2. Universidad Cardenal Herrera-CEU

We used anonymous questionnaires to assess the hygienic and sanitary aspects of game meat self-consumption in Eastern Spain as the first step towards a health risk assessment. The survey yielded 472 valid interviews from active hunters. The maximum possible score was 65 points (average 29 8; range 1–52). Most participants were men (95%), but women achieved significantly better scores (p = 0.003). Hunters above 65 years old scored significantly lower results than younger groups (p = 0.007). The score increased with the educational level (p = 0.046). A 92% of the collaborators consumed game meat. Veterinary inspection and freezing were irregular among the participants. Most respondents declared carrying the animals in their personal vehicles. Of the dressing process, 61% of sites were outdoors, 68% of the participants declared using specific knives, 64% used the same clothes as in the field, and 42% used disposable gloves. The most usual way to dispose of the remains was garbage containers (41%); offal abandonment in the field was 33%, and 13% fed domestic animals using the remains. We conclude that public health authorities should increase their interest in the self-consumption of game meat. Clear guidelines about domestic dressing facilities and hygienic habits should be published, these being essential when looking for synergies with hunter associations.

Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, with approximately one third of the population around the world seropositive. The consumption of contaminated food is the main source of infection. These include meat products with T. gondii tissue cysts, and dairy products with tachyzoites. Recently, contamination has been detected in fresh products with oocysts and marine products. Despite the great health problems that are caused by T. gondii, currently there are no standardized methods for its detection in the food industry. In this review, we analyze the current detection methods, the prevalence of T. gondii in different food products, and the control measures. The main detection methods are bioassays, cell culture, molecular and microscopic techniques, and serological methods, but some of these do not have applicability in the food industry. As a result, emerging techniques are being developed that are aimed at the detection of multiple parasites simultaneously that would make their application more efficient in the industry. Since the prevalence of this parasite is high in many products (meat and milk, marine products, and vegetables), it is necessary to standardize detection methods, as well as implement control measures.

Background: Campylobacter is the main pathogen involved in zoonotic gastrointestinal diseases. Last year, the European regulation 2017/1495 on Campylobacter in broiler carcasses came into force. In this context, the aim of this study was to assess the potential risk factors associated with exceeding the 1,000 CFU/g criterion set by the European Commission in several slaughterhouses in Spain. Methods: Information relating to 12 factors were collected using questionnaires. Samples were collected from 12 Spanish abattoirs during June, July and August 2017 (n=1,725) and were analysed following ISO/TS 10272-2:2006 method. Results: The proportion of Campylobacter-positive samples was 23.7% (n=409). Analysis of the flock age (41-50 days) revealed a significantly increased OR in Campylobacter enumeration (OR=7.41). Moreover, scalding temperature (51.9.54 ºC) was positively associated with an increase in OR (OR=2.75). Time in transit to slaughter (1-1.5h), showed a significant OR decrease (OR=0.25). However, when processed for more than 2 hours, presented an increase in OR (OR=4.44). Regarding carcass weight, the range from 3.21-3.58 presented a decrease in OR (OR=0.01). Conclusion: The outcomes of this study suggest that although most chickens are contaminated by the bacterium, the prevalence that exceeds the limit of 1,000 CFU/ is not so high as we thought.

In this study, the possible influence of the food matrix and the interfering population of microorganisms on the detection and count of Listeria monocytogenes in three common foods of the Spanish diet (Spanish omelette, fresh cheese and vegetable salad) was determined. Four groups were assayed: one control, two groups with interfering microorganisms (Salmonella Enteritidis, Staphylococcus aureus and Proteus mirabilis) with different levels of L. monocytogenes and a final group only contaminated with L. monocytogenes. The samples were analyzed with the normalized method (UNE-EN ISO 11290:2018) and with an alternative technique (VIDAS). The results show that the presence of interfering microorganisms did not seem to interfere with the determination of L. monocytogenes. Furthermore, the type of food did not seem to influence the determination of L. monocytogenes, but the culture media used showed differences. In fact, regardless of the type of food, the ALOA medium showed higher sensitivity than the other media, with higher recovery in 100% of samples (only for the Spanish omelette in Group B was the result the same as that for PALCAM, 􀀀8.11 log cfu/g). The results obtained using the VIDAS were not influenced by any of the factors or conditions used and show 100% efficiency.

Anisakidosis is a parasitic zoonotic disease which can cause gastroallergic reactions in humans. In 2010, the European Food Safety Agency estimated that approximately 20,000 cases of anisakiasis had been reported across the world, with Spain having the highest number of infections in Europe. The blue whiting (Micromesistius poutassou, Risso, 1827) is one of the most widely fished species worldwide and represents around 25% of the white fish eaten in Spain. The Spanish Food Safety Authority requires obligatory evisceration of certain fish species before commercialization, but not for blue whiting. Nevertheless, some supermarkets carry this out themselves to prevent human infections and negative customer reactions deriving from the presence of ascaridoid larvae. To assess the effectiveness of eviscerations at supermarkets, a total of 320 blue whiting specimens were examined. The risk of larval migration from the visceral cavity to the musculature in gutted and ungutted fish was also assessed. Our results showed a total prevalence (25%) of ascaridoids in fish gutted at the supermarket, and a direct relationship was found between the presence of larvae in the muscle and time until evisceration. In ungutted fish, the standard length and weight were higher for infected than for non-infected fish. Also, massive infections had a higher prevalence in these larger specimens, in which the viability of larvae was also high. Larval viability was not found to be affected by a 24-h refrigeration period. Anisakis was the most prevalent genus identified in the fish examined. The results indicate that gutting at the supermarket is not an effective method for the total removal of ascaridoid larvae and that additional safety measures are advisable.

Variation in methylxanthines (theobromine and caffeine) and flavanols (catechin and epicatechin) was studied in a large set of cocoa powders (covering different origins, processing parameters and alkalization levels). The content of these compounds was established by high-performance liquid chromatography (HPLC), whose results showed that the alkalisation process lowered the content of all analytes, whose loss was more evident in flavanols. Therefore, the determination of these analytes in a huge set of samples allowed not only better knowledge of the concentration variability in natural commercial cocoas from different origins, but also the understanding of the effect that industrial alkalisation has on these contents. The feasibility of reflectance near-infrared spectroscopy (NIRS) combined with partial least square (PLS) to non-destructively predict these contents, was also evaluated. All the analytes were generally well predicted, with better predictions for methylxanthines (R2 P 0.882 for both analytes; RMSEP 0.020-0.061%, bias -0.027-0.006) than for flavanols (R2 P 0.818-0.863; RMSEP 6.63-15.87%, bias 1.942-3.056). Thus NIRS could be an alternative fast reliable method for the routine assessment of these analytes in the cocoa industry.

Cocoa shell must be removed from the cocoa bean before or after the roasting process. In the case of a low efficient peeling process or the intentional addition of cocoa shell to cocoa products (i.e. cocoa powders) to increase the economic benefit, quality of the final product could be unpleasantly affected. In this scenario, the Codex Alimentarius on cocoa and chocolate has established that cocoa cake must not contain more than 5% of cocoa shell and germ (based on fat-free dry matter). Traditional analysis of cocoa shell is very laborious. Thus, the aim of this work is to develop a methodology based on near infrared (NIR) spectroscopy and multivariate analysis for the fast detection of cocoa shell in cocoa powders. For this aim, binary mixtures of cocoa powder and cocoa shell containing increasing proportions of cocoa shell (up to ca. 40% w/w based on fat-free dried matter) have been prepared. After acquiring NIR spectra (1100-2500 nm) of pure samples (cocoa powder and cocoa shell) and mixtures, qualitative and quantitative analysis were done. The qualitative analysis was performed by using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), finding that the model was able to correctly classify all samples containing less than 5% of cocoa shell. The quantitative analysis was performed by using a partial least squares (PLS) regression. The best PLS model was the one constructed using extended multiple signal correction plus orthogonal signal correction pre-treatment using the 6 main wavelengths selected according to the Variable Importance in Projection (VIP) scores. Determination coefficient of prediction and root mean square error of prediction values of 0.967 and 2.43, respectively, confirmed the goodness of the model. According to these results it is possible to conclude that NIR technology in combination with multivariate analysis is a good and fast tool to determine if a cocoa powder contains a cocoa shell content out of Codex Alimentarius specifications.

Cocoa powder is a global product of great value that can be adulterated with low-cost raw materials such as carob flour without changing the characteristics of color, aroma and flavor of the product. The use of rapid methods, as a NIR technology combined with multivariate analysis, is of interest for this detection. In this work, 216 adulterated samples prepared by blending commercial cocoa powders with different alkalization levels (n = 12) with commercial carob flour (n = 6) in different proportions (0-60% of adulteration) were analyzed. The diffuse reflectance spectra of the samples were acquired from 1100 to 2500 nm using a Foss NIR spectrophotometer. A qualitative and quantitative analysis was done. For the qualitative analysis, a principal component analysis (PCA) and a partial least squares discriminant analysis (PLS-DA) was performed. The coefficient of determination (R2) of the model PLS-DA was 0.969 and the coefficient of determination of the validation (R2CV), based on a full cross validation was 0.901 indicating good calibration with good predictability. These results indicate that it is possible to distinguish between pure cocoa powders from the adulterated samples. For the quantitative analysis a partial least squares (PLS) regression analysis was performed. The most robust model of PLS prediction was obtained with 1 factors (LV) at coefficient of determination (R2) of 0.980 and a root mean square error of prediction (RMSEp) of 3.237 % for the external validation set. These data lead to the conclusion that NIR technology combined with multivariate analysis allows the identification and determination of the amount of natural cocoa powder present in a mixture adulterated with carob flour.