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I. Doskocil, B. Lampova, P. Smid, A. Kopec
Effect of heat treatment of sardines and sprats on immunomodulatory activity in vitro

Fish, rich in polyunsaturated fatty acids, is among the foods that may have a positive impact on human health. These include sardines and sprats, which are rich sources of PUFA. It is known that they can also play a role in inflammatory reactions in the body, where their long-term administration reduces the levels of several cytokines, such as interleukins, TNF-a, and others. Sprats were caught in the Baltic Sea, and sardines in the Mediterranean Sea. After removing inedible parts, washing fish were thermally treated using the following processes: cooking, steaming, baking, or deep frying. In the next part, the samples were digested using the in vitro digestion model INFOGEST, simulating digestion in oral, gastric, and intestinal phases. At the end of intestinal digestion, samples were centrifuged, filtered through a 0.22 µl filter, and then frozen at -80 °C until further testing. Samples were tested for inhibiting nitric oxide (NO) production on the RAW264.7 cell line when tested at a concentration of 20-0.009% digestion at 24 h incubation along with 1µg LPS. After 24h incubation, 50 µl of supernatant was taken, mixed with 50 µl Griess reagent, and measured at 540 nm. MTT was added to the cells, and after 2h incubation, the IC95value was determined. Subsequently, TNF-a was determined using the commercial ENZO kit. The IC95 was determined at 0.04% of the grass except for Steamed Sardines and sprats, where even a dose of 20% was not toxic with this treatment. Steamed Sardines and Sprats resulted in a 100% increase in NO production at a 20% grass concentration. Conversely, RAW Sardines led to a 30% decrease in NO production, as did Steamed Sardines and Baking Sprats, when there was a 10% decrease. On the other hand, increased production occurred in Cooking Sardines. TNFBaked, and Raw Sprat Sardines showed almost double the production compared to the LPS-treated control (p < 0.001). Heat treatment has a positive effect on increasing TNF-a production and thus enhancing anti-inflammatory activity. This research was funded by the Grant Agency of the Czech Republic, grant no. GA 21-42021L. And the National Science Centre Poland, grant no. 2020/39/I/NZ9/02959.

B. Lampova, I. Doskocil, P. Smid, A. Kopec
Effect of heat treatment of sardines and sprats after fattening on cellular integrity

Currently, studies focus on the effect of individual fatty acids on the integrity of the intestinal barrier. Sardines and sprats are essential sources of fatty acids and are also present in the human diet. However, it is common for these and other fish rich in fatty acids to be cooked. This treatment could also affect intestinal integrity. Sprats were caught in the Baltic Sea, and sardines in the Mediterranean Sea. After removing inedible parts, fish washed were thermally treated using the following processes: cooking, steaming, baking, or deep frying. Subsequently, the samples were digested using the in vitro digestion model INFOGEST. At the end of intestinal digestion, samples were centrifuged, filtered through a 0.22 µl filter, and then frozen at -80 °C until further testing. Intestinal barrier integrity testing was performed on the C2BBe1 (clone of Caco-2) cell line, cultured in 24-well inserts with complete media for 28 days. On the day of testing, the plates were washed with HBSS, and then TEER was measured. Then a sample of the prepared digest was added to the apical side with a final concentration of 1% along with 25 µm Lucifer Yellow (LY). On the basolateral side, only HBSS was included. Subsequently, 50 µl of the sample was collected from the basolateral side at defined time points and measured. Subsequently, the permeability was determined as the ratio between LY on the apical and basolateral sides. From the results for all heat treatments, permeability increased by an average of 8% after 0.5 h. However, after 1 h, permeability increased by 16% for Cooked Boiled Sardines and 17% for Baked Sardines. After 3 h of incubation, permeability increased by 53% overall for raw Sardines and almost 60% for Cooked Boiled Sardines. On the other hand, the smallest effect on permeability was observed after 3h for Steamed Sprats when the value reached 34%. The results show that the different treatments influence intestinal permeability as measured by LY, and prolonged exposure to digestion negatively affects cellular integrity. This research was funded by the Grant Agency of the Czech Republic, grant no. GA 21-42021L. And the National Science Centre Poland, grant no. 2020/39/I/NZ9/02959.

N. Berrighi, K. Benchaa, S. Bakhti, M. Belkada
Effects of the brewers' spent grain on milk quality of dairy cows reared on the Algerian pastures

Each year, considerable quantities of agro-industrial co-products are rejected as is by the food industry (253 tons of brewery grains). However, these discharges present a considerable potential energy source for animal feed. In this contribution, we evaluated the effect of two Algerian feeding systems consisting of three main natural sources: green grass, concentrate and brewers' grains on the biochemical and nutritional characteristics of cow milk.
According to the type of diet, a significant effect (p<0.05) was found in all physicochemical parameters, except for density. Acidity was significant in the milk of cows fed with spent grain (21.66°D).
The results obtained show variations in the composition of milk depending on the type of diet. The R1 diet induces the highest levels of total nitrogen matter (TNM) with 27.69% of DM and total lipids (TL) with 06.95% of DM, this being related to the higher energy intake provided by the brewers' grains compared to the R2 diet. The introduction of brewery dregs stimulated milk production and induced significant variations in milk composition. Similarly, statistical analysis showed significant differences (p<0.05) and predominant for the group of cows fed the R1 diet compared to the group fed concentrate and pasture (R2), in particular total dry matter (69.33% vs. 53.67%) and fat free dry matter (41.33% vs. 18.67%), respectively. The results reported on the protein analysis indicate significant (p<0.05) and predominant differences for milk from cows fed the R1 diet compared to the R2 (3.20% vs. 2.60%, p<0.05). The results showed more fat in the milk from cows fed with spent grain compared to the milk from cows fed with pasture and concentrate in their rations (3.58% vs. 3.47), respectively.
The use of spent grain is very interesting for the breeding of dairy cattle and allows to obtain a milk recommended by nutritionists and of an attractive sensory quality.

C. Zoani, V. Poscente, E. Pucci, G. Serafini, K. Presser, J. Czach, A. Zolynia, K. Hermsen
DRG4Food - Achieve Trust in a data-driven Food System by Implementing DRGs for the Food Sector

The importance of the food system makes it necessary to implement digital use cases and do so very responsibly, to drive innovation and address key issues such as food security, sustainability, personalized nutrition, food waste reduction, and equitable conditions throughout the food chain. An important concept to implement data rights in the food chain are the Digital Responsibility Goals (DRGs), developed by Identity Valley to promote digital technologies grounded on democratic rights and values. The goals propose concrete measures for seven key focus areas that have the potential to shape the digital economy in a way that is both ethical and human-centred. DRG4Food is an EU project about achieving trust in a data-driven food system by implementing Digital Responsibility Goals for the food sector. To create a data-driven food system, the project aims to set new standards by aligning with the European Digital Rights and Principles and the Digital Responsibility Goals. The project will enable new levels of innovation in food safety, sustainability, personalised nutrition, reduction of food waste and fair conditions throughout the entire food chain. In particular, some activities are focused on the implementation of a strategic roadmap (a new virtual food system) that will guide creation of a new food system apps. The roadmap should impact policy makers and funding providers to steer future development in a trustworthy, user-centred future where apps allowing users to execute their data rights are implemented with trust by design. The (road)map should represent different dimensions, including technological, architectural, and general approaches, in alignment with the European Green Deal priorities, and should help to classify solutions. One of the main aspects and outcomes will be the possibility to enable users to keep control over their personal data and allowing users to apply, at any time, their right to limit or revoke their personal data. The impacts of the "Digital Responsibility Roadmap" could be categorized into scientific (data-driven approaches leading to new technologies), economic/technological (development of enabling technologies that may lead to new business models), and social domains (enable users to execute their data rights).

C. Van Camp, W. H. Van Hassel, M. F. Abdallah, J. Masquelier
Development of a novel method for the simultaneous quantification of cyanotoxins and aflatoxin M1 in milk by LC-MS/MS

Cyanotoxins are secondary metabolites produced by cyanobacteria, a varied group of Gram-negative photosynthetic bacteria, also known as blue-green algae. Under specific conditions (i.e. water temperature, light intensity, and nutrient levels), cyanobacteria can form blooms. This natural phenomenon is characterised by an accumulation of algal biomass and the possible release of cyanotoxins into aquatic ecosystems. Human exposure to cyanotoxins through contaminated food or contaminated drinking water can be associated with symptoms such as fever, convulsion, gastroenteritis, headache and, in rare cases, death. Global warming has led to an increase in the duration and frequency of blooms and the use of contaminated water as drinking water for cattle could lead to the production of contaminated milk. Aflatoxin M1 (AFM1) is a well-known mycotoxin, found as a metabolite of aflatoxin B1 (AFB1), present in the milk of animals after ingestion of feed contaminated with AFB1. Unlike cyanotoxins, for which there is no legislation, AFM1 levels in milk are regulated under Commission Regulation (EC) No 1881/2006. To our knowledge, there is no existing LC-MS/MS method for the quantification of multiple cyanotoxins in milk. In this study, a simple and sensitive analytical method based on ultra-high-pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) was developed, optimised and validated for the simultaneous quantification of AFM1, eight microcystins congeners, and nodularin. After improving the extraction and purification conditions (e.g. solvents, solid phase extraction), the parameters of the validation were assessed. Specificity and selectivity, limits of detection and quantification, linearity, matrix effect, recovery, reproducibility, repeatability and measurement uncertainty were determined in accordance with the main analytical guidelines.

B. Aiuto, S. Cirrincione, C. Lamberti, L. Cavallarin, C. Portesi, A. M. Rossi, F. Romaniello, M. G. Giuffrida
Development of a micro UHPLC-HRMS/MS method for the absolute quantification of adjuvants in wine

Proteinaceous products are frequently employed as adjuvants during winemaking to improve the wine's clarity, color, flavor, and structural stability. For allergic subjects, the presence of this fining agent traces (such as egg albumen and animal gelatine) in the finished product poses a risk. For this reason, several Directives of European Union require the labeling of allergenic ingredients whenever if they are only used for food making, regardless of their level of inclusion. The International Organization of Vine and Wine (OIV) had first established the limit of detection (LOD) and quantification (LOQ) for milk and egg clarifiers for immunoassay at 0.25 mg/L and 0.50 mg/L, respectively (regulation 579/2012). Quantification of allergen traces in food can be achieved using different techniques, including PCR and ELISA, but both methods do not achieve the results that are promised by reference methods. EU legislation with the Council Directive 96/23/EC's implementation indicated MS techniques as best candidates for this purpose. Our research's objective is to provide an accurate reference approach for the absolute quantification of adjuvants in red wine using MS techniques. In order to assess the selected peptides for fining agent detection, a HPLCHRMS DDA (Data Dependent Analysis) on albumen and gelatine standards was carried out, and 5 distinct and specific peptides (3 from albumen and 2 from gelatin) were chosen. The peptides were selected for the absence of both missed tryptic cleavage sites and post-translational modifications. Moreover, they have to be speciesspecific for each fining agents, as determined by BLAST. In the current investigation, a set of five signature peptides were preliminary quantified by LC-HRMS/MS up to 0,25 ppm. The workflow will proceed with a) the synthetization of the labeled and unlabeled signature peptides; b) the creation of a calibration curve using synthesized peptides (internal standard) in the range of 0,1 to 10 ppm; c) the validation of this procedure. The synthetic peptides will be employed as calibrants, and the Amino Acid Analysis (AAA) will assign an uncertainty value for each peptide to achieve metrological traceability to the SI.

G. Carne, D. Makowski, S. Carrillo, T. Guérin, P. Jitaru, J.-C. Reninger, G. Rivière, N. Bemrah
Development of a method to define a maximum health level in food: cadmium and the emergent consumption of edible seaweed in France

The consumption of edible seaweed is an emerging and growing phenomenon in Europe and France. Seaweed are a bioindicator of marine environmental quality and have a high propensity to be linked to metals due to the presence of polysaccharides in its structure. Specifically, higher levels of cadmium exceeding the French recommendation of 0.5 mg.kg-1 dry matter (DM) are observed. Cadmium (Cd) is a trace element of concern found in various environmental compartments (soil, water, air) due to its natural (Earth's crust) and anthropogenic origin (industrial and agricultural activities). Cadmium is recognized to induce nephropathy, bone pathologies, cancer and disorders for the reproduction. European and French populations are overexposed to cadmium by food. To add potentially significant cadmium intakes by an emerging consumption of seaweed can increase the exposure of consumers. The objective was to develop a methodology allowing to derive a low and protective cadmium concentration in edible seaweeds to limit cadmium overexposure in consumers. A probabilistic approach was developed including analytical data on concentration levels of cadmium in seaweed and consumption of seaweed by French consumers, taking into account other dietary sources of exposure to cadmium. A maximum cadmium limit in edible seaweed was calculated from the approach so as not to increase the initial dietary cadmium exposure of seaweed consumers compared to oral health-based guidance value (HBGV) for cadmium. Considering that the seaweed-consuming population did not exceed the HBGV in 95% of cases, when simultaneously exposed to other dietary sources of cadmium, a cadmium concentration of 0.35 mg.kg-1 DM of unprocessed seaweed (confidence interval [0.18,1.09]) has been calculated (Carne et al., 2022). The new methodology revises the fixation of maximum limits in the regulatory system. It is a support to decision, especially in a context of future regulation on establishment of contaminants limits for seaweed in Europe. The approach could be applied to other relevant food / substance pairs. Taking the various sources of exposure into account, this methodology ensures better health consumer protection.

N. Marchond, N. Zephyr, C. Mazurais, A. Leufroy, P. Jitaru
Development of a multi-elemental method for foodstuffs analysis by inductively coupled plasma-mass spectrometry following microwave digestion and validation by using the accuracy profile approach

This work describes an optimization and validation process for the simultaneous determination of 35 elements - lithium (Li), boron (B), sodium (Na), magnesium (Mg), aluminium (Al), potassium (K), calcium (Ca), titanium (Ti), vanadium (V), chrome (Cr), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), gallium (Ga), germanium (Ge), arsenic (As), selenium (Se), strontium (Sr), molybdenum (Mo), rhodium (Rh), silver (Ag), palladium (Pd), cadmium (Cd), tin (Sn), antimony (Sb), tellurium (Te), barium (Ba), platine (Pt), mercury (Hg), thallium (Tl), lead (Pb) and uranium (U) - in food samples by inductively coupled plasma-mass spectrometry (ICP-MS) after closed-vessel microwave digestion or multiwave with pressurized digestion cavity. In order to improve the limits of quantification of certain elements for risk assessment in the context of the third French Total Diet Study (3rdTDS), the analytical conditions of the multi-elemental method were optimized. The method was validated based on the accuracy profile approach according to the NF V03-110 French standard, which takes into account the simultaneous assessment of the accuracy and precision of the method. The accuracy profile is an expression of the combination of the systematic (trueness) and the random error (repeatability and/or intermediate precision) for a series of analyst's levels in various matrices in range of concentrations called validity domain. For this purpose, five to six measurement series were repeated in duplicate on different days, over a timespan of three months for constructing the accuracy profile with six levels of concentration. Several performance criteria such as limits of quantification (LOQ), specificity, linearity, precision under repeatability conditions, intermediate precision reproducibility with the use of the accuracy profile. Furthermore, the method accuracy was assessed by means of several external quality controls (EQC). Results indicate that this method could be used for the determination of these 35 essential and non-essential elements in foodstuffs at trace and ultra-trace levels, including for 3rd TDS, with acceptable analytical performance.

D. Ghaffour, V. Sirot, M. Champion, A. Leufroy, P. Jitaru
Development & validation of a method for Arsenic speciation analysis in food by HPLCICP-MS and application to the 3rd (French) Total Diet Study

Total Diet Studies are one of the most efficient tools for national assessment of chemical contamination of food prepared as consumed. They are endorsed by the World Health Organisation (WHO) and Food and Agriculture Organization of the United Nations (FAO). For assessing potential risks of dietary exposure, a simple, fast and a robust method for routine speciation analysis of arsenic species was optimized and validated, this method is to be applied for the analysis of a large panel of foodstuffs samples within the framework of the 3rd (French) total Diet Study (3rd TDS). In this method, multiple arsenic species including, arsenobetaine (AsB), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenite (AsIII) and arsenate (AsV) were extracted using two extraction approaches, employing a closed microwave system and a heating block, respectively (only N-ethylmaleimide in Ultrapure water was used, as extraction solution, to prevent the interconversion of AsIII and other AsSugars). The separation was achieved in less than 10 min by an anion exchange high-performance liquid chromatography(HPLC) with a mobile phase consisting of a mixture of methanol and ammonium carbonate ((NH4)2CO3). The separated species were detected online by inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated based on the accuracy profile approach according to the NF V03-110 French standard, which takes into account the simultaneous assessment of the accuracy and precision of the method. The accuracy profile is an expression of the combination of the systematic (trueness) and the random error (repeatability and/or intermediate precision) for a series of analyst's levels in various matrices in range of concentrations called validity domain. For this purpose, six measurement series were repeated in duplicate on (6) different days, over a time span of 6 weeks for constructing the accuracy profile. The method was applied to the speciation analysis of AsB, DMA, MMA, AsIII and AsV in a variety of foodstuffs of the 3rd (French) Total Diet Study.

P. Guichard, Y. Pirotais, S. Gautier, M. Bessiral, D. Hurtaud-Pessel, E. Verdon
Confirmation of total florfenicol residues by LC-MS/MS: risk assessment based on falsenegative results when non-hydrolysis was performed

Florfenicol (FF) is a synthetic, broad-spectrum systemic antibiotic. The metabolic pathways are well documented, with the main route of elimination being in the urine. Is mainly metabolized to florfenicol amine (FFA) as final marker residue via three main intermediate metabolites: flofenicol alcohol, florfenicol oxamic acid and monochloroflorfenicol and can therefore be found in the muscle of animals intended for human consumption (1). Listed in Table 1 of Regulation (EU) 37/2010, its veterinary medicine use is authorized in the European Union (EU) in all food-producing species, except for animals from which milk and eggs are produced for human consumption. Maximum residue limits (MRLs) in muscle are set to 100 µg.kg-1 for poultry; 200 µg.kg-1 for bovine, ovine and caprine; 300 µg.kg-1 for porcine; and 1000 µg.kg-1 for fin fish. MRL for all other food producing species is established to 100 µg.kg-1. Its determination is based on the marker residue defined as the sum of the FF and its metabolites measured as FFA. There are two possibilities: determine the concentration of each metabolite and express it in FFA equivalents; or perform an acid hydrolysis to convert all FF residues into FFA. This second option is recommended, because a significant proportion of FF residues are proteinbounded and therefore not extractable when hydrolysis is not used (2). A reliable quantitative LC-MS/MS method was fully developed and validated, according to the new Regulation (EU) 2021/808, for determination of total FF residues as FFA residue marker using hydrolysis step in all muscle food-producing species. Chromatographic conditions enables the monitoring of FFA and its parent compound to ensure the total conversion during hydrolysis step. Furthermore, mass spectrometry conditions include an isotopically labelled internal standard to minimize matrix effects and obtain an accurate quantification with regard to the different species and the concentration levels studied, ranging from 0.1 to 1.5 MRLs.This method was compared to a similar protocol but without this specific step, focusing exclusively on the FF and its main metabolite: FFA. Carried out on naturally contaminated samples, results demonstrated a proven risk of false-compliant results when a hydrolysis step is not applied.

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