Phenolic Composition, Antiradical, Antimicrobial, and Anti-Inflammatory Activities of Propolis Extracts from North East Spain.

Antioxidant-related parameters and anti-inflammatory and antimicrobial activities against Listeria monocytogenes were assessed in eight North East Spain poplar propolis samples. Propolis extracts (PEs) were obtained using 70% ethanol (PEE) and methanol (PME). Yield and total phenol compounds were higher in PEE. Phenolic acids were analyzed by a high-performance liquid chromatograph-diode array detector. Caffeic and ferulic acids were quantified in all PEE and PME. All samples contained p-coumaric acid (quantified in 6 PEE and in 3 PME). Ascorbic acid was detected in all propolis, but mainly quantified in PME (≤0.37 mg/g PE). Biological properties were tested on PEE. As for antiradical activities, trolox equivalent antioxidant capacity (TEAC) [against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)•+], ranged between 578 and 4620 µmol trolox/g, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (against DPPH free radical), between 0.049 and 0.094 mg/mL, antioxidant activity against hydroxyl (•OH) radical (AOA), between 0.04 and 11.01 mmol uric acid/g, and oxygen radical absorbance capacity (ORAC) against peroxyl (ROO•) radical between 122 and 3282 µmol trolox/g. Results of TEAC, AOA, and ORAC were significantly correlated. IC50 anti-inflammatory activity ranged from 1.08 to 6.19 mg/mL. Propolis showed higher inhibitory activity against L. monocytogenes CECT934 and L. monocytogenes CP101 by agar well diffusion (P < .05) (10.5 and 10.2 mm, respectively) than against L. monocytogenes CP102 (7.0 mm). Data of this research show that North East Spain propolis may be of interest for pharmaceutical and food industry use.

Effect of Domestic Freezing on the Viability of Toxoplasma gondii in Raw and Dry-Cured Ham from Experimentally Infected Pigs.

ABSTRACT: Toxoplasma gondii is the causative agent of the parasitic disease toxoplasmosis, which is an important foodborne zoonosis. Eating undercooked meat of infected animals has been considered the major transmission route of T. gondii to humans. The present study was conducted to evaluate the efficacy of domestic freezing for inactivation of T. gondii bradyzoites in ham. Raw and dry-cured ham from a pig experimentally inoculated orally with 4,000 oocysts of T. gondii VEG strain was subjected to domestic freezing at -20°C for up to 14 days. The effect was evaluated by bioassay in mice and a quantitative PCR assay. In raw and dry-cured ham, -20°C for 7 and 14 days, respectively, did not inactivate T. gondii. More studies are needed to find the correct temperature and time needed to render the bradyzoites noninfectious for humans. Meanwhile, the recommendations for freezing to inactivate T. gondii in raw or dry-cured meats must be revisited because domestic freezing conditions do not reduce the risk of infection.

Occurrence of Pesticide Residues in Spanish Honey Measured by QuEChERS Method Followed by Liquid and Gas Chromatography-Tandem Mass Spectrometry.

In the current study, the QuEChERS extraction method with slight modifications, followed by liquid and gas chromatography-tandem mass spectrometry, was applied for the determination of 399 pesticide residues in 91 raw honey samples from northeastern Spain. The quality control procedure established in Document No. SANTE/12682/2019 was successfully followed: the responses in reagent blank and blank honey samples were below 30% of the reporting limit (0.01 mg kg-1) for all analysed compounds, the correlation coefficients (R2) were higher than 0.99 in most calibration curves, the deviation of back-calculated concentration from the true concentration was below ±20% (using the standard of 50 µg L-1 concentration), and the recoveries of spiked samples on matrix were within the range of 70-120% for almost all analytes. Only chlorfenvinphos (2-7.8 ng/g) and coumaphos (8.8-37 ng/g) were detected in 13 samples, and neither were observed to exceed their maximum residue limits (MRLs). Dietary risk assessment for pesticide residues in honey above their lowest calibrated level (LCL) was performed, and two different age groups, adults and infants, were considered as populations at risk. The contribution of honey lay far below the acceptable daily intake (ADI) for both pesticide residues. Therefore, according to our results, honey is unlikely to pose concerns for consumer health in terms of its contribution to dietary long-term exposure. However, to maintain the level of compliance, pesticide residues in honey should be continuously monitored.

Evaluation of a Method for Rapid Detection of Listeria monocytogenes in Dry-Cured Ham Based on Impedanciometry Combined with Chromogenic Agar.

The food industry is in need of rapid, reliable methodologies for the detection of Listeria monocytogenes in ready-to-eat products, as an alternative to the International Organization of Standardization (ISO) 11290-1 reference method. The aim of this study was to evaluate impedanciometry combined with chromogenic agar culture for the detection of L. monocytogenes in dry-cured ham. The experimental setup consisted in assaying four strains of L. monocytogenes and two strains of Listeria innocua in pure culture. The method was evaluated according to the ISO 16140:2003 standard through a comparative study with the ISO reference method with 119 samples of dry-cured ham. Significant determination coefficients ( R2 of up to 0.99) for all strains assayed in pure culture were obtained. The comparative study results had 100% accuracy, 100% specificity, and 100% sensitivity. Impedanciometry followed by chromogenic agar culture was capable of detecting 1 CFU/25 g of food. L. monocytogenes was not detected in the 65 commercial samples tested. The method evaluated herein represents a promising alternative for the food industry in its efforts to control L. monocytogenes. Overall analysis time is shorter and the method permits a straightforward analysis of a large number of samples with reliable results.

Toxoplasma gondii in raw and dry-cured ham: The influence of the curing process.

The aim of this work was to analyze Toxoplasma gondii in raw hams by mouse bioassay and to evaluate the effect of curing on the viability of the parasite to assess the risk of infection from eating dry-cured ham. After a serology study of 1200 pigs in Aragón (Spain), forty-one naturally infected pigs with different serological titers against T. gondii were selected. Two cured periods (9 and 12 months) were evaluated as well as the influence of the physicochemical composition of hams on T. gondii survival. Although the parasite burden was low, a high number of seropositive pigs with Toxoplasma tissues cysts in raw hams were found (31.6%). Viability of T. gondii was influenced by the curing, with statistically significant differences between fresh and cured hams (p < 0.001). The viability was higher in hams cured for 9 months compared to those cured for 12 months. However, this period of curing resulted in the reduction but not in a complete elimination of the risk. Thus, from a public health point of view, under the conditions of this study it is safer to consume dry-cured ham with periods of curing higher than 12 months. Analysis of physicochemical results did not identify any variable with significant influence on the presence and viability of T. gondii in cured ham, but loss of viability of T. gondii was observed in hams with a lower fat content. Further research is required to validate combinations of salts concentration and time of curing that can be used as preventive measures in the HACCP system of dry-cured ham industry.

Toxoplasma gondii: Pig seroprevalence, associated risk factors and viability in fresh pork meat.

This study was conducted on 161 fattening pig farms located in Aragón (Northeast Spain). Serum samples from 1200 pigs were tested for antibodies against T. gondii by indirect immunofluorescence assay (IFA). Antibodies to T. gondii (≥1:20) were detected in 301 pigs (24.52%). The seroprevalence observed in the present study indicates a widespread exposure to T. gondii, as seropositive pigs were found in 96.67% of the farms studied although low pig titers were determined. Risk factors associated with T. gondii seroprevalence were presence of cats in or around the farms, presence of dogs around the facilities, low number of animals in the farms, poor hygiene and bad maintenance of the farms. Finally, it was observed that where rodent baits were used, Toxoplasma prevalence was lower. Risk management measures including control of cats and rodents on the farms, among others, could help to reduce the observed prevalence levels. By mouse bioassay, T. gondii was detected in 73.7% and isolated from 42.1% of seropositive pigs and a significant relation between the titers of pigs and the presence and viability of T. gondii in the tissues was found. The detection of T. gondii is not possible by currently practiced meat inspection. Nevertheless, the increased probability of detecting viable forms of T. gondii in tissues of pigs with titers ≥1: 80 could be used as the cutoff for discriminating higher risk animals, and could be used as an effective control tool for the industry of cured meat products. In practical terms, we propose that this value could be used as a critical limit in the HACCP system.

Bioactive properties of a propolis-based dietary supplement and its use in combination with mild heat for apple juice preservation.

This study characterizes the antioxidant and antibacterial properties of a propolis-based dietary supplement (PDS) and investigates its incorporation into apple juice to decrease the intensity of the heat treatment required to inactivate 5 log10 cycles of Escherichia coli O157:H7. As the source of propolis, we used a PDS containing 0.2 mg/µL of propylene glycol-extracted propolis (propolis). The total phenolic content and antioxidant activity (IC50) of the PDS were 82.15±3.53 mg/g and 0.055±0.003 mg/mL, respectively. Regarding antimicrobial activity, propolis (0.2 mg/mL) was very effective under acidic pH against Listeria monocytogenes EGD-e, inactivating more than 5 log10 cell cycles in 1h, but hardly inactivated or sub-lethally injured E. coli O157:H7 Sakai. However, incorporating propolis (0.2 mg/mL) into acidic buffer decreased the time needed to inactivate 5 log10 cycles of E. coli O157:H7 Sakai at 51 °C by more than 40 times. Moreover, when combined with heat in apple juice, propolis (0.1mg/mL) reduced the thermal treatment time and temperature needed to inactivate 5 log10 cycles of E. coli by 75% and 3 °C, respectively. The corresponding PDS concentration did not decrease the organoleptic properties of the apple juice, which implies the possibility of obtaining a sensorially appealing, low-pasteurized apple juice with the functional properties provided by propolis.

Evaluation of heavy metals and polycyclic aromatic hydrocarbons in honeys from different origins.

A survey of honey samples from different geographical and botanical origins, including some samples collected from a fire-affected area in Spain, was conducted to assess their content of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The levels of the determined toxic elements (Pb, Cd, As, and Sn) were low and were in the range of those reported by other studies. In our work the total amount of heavy metals and Pb was higher in dark honeys than in pale honeys. In the collected samples, no detectable levels of the 15 PAHs studied were found. The obtained data served to assess the levels of heavy metals and PAHs in honey samples from different geographical and environmental origins and to contribute to the scarce data about pollutant content of this matrix. In light of these results, the analyzed samples do not pose any serious concern to human health, and the data obtained in this study could serve to contribute to the establishment of specific maximum limits for honey.

Toxoplasma gondii in commercially available pork meat and cured ham: a contribution to risk assessment for consumers.

Toxoplasmosis is an infection caused by Toxoplasma gondii, whose transmission has usually been attributed to ingestion of undercooked or raw meat. Dry-cured ham is a high-quality meat product of increasing economic relevance, and epidemiological studies point to cured meat products as a risk factor for acquiring toxoplasmosis. With the aim of contributing to the risk assessment process, 50 samples of fresh pork meat and commercial cured ham were collected in the city of Zaragoza (northeastern Spain), and the presence of viable forms of T. gondii was analyzed. A mouse concentration bioassay technique was used, and the presence of the parasite in mice was determined by indirect immunofluorescence assay. T. gondii was detected in two samples of rib, reflecting a frequency of 8% positive fresh pork meat (4% positivity of total samples analyzed). Brains of seropositive mice were analyzed by histology and PCR, although the parasite was not isolated in the seroconverted mice. No viable forms were detected either in other types of fresh meat or in the samples of cured ham.

Multiresidue determination of carcinogenic polycyclic aromatic hydrocarbons in honey by solid-phase extraction and high-performance liquid chromatography.

An analytical procedure based on solid-phase extraction, using ethyl acetate as the elution solvent, and high-performance liquid chromatography with fluorescence and diode array detection was developed for the identification and quantification of polycyclic aromatic hydrocarbons (PAHs) in honey. The method has been optimized and validated in accordance with Commission Regulation 333/2007 and Commission Decision 2002/657/EC. This method allows the identification of the 15 PAHs that should be monitored in food matrices, as proposed in 2002 by the Scientific Committee on Food and later by the European Union in the Commission Recommendation 2005/108/EC, because of their genotoxic and carcinogenic properties. The results of the validation study were in agreement with quality criteria described in European legislation in terms of sensitivity, accuracy, and ruggedness, and the method was applied to the analysis of 42 honey samples (21 from Spain and 21 from other regions). The honey samples were not contaminated by PAHs at detectable levels and thus could be marketed without health risk.

Determination of the viability of Toxoplasma gondii in cured ham using bioassay: influence of technological processing and food safety implications.

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii and distributed worldwide. Ingestion of viable cysts from infected raw or undercooked meat is an important route of horizontal transmission of the parasite to humans. Little information is available concerning the effect of commercial curing on cysts of T. gondii. This study is the first in which the influence of processing of cured ham on the viability of T. gondii has been evaluated, using bioassay to assess the risk of infection from eating this meat product. Naturally infected pigs were selected for the study, and a mouse concentration bioassay technique was used to demonstrate viable bradyzoites of T. gondii in porcine tissues and hams. No viable parasites were found in the final product (14 months of curing) based on results of the indirect immunofluorescence assay and histological and PCR analyses. Our results indicate that the consumption of hams cured as described here poses an insignificant risk of acquiring toxoplasmosis. However, additional studies are required to evaluate the safety of ham products cured under different conditions of curing time, salt, and nitrite concentration.

Impact of the use of fluvalinate on different types of beeswax from Spanish hives.

Acaricides are applied in agriculture as phytosanitary products against pests and in apiculture to control the bee parasite Varroa destructor. Poor apicultural practices could result in an accumulation of residues in honeybees, in the environment, and in beeswax and other bee products by migration from the wax comb into stored honey through a process of diffusion and consequently constitute a potential risk for humans. In this study, six different types of beeswax samples were analysed for the determination of residues of fluvalinate, coumaphos, and bromopropylate and its metabolite 4,4'-dibromobenzophenone, all of which are the most commonly acaricides used by Spanish beekeepers against V. destructor. The analytic method consists of solid-phase extraction on a SPE Florisil cartridge and high-performance liquid chromatography separation using a photo diode array detector. The results show that fluvalinate residues were detected in 36.3% of samples, ranging from 1.2 to 6.6 microg/g wax. Residues of coumaphos, bromopropylate, and 4,4'-dibromobenzophenone were not found to be greater than their detection limits. This study indicates that the analysis of these compounds in beeswax samples could be used as bioindicators of fluvalinate sanitary treatment and handling practices applied by beekeepers.

Evaluation of daily intake of PCDD/Fs and indicator PCBs in formula-fed Spanish children.

Human exposure to polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) occurs predominantly via food intake. In this study, the exposure assessment of these contaminants has been estimated for infant formula-fed children up to 1 year of age. PCDD/F concentrations in the infant formulae was low, ranging between 0.09 and 0.17 pg WHO-TEQ g(-1) fat and between 0.30 and 0.46 pg WHO-TEQ g(-1) fat when results were calculated with the lower and medium bound values, respectively. Indicator PCB contamination levels were below 1 ng g(-1) fat in all cases. Thus, the estimated daily intake of PCDD/Fs and indicator PCBs for infants has been assessed taking into account the above-mentioned contamination levels as well as different scenarios of body weight and food consumption data for babies aged 0-12 months. The results vary in the different scenarios considered but, on the whole, the daily estimated dioxin and indicator PCBs intake of the average infant population due to the consumption of infant formulae does not exceed the tolerable daily intake (TDI) of 2 pg WHO-TEQ kg(-1) bw day(-1) recommended by the Scientific Committee on Food (available at http://ec.europa.eu/food/fs/sc/scf/out90_en.pdf) nor the threshold value of 10 ng kg(-1) bw day(-1) proposed by the Dutch National Institute of Health and Environment (RIVM) (Baars et al. 2001. Report no. 711701025, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands).

Evaluation of GC-ion trap-MS/MS methodology for monitoring PCDD/Fs in infant formulas.

The application of high resolution gas chromatography in combination with low resolution mass spectrometry with electron ionization and MS/MS detection (HRGC-MS/MS) is tested for its use in the analysis of PCDD/Fs in infant formulas. Development of the analytical method was based upon EPA directrices and international recommendations. Calibration linearity was tested and average relative response for any native and labelled compound over the five-point calibration range below 14% was found. The precision and accuracy of the proposed analytical procedure are also presented. Results obtained are in agreement with EPA criteria. The method is applied to the analysis of a number of initial and follow-on milk based infant formulas. In general, HRGC-MS/MS constitutes an interesting method for the analysis of dioxins in such matrices.

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS.

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.

Rapid method for trace determination of organochlorine pesticides and polychlorinated biphenyls in yogurt.

A new multiresidue method was developed for the analysis of 19 organochlorine pesticides and 6 polychlorinated biphenyls in yogurt. The sample was extracted twice with acetone by homogenization with an Ultra-Turrax dispersing unit, and the combined extracts were filtered. The extract was then purified by reversed-phase C18 columns and subjected to further cleanup with neutral alumina columns. The residues were determined by gas chromatography with electron capture detection. After the method was optimized, it was validated by determination of recovery percentages, precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) with yogurt samples fortified at 10 and 1 microg/kg concentration levels. The recovery of 23 organochlorine residues ranged from 77 to 95% at a level of 10 microg/kg, from 74 to 102% at a level of 1 microg/kg, and between 54 and 61% for dieldrin and alpha-endosulfan. The method is repeatable and reproducible, with relative standard deviation values <19% for all residues except dieldrin. Detection and quantitation limits were between 0.02 and 0.62 microg/kg. The analytical method proposed was quick, accurate, repeatable, and reproducible for the determination of organochlorine residues in yogurt samples.

Organochlorine Pesticide Residues in Spanish Meat Products and Meat of Different Species.

The level of organochlorine pesticides in 229 samples of Spanish meat and meat products of different species (lamb, pork, beef and poultry) was investigated. Chlorinated residues were quantitated by gas-liquid chromatography with electron capture detector using packed and capillary columns. Hexachlorobenzene (HCB) and hexachlorocyclohexane (HCH) were detected in all samples. In general, lamb appeared to be more heavily contaminated by HCB and HCH, which reached maximums of 178 ppb (µg/kg on a fat basis) and 505 ppb, respectively. The level of HCB averaged 49 ppb in lamb; varied between 8-18 ppb in pork and beef products; and amounted to 26 ppb in fresh poultry sausages. Of the three isomers of HCH determined, the γ-HCH (lindane) was most frequently detected; 100% in lamb and pork (both meat, cured sausage and pork bologna), and 64 to 94% in fresh sausages of poultry and beef. The level of the HCH group averaged 112 ppb in lamb, 85 ppb in poultry, nearly half that much in pork and pork products, and around 20-40 ppb in beef products. Dieldrin was the only chlorocyclodiene detected: 8 to 15% in pork products, and 28% in fresh poultry sausage. The DDTs in lamb showed 83% of detection, especially in the pp' form of DDE and DDT. The overall contamination with DDT and its metabolites was found to be very moderate averaging 25 ppb, with a maximum of 91 ppb. No residues of aldrin, endrin, heptachlor, heptachlor epoxide, chlordane, methoxychlor, endosulfan or trans-nonachlor were detected.