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In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute res

Protein modifications (PMs) are covalent changes occurring on amino acid (AA) side chains of proteins via enzymatic action or spontaneously. They increase the protein structure complexity from the level of the genome to the proteome, and can determine their activity and, interactions1. It has been shown, on myofibrillar fish proteins that oxidative modifications depending on their saturation can decrease or enhance protein digestability2. Thus in this study shellfish meat samples were subjected to cooking followed by simulated INFOGEST in vitro gastrointestinal digestion protocol. Mass Spectrometry and in gel based proteomics, were used to detect PMs and digestion resistan peptides of raw and cooked shellfish meat samples with a foucs on major shellfish allergens: sarcoplasmic calcium-binding protein (SBP) and tropomyosin (TPM). Lastly, identified PMs were marked on TPM alignment from shrimp, oyster and abalone species, to gain insight into their pattern on the known IgE binding epitopes. Focusing on oxidative PMs, mostly on oxidation of Met, it was confirmed that, in all shellfish samples, PMs on TPM and SBP were more predominant after cooking. For instance in abalone, double oxidation of Met was only detected in cooked samples (sulfone M68 and M81). Another indicator of oxidative stress, 4-hydroxynonenal (HNE), was identified on shrimp TPM and, notably HNE was 21 times more abundant in the cooked sample. Study highlights the susceptibility of immunodominant epitopes in major shellfish allergens to oxidative PMs, which could impact interactions with the immune system in sensitive individuals. Significant resistance to digestion was demonstrated by paramyosin fragments in abalone and oyster suggesting the need for further analysis of their allergenicity. SBP of raw shrimp was strikingly resistant to both gastric and intestinal digestion. Moreover, our results indicate that oxidative modifications may decrease the stability of proteins like shrimp TPM, making them mo

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno- PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

Accurately diagnosing people with suspected SARS-CoV-2 infection is essential to help manage COVID-19. Currently available SARS-CoV-2 diagnostics detect either RNA of the virus by RT-PCR or the presence of viral antigens in biological fluids by ELISA or similar techniques. Low sensitivity of antigen tests could lead to the risk of false negative results. Therefore, this study aimed to develop a highly sensitive immuno-PCR method for quantifying SARS-CoV-2 nucleocapsid (N) protein that combines the specificity of sandwich ELISA with the sensitivity of PCR. Recombinant N protein fragment was produced in E. coli as an expression system and purified using immobilized metal ion affinity chromatography. The antibodies against the N protein were raised in rabbits and mice. High-affinity polyclonal mice and rabbit N protein-specific antisera were purified using ammonium sulfate precipitation and used to develop sandwich ELISA for the quantification of N protein. Mice polyclonal serum was used as a capture for N protein. N protein bound to mice antibodies was detected with rabbit polyclonal sera. A double-stranded amino-DNA molecule of 77 base pairs was PCR-synthesized, covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. The results were compared to analogous sandwich ELISA consisting of alkaline phosphatase-labeled goat anti-rabbit antibody. The sensitivity of immuno-PCR for quantification of N protein was increased by up to 7-fold compared to analogous ELISA, having a limit of detection of 92 pg/mL and a limit of quantification of 840 pg/mL. The developed immuno-PCR method thus has the potential to be used as a new antigen test for COVID-19 and beyond.

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno-PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating accuracy as low as 19.8 pg/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.

Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno- PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating a ccuracy a s l ow a s 1 9.8 p g/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.

Experimental data for the results shown in the manuscript Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR are given in the attachment.

This study investigated the effectiveness of the ultraviolet irradiation of the C region (UV–C) for the decontamination of Aspergillus flavus and aflatoxin B1 (AFB1) on artificially inoculated maize and peanut using innovative decontamination equipment that supports vibrations to achieve semi-fluidization of the grain/kernel material and getting equal irradiation of all surfaces of irradiated foods. UV irradiation is a non-thermal alternative to thermal processes commonly used in food processing with a well-known effect on AFB1 degradation. Samples of maize and peanut were exposed to UV-C irradiation with a total dose in the range of 1080–8370 mJ/cm2. Analysis by Tracker and ImageJ software confirmed the even distribution of irradiation on all surfaces during the entire duration of exposure. The highest reduction of A. flavus count was observed after ten days of incubation and irradiation treatment delivering a dose of 8370 mJ/cm2 achieving A. flavus count reduction of 4.4 log CFU/g in maize and 3.1 log CFU/g in peanut. Depending on the treatment, AFB1 reduction level in maize ranged from 17 to 43% and in peanut ranged from 14 to 51%. Sensory and physical testing of the peanut samples showed only minimal changes in the evaluated characteristics caused by different levels of the UV-C treatment. Presented results demonstrate a potential for the use of the presented approach as an effective reduction strategy for both A. flavus and AFB1 in maize and peanut.

Tropomyosin (TPM) is considered as a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products iscrucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDSPAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content., Tropomiozin (TPM) se smatra glavnim alergenom morskih plodova. Razvoj metoda za kvantifikaciju TPM-a u prehrambenim proizvodima je ključan za alergične osobe. Nekoliko pufera za ekstrakciju je upoređeno u pogledu efikasnosti ekstrakcije protein iz sveže zamrznutih i kuvanih gambora, tokom 2 i 24 časa. Sadržaj proteina u solubilnim ekstraktima je određen Bredfordovom metodom. Proteinski profil ekstrakata je analiziran SDS-PAGE metodom. Za kvantifikaciju TPM-a je razvijena sendvič ELISA metoda. Nijedan puffer nije pokazao značajnu razliku u količini ekstrahovanih protein nakon 2 i 24 časa ekstrakcije. U poređenju sa sirovim, značajno manje proteina je ekstrahovano iz kuvanih gambora. Kvantifikacija TPM-a ELISA metodom je pokazala da se fosfatom puferisanim fiziološkim rastvorom (PBS) koji sadrži 1 M NaCl (PBSN) i karbonatnim puferom, pH 10, ekstrahuje oko 6 puta više TPM-a u poređenju sa PBS-om. Ovo ukazuje na značaj odabira adekvatnog pufera za ekstrakciju TPM-a, jer se upotrebom tradicionalno korišćenih pufera može potceniti koncentracija TPM-a u gamborima.

Tropomyosin (TPM) is considered a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products is crucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDS-PAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content.

Tropomyosin (TPM) is considered a major allergen among different shellfish species. Developing sensitive, specific, and reliable methods for quantifying TPM in food products is crucial for persons allergic to shellfish. We have previously developed a highly sensitive sandwich ELISA method for quantifying shrimp TPM. Despite high amino acid sequence homology between shrimp and mussels TPM, the method has not been reliable for quantifying TPM from mussels, underestimating its concentration up to three orders of magnitude. Therefore, this work aimed to develop alternative immunological methods for mussel TPM quantification. Western blot, dot blot, and indirect ELISA using monoclonal anti-TPM antibody and alkaline phosphatase-labeled secondary antibody were developed and compared in terms of their sensitivity. Tropomyosin in mussels extracts was quantified using highly purified natural shrimp tropomyosin as standard. The linear range for TPM quantification using dot blot was between 5 and 50 µg/ml, while Western blot has slightly increased sensitivity, with a linear range between 1.25 and 12.5 µg/ml. Indirect ELISA has further improved the sensitivity of TPM quantification, with a 0.04-0.4 µg/ml linear range. Additional work will be performed to enhance the sensitivity of the presented methods, with the final aim of reducing risks of inadvertent food contamination.

With most of the plastics ever produced now being waste, slowly degrading and fragmenting in the environment, microplastics (MPs) have become an emerging concern regarding their presence in food and influence on human health. While many studies on marine ecotoxicology and the occurrence of MPs in fish and shellfish exist, research on the occurrence of MPs in other foods and their effect on human health is still in early-stage, but the attention is increasing. This review aimed to provide relevant information on the possible health effect of ingested MPs, the occurrence, and levels of MPs contamination in various foods and estimated exposure to MPs through food. Potential toxic consequences from exposure to MPs through food can arise from MPs themselves, diffused monomers and additives but also from sorbed contaminants or microorganisms that colonise MPs. Recent publications have confirmed widespread contamination of our food with MPs including basic and life-essential constituents such as water and salt providing the basis for chronic exposure. Available exposure assessments indicate that we ingest up to several hundred thousand MPs particles yearly.

Within this study, food safety management systems for wheat and maize, and derived white wheat flour and maize flour, have been evaluated, considering different prevention and practical intervention measures to be applied during storage and processing. For this purpose, through an operational flow chart, authors have performed mycotoxin-hazard analysis in the pre- and post-harvest phases and applied Food Safety Objective (FSO) and Performance Objective (PO) concept. Mycotoxin-hazard and analysis of control measures in the pre-harvest operations indicated that the most important practices at pre-harvest level for the control of mycotoxins in wheat/maize chain may include: (i) land preparation, such as tillage, cover crop and crop rotation, (ii) planting and antifungal mulch treatment, application of botanical extracts and intercropping, (iii) application of fungal biocontrol agents to reduce aflatoxins and (iv) application of fungal and bacterial biocontrol agents. For the post-harvest operations one Critical Control Point (CCP) and five Control Points (CPs) have been determined. The CCP includes an intervention measure that can be applied during storage, such as the application of treatments with volatile bioactive compounds, cold plasma, ozonisation or UV treatment. The results of FSO/PO analysis applied to wheat/maize chain revealed that FSO (the maximum level of mycotoxins in final food products given in European legislation) could be reached by combining conventional production steps such as cleaning and milling with different intervention strategies. For all strategies presented here, application on actual food production site must take into account local specificities and its interaction with climatic conditions.

Classified as Group 1 carcinogens aflatoxins (AFs) are of major concern for food safety and public health. Within the AFs group, aflatoxin B1 (AFB1) occurs at the highest levels in different food products and is considered as the most potent representative. A total of 463 samples of products susceptible to contamination with AFB1 and commonly consumed in Serbia were collected and analyzed. Consumption surveys were performed using a food frequency questionnaire (FFQ) and 24-h recall-based method. Monte Carlo simulation of 100,000 iterations was performed to estimate the intake of AFB1 through each food category, while a simple distribution approach was performed to estimate overall exposure of tested population groups. Risk characterization was performed by calculation of the Margin of Exposure (MOE) and by calculation of the number of possible hepatocellular carcinoma (HCC) cases. The highest exposure was observed in children with the mean overall estimated daily intake (EDI) in the range of 0.79–1.10 and 1.20–1.66 ng kg−1 bw day−1, followed by adolescents 0.56–0.81 and 0.94–1.32 ng kg−1 bw day−1, adult females 0.52–0.72 and 0.56–0.76 ng kg−1 bw day−1 and adult males with the mean overall EDI in the range of 0.39–0.56 and 0.47–0.66 ng kg−1 bw day−1 depending on consumption survey method and for the lower (LB) and upper bound (UB) scenario. MOE values that resulted from the mean values of the overall EDI of AFB1 were low, indicating high concern for all population groups. Based on obtained mean values of the overall EDI higher estimates of possible HCC cases caused by exposure to AFB1 were in the range of 0.01–0.02 cases/year/105 individuals, depending on the population group.

Za sada ne postoji dovoljno informacija o riziku koji za bezbednost hrane predstavljaju aflatoksini (AFs) prisutni u namirnicama na tržištu Republike Srbije. Fragmentirane studije i ukupan broj dostupnih podataka nisu dovoljni da se izvrši formalna procena rizika i procena izloženosti različitih grupa potrošača. Iz tih razloga su kao ciljevi ove doktorske disertacije postavljeni procena izloženosti aflatoksinu B1 (AFB1), karakterizacija rizika i ocena efikasnosti dekontaminacije AFB1 pomoću ultraljubičastog (UV) zračenja, kao jedne od mera fizičke dekontaminacije mikotoksina. Na osnovu analize proizvoda na bazi kikirikija i kukuruza, proizvoda mešanog sastava, jezgrovitog voća, pirinča, prosa, suve smokve i suve mlevene paprike, imuno-enzimskom (ELISA) metodom i metodom tečne-hromatografije sa masenom spektrometrijom (LC-MS/MS), zabeležena je učestalost pojave AFB1 u svim uzorcima od 29,2% (n=463). Podaci o unosu hrane su prikupljani metodama 24 h anketa ishrane i upitnik o učestalosti unosa hrane (FFQ). Procena izloženosti AFB1 je izvršena determinističkim pristupom, probabilističkim pristupom (Monte Karlo simulacijom) i pristupom proste distribucije. Ukupni prosečni procenjeni dnevni unos (PDU), na osnovu metode 24 h anketa ishrane, se kretao u rasponima od 1,203-1,659, 0,935-1,324, 0,523-0,721 i 0,466-0,662 ng/kg TM/dan za populaciju dece, adolescenata, odraslih žena i odraslih muškaraca, ponaosob. Karakterizacija rizika je izvršena određivanjem granice izloženosti (MOE) i broja mogućih slučajeva hepatocelularnog karcinoma (HCC). MOE vrednosti povezane sa prisustvom rizika usled unosa AFB1 su najčešće nastale kao posledica konzumiranja prženog kikirikija, kikiriki putera, kukuruznog hleba, proje, palente, tortilja čipsa, suvih smokvi, badema, oraha i pirinča. Na osnovu ukupnih prosečnih PDU, dobijenih metodom 24 h anketa ishrane, procenjeni broj mogućih HCC slučajeva/godišnje/105 osoba se kretao u rasponima od 0,016-0,022, 0,013-0,018, 0,009-0,013 i 0,008-0,012 z, Information on food safety risk of aflatoxins (AFs) in food in the Republic of Serbia are currently scarce. Fragmented researches and total number of available data are insufficient to perform formal risk and exposure assessment of different consumers groups. For these reasons, main goals of this doctoral dissertation include exposure assessment to aflatoxin B1 (AFB1), risk characterization, and efficiency evaluation of AFB1 decontamination by ultraviolet (UV) irradiation, appearing as one of physical mycotoxin decontamination methods. Based on analysis of peanut and maize based products, mixed composition products, nuts, rice, millet, dried figs and dried ground red paprika, by immuno-enzyme (ELISA) and liquid chromatography with mass spectrometry method (LC-MS/MS), the occurrence of AFB1 in all samples was 29.2% (n=463). Consumption surveys were performed using a 24h recall method and food frequency questionnaire (FFQ). Exposure assessment to AFB1 was performed with a deterministic approach, probabilistic approach (Monte Carlo simulation) and simple distribution approach. The mean overall estimated daily intake (EDI), based on 24h recall method, ranged between 1.203-1.659, 0.935-1.324, 0.523-0.721 and 0.466-0.662 ng/kg bw/day for population of children, adolescents, adult females, and adult males, respectively. Risk characterization was performed by calculation of the Margin of Exposure (MOE) and by calculation of the number of possible hepatocellular carcinoma (HCC) cases. Risk-related MOE values due to AFB1 intake appeared commonly as the consequence of roasted peanuts, peanut butter, maize bread, traditional maize bread, polenta meal, tortilla chips, dried figs, almond, walnut, and rice consumption. Based on the overall mean EDI, from 24h recall method, assessed number of possible HCC cases/year/105 persons was in the range of 0,016-0,022, 0,013-0,018, 0,009-0,013 and 0,008-0,012 for population of children, adolescents, adult females, and adult males, respectiv

In this work, we observed the effect of grape juice (% concentrated juice/% concentrated camel milk: GJ20/80, GJ50/50) and pomegranate juice (PJ20/80, PJ40/60) fortification on camel milk (CM) protein solubility and digestibility. Proteins were dissolved in sodium phosphate buffer to 50 mg/ml and defatted prior Bradford assay of protein concentration, then analyzed by Size Exclusion-Ultra High-Performance Liquid chromatography (SE-UHPLC). The CM protein aggregation and their stability were further monitored at different pH 2.0, 4.0, and 7.5 via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Freeze dried CM (FDCM) was the reference sample and our results showed that GJ50/50 and PJ40/60 with the highest fruit juice ratio had the lowest protein content in the supernatant, hence the decreased solubility. SE-UHPLC of supernatants showed a slight decrease in retention times of 11 kDa and 62 kDa proteins for GJ50/50 and PJ40/60 suggesting a possibility of adduct formation due to fortification leading to higher molecular weight. The simulated static in vitro gastrointestinal digestion of samples revealed that most soluble proteins were readily digested by pepsin, trypsin and chymotrypsin enzymes leading to small peptides. However, the SDS PAGE of pellets showed the partial resistance of casein and α-lactalbumin against peptic digestion.

Bivalves are a good source of nutrients but also a potential source of environmental contaminants, which could pose a risk for consumers. The aims of this study were: the determination of 16 elements by ICP-MS in 48 samples of five bivalve species purchased from market in Korea; the identification of elements useful for species classification using multivariate analyses; and the benefit-risk evaluation associated to the consumption of these bivalves. The highest difference among content of elements between species was found for Cd, Mn, Ni, Zn, and Fe. Partial last squares discriminant analysis revealed elements with a VIP score >1 which were considered as the most relevant for explaining certain species. As, Cd, Co, and Ni were found as taxonomical markers of V. philippinarum; Mn, Zn, Mg, and Na of A. irradians; and Cd, Ni, and Fe of M. yessoensis. These species could serve as good dietary sources of essential elements. Cd exposure by consumption of Manila clams is not representing a health risk for the Korean population; however, through consumption of Yesso scallops, 5.3% of the Korean population has a potential health risk. Removal of the digestive gland before eating will drastically reduce the amount of Cd ingested.

The French paradox describes a lower incidence of cardiovascular problems despite a high intake of saturated fats. This phenomenon was associated with higher consumption of red wine, only to be later discovered that the presence of several antioxidants, including resveratrol, are responsible for it. We investigated if resveratrol has a more direct role in protection from harmful oxidation and development of thrombosis, presumably through binding to important proteins of the blood coagulation process. Spectrofluorimetric analysis demonstrated binding of resveratrol to fibrinogen, the main protein in the coagulation process, which also has an important application as a food additive in making of fibrin gels. Various spectroscopic methods have demonstrated that binding of resveratrol does not unfold or destabilize fibrinogen since both near and far-UV CD spectra as well as its melting temperature remained unchanged. A mutually protective effect against the free radical-induced oxidation of resveratrol and fibrinogen was found. The presence of fibrinogen caused a very small masking effect of the antioxidative potential of resveratrol, measured by a reduction of hexacyanoferrate (III), while greatly increasing its solubility in an aqueous environment, thus increasing potential bioavailability and activity of resveratrol in circulation. By direct interaction and protection of fibrinogen, resveratrol may serve as an important antioxidant for prevention of thrombosis. The antioxidative effect of resveratrol may also protect and thus keep the desired characteristics of fibrinogen during the application of this protein as a food additive.

Food allergies affect up to 10% of the general population and represent an important health problem in the field of food safety in industrialized countries. Hence, developing reliable, specific, and sensitive methods for detecting and quantifying allergens in food products is of high importance. Shellfish have been recognized as one of the eight most common sources of allergens, with tropomyosin (TPM) being considered a major heat-stable allergen, having a highly conserved amino acid sequence among different shellfish species. Allergenicity of TPM may change during food processing, such as cooking. The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of shellfish tropomyosin in food samples. Two different extraction buffers - phosphate-buffered saline (PBS) and PBS containing 1 M sodium-chloride (PBSN), were compared for their ability to recover proteins from pre-cooked frozen Mediterranean mussel (Mytilus galloprovincialis) and fresh frozen razor mud shrimp (Solenocera melantho). The samples were additionally cooked according to the manufacturer's instruction and analyzed as such. The protein content was quantified using Bradford protein assay, and the protein components of soluble extracts were profiled using SDS-PAGE. TPM presence was confirmed using Western blot. Sandwich ELISA was developed using a monoclonal anti-TPM antibody as a capture antibody, while polyclonal anti-TPM antibody served as a detection antibody and was coupled to the biotinylated secondary antibody and streptavidin-alkaline phosphatase conjugate. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The profile of extracted proteins was changed when using PBSN instead of PBS. A higher concentration of proteins was recovered from raw shrimp using PBSN instead of PBS. At the same time, the type of extraction buffer did not affect protein recovery either from heated shrimp or pre-cooked/he

Food allergies affect up to 10% of the general population and represent an important health problem in the field of food safety in industrialized countries. Hence, developing reliable, specific, and sensitive methods for detecting and quantifying allergens in food products is of high importance. Shellfish have been recognized as one of the eight most common sources of allergens, with tropomyosin (TPM) being considered a major heat-stable allergen, having a highly conserved amino acid sequence among different shellfish species. Allergenicity of TPM may change during food processing, such as cooking. The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of shellfish tropomyosin in food samples. Two different extraction buffers - phosphate-buffered saline (PBS) and PBS containing 1 M sodium-chloride (PBSN), were compared for their ability to recover proteins from pre-cooked frozen Mediterranean mussel (Mytilus galloprovincialis) and fresh frozen razor mud shrimp (Solenocera melantho). The samples were additionally cooked according to the manufacturer's instruction and analyzed as such. The protein content was quantified using Bradford protein assay, and the protein components of soluble extracts were profiled using SDS-PAGE. TPM presence was confirmed using Western blot. Sandwich ELISA was developed using a monoclonal anti-TPM antibody as a capture antibody, while polyclonal anti-TPM antibody served as a detection antibody and was coupled to the biotinylated secondary antibody and streptavidin-alkaline phosphatase conjugate. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The profile of extracted proteins was changed when using PBSN instead of PBS. A higher concentration of proteins was recovered from raw shrimp using PBSN instead of PBS. At the same time, the type of extraction buffer did not affect protein recovery either from heated shrimp or pre-cooked/he

The aim of this study was to compare the sensory quality and acceptance of dried ready-to-eat beetroot snacks as a result of different drying methods applied: supercritical CO2-drying (scCO(2)-drying), frying, and freeze-drying. Descriptive sensory analysis, quality rating (10 assessors), and consumer acceptance testing (n= 102) were performed. Mean overall quality scores within the range of "very good" quality were found only in non-precooked scCO(2)-dried samples which were characterized by typical magenta color, low level of shape and surface deformations, pronounced brittleness and crispiness, and good rehydration during mastication. The other samples were in the range of "good" quality. The pre-cooking step before scCO(2)-drying negatively influenced the sensory quality parameters, particularly appearance. Around 60% of tested consumers showed a preference for the fried and non-precooked scCO(2)-dried samples. The drivers of liking were mostly related to the characteristics of the product, which was salted, fried, and crispy, with an oily and overburnt flavor, i.e., the product most similar to commercial potato chips products. Freeze-drying had a negative effect primarily on appearance and flavor. According to the sensory evaluation conducted, direct scCO(2)-drying without a pre-cooking step showed itself as a promising alternative drying technology in the production of dried beetroot snacks.

There is increasing interest in methodologies for the simultaneous concentration and detection of multiple targets in individual samples. The aim of this study was to investigate the potential presence of E. coli DNA in beef extract powder used as part of a procedure to concentrate water samples for the simultaneous detection of bacteria, viruses and protozoa. DNA from E. coli was detected in five out of six beef extract lots tested, demonstrating the limitations of its inclusion when being used in assays that will be used for the detection of E. coli in water samples. Further work is required to clarify if this phenomenon also occurs for other microorganisms of interest in water.