sativum L., PEA) on the carcass and meat quality traits of two medium-growing broiler strains (Kabir Rosso Plus, KB; New Red, NR). Birds were housed in 20 pens (five replications/groups, six birds each). At 83 days of age, 40 birds (2/replication) were slaughtered and the pectoral muscle (PM) was removed for analyses. Diet did not affect slaughter weight, carcass traits and meat quality. A pea diet determined a significant increase of MUFA and a decrease of PUFA, n-3 and n-6 PUFA; hence, the pea-fed group had a lower PUFA/SFA and a higher n-6/n-3 ratios compared to the soy-fed. NR chickens were heavier, with higher carcass and cut weights (p < 0.01) compared to KB chickens. Interactions (p < 0.05) between factors were found for PM weight and yield. Meat from NR had a higher (p < 0.05) pH. Fatty acids were slightly affected by genotype. Replacing soybean with pea adversely affects meat fatty acid composition in terms of nutritional profile.

Antibacterial potential of donkey's milk disclosed by untargeted proteomics.

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM. The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H2O2. κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins. Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SIGNIFICANCE: Donkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy. DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring. DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H2O2. In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits. Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population. Data of this study represent the largest inventory of proteins identified in Equidae milk, so far.

Measurement of thyroid hormones in donkey (Equus asinus) blood and milk: validation of ELISA kits and evaluation of sample collection, handling and storage.

Donkey's milk is well tolerated by human infants with cow's milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (-20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at -20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.

Equine Milk Production and Valorization of Marginal Areas-A Review.

The equine dairy chain is renewing the interest toward horse and donkey breeding for the production of milk with potential health promoting properties. The dairy equine chain for human consumption could contribute to the rural eco-sustainable development for the micro-economies of those areas threatened by marginalization. As a part of the whole equine industry, and its possible impact in the modern and future society, the main traits of the equine dairy enterprise are reviewed with a special focus on management of animals and milk. Equine milk compositional and nutritional peculiarities are described as also related to milk hygiene and health issues. Scientific and technical aspects of the feeding management are considered in the frame of the emerging dairy equine enterprise, where pasture is an essential element that allows to match production goals for horses and donkeys, biodiversity preservation, as well as landscape safeguard.

Study on analytical characteristics of Nicotiana tabacum L., cv. Solaris biomass for potential uses in nutrition and biomethane production.

In order to limit the smoking tobacco sector crisis, a new non-GMO Nicotiana tabacum L. cv. Solaris was proposed as oil seed crop. Residues of oil extraction were successfully used in swine nutrition. The aim of this study was to explore the full potential of this innovative tobacco cultivar as multitasking feedstock non interfering with the food chain. In the triennium 2016-2018, samples from whole plant, inflorescence and stem-leaf biomass were collected in three experimental sites and analysed for chemical constituents, including fibre fractions, sugars and starch, macro-minerals and total alkaloids. The KOH soluble protein content and the amino-acid profile were also investigated as well as the biochemical methane potential. All the analyses were performed according to official methods and results were compared with values reported in literature for conventional lignocellulosic crops and agro-industry residues. The average protein content, ranging from 16.01 to 18.98 g 100 g-1 dry matter respectively for stem-leaf and whole plant samples, and their amino-acid profile are consistent with values reported for standard grass plant. These findings suggest the potential use of cv. Solaris in industrial food formulations. Moreover, considering the average content of both fibre available for fermentations (72.6% of Neutral Detergent Fibre) and oils and fats (7.92 g 100 g-1 dry matter), the whole plant biomass of cv. Solaris showed good attitude to anaerobic fermentation, confirmed by the biochemical methane potential of whole plant (168 Nm3 t-1 organic matter). Similarly, results allow to define the cv. Solaris biomass as a good quality forage apt to ensiling for its chemical composition. The low total alkaloids content of cv. Solaris, in average 0.3 g 100 g-1 dry matter, was previously reported not to affect growth performances and welfare traits of dairy heifers. These are the first results showing the multitasking potential use of cv. Solaris biomass, that could allow the recovery of tobacco cultivation know-how especially in marginal areas.

Lactobacillus rhamnosus as additive for maize and sorghum ensiling.

The effects of Lactobacillus rhamnosus AT195, a potential probiotic microorganism cultured in buffalo "scotta" whey, on chemical and microbiological composition in maize and sorghum ensiling were evaluated. Both crops were harvested, chopped, and treated or not with the selected strain prior to ensiling in fiberglass vertical silos; 90 days after ensiling, silages were sensorially evaluated and sampled. Different chemical components were evaluated both on fresh crops and silages: in particular, the water-soluble carbohydrates content was investigated by high-field NMR spectroscopy and the carbohydrate fermentation profile was performed by GC. Besides phenotypic identification and typing, microbiological studies included Lb. rhamnosus genotype typing by RAPD-PCR. All silages, inoculated or not, were well preserved, as their chemical and microbiological data along with the fermentation profiles showed. The selected strain used as inoculum influenced the lactic acid population of silages and evidenced a good survival performance during the ensiling process of both maize and sorghum. Moreover, the use of Lb. rhamnosus strain efficiently improved the quality of the multifactorial ensiling process by significantly reducing the ammonia nitrogen content of both maize and sorghum silages.

Hygienic characteristics and microbiological hazard identification in horse and donkey raw milk.

Today the interest toward horse (Equus caballus) and donkey (Equus asinus) milk for human consumption is receiving a renewed attention because of its particular composition, hypoallergenicity, and nutraceutical properties. The realistic perspective of global use of this aliment in balanced diets, especially for infancy and geriatrics, poses the need for a more in depth knowledge on milk hygiene and on the health status of dairy animals, as a prerequisite of consumers' safety. The aim of this paper was to review the available literature on the health and hygiene parameters as well as on the potential microbiological hazards in horse and donkey milk and the risks related to their consumption. Both microbial contamination and somatic cell count are reasonably low in equine milk and also the presence of pathogens, like Escherichia coli O157, Salmonella spp., Campylobacter spp., Yersinia enterocolitica, Brucella spp., Mycobacterium spp., Bacillus cereus, Cronobacter sakazakii, Streptococcus equi subsp. zooepidemicus, Rhodococcus equi, Streptococcus dysgalactiae subsp. equisimilis, Clostridium difficile and Burkholderia mallei is low. However, in those regions of the world where the prevalence of Brucella spp. and Rhodococcus equi is high, the alimentary risks could increase. Similarly, in areas with higher incidence of immunocompromised people, the increased risks should be warned not only for pathogens but also for opportunistic microbiota.

Isoelectric focusing and ELISA for detecting adulteration of donkey milk with cow milk.

Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey-cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1-28 sequence stretch of cow ß-casein was also developed for an accurate definition of composition of donkey-cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey-cow milk mixtures prepared at laboratory scale and assayed by ELISA.

Determination of triacylglycerols in donkey milk by using high performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry.

The separation and determination of triacylglycerols (TAGs), which are the main components of naturally occurring fats and oils, in milk fat is a challenging task due to the very complex nature of this matrix. In the present study the TAG fraction of donkey milk lipids has been characterized by using high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). HPLC in reversed phase mode has been used for TAG separation and silver ion (Ag+) HPLC has been used as a second dimension to clarify and confirm the identification. The RP-HPLC eluate was fractionated and the fractions of interest were injected onto the Ag+-HPLC column. In both cases peak assignment was carried out by combining retention data with APCI-MS spectra information. In total, 55 TAGs in donkey milk fat were identified (without considering the positional isomers) and quantified on the basis of percentage peak areas in the RP-HPLC chromatogram (without the use of correction factors). Amongst the identified triacylglycerols, POLn, POO, PPO, CaPO, POL, and PPoO proved to be the main components of the TAG fraction of donkey milk.

Major constituents, leptin, and non-protein nitrogen compounds in mares' colostrum and milk.

Five Haflinger mares were hand-milked at 0 h (pre-suckle) and 6 h (postsuckle), 12, 24, 48, 72 and 96 h after parturition. Total solids, protein, fat, lactose, calculated gross energy content, leptin and non-protein nitrogen components (urea, alpha-amino nitrogen, creatinine and allantoin) were determined. The levels of the major constituents differed significantly in pre-suckle colostrum from subsequent samples. Leptin levels were the highest in whole (9 ng x mL(-1) of immunoreactive human equivalent HE +/- 0.48 ng x mL(-1), SEM) and skimmed (7.8 ng HE x mL(-1) +/- 0.52 ng x mL(-1), SEM) pre-suckle colostrum, declined sharply at 6 hours postpartum, and more slowly subsequently. Mean urea concentration was constant at around 5.0 mM, while a-amino N increased over the observation period and creatinine and allantoin decreased. These findings provide a further indication that mares' milk can be regarded as a functional food.