Anisakis spp. Larvae in Deboned, in-Oil Fillets Made of Anchovies (Engraulis encrasicolus) and Sardines (Sardina pilchardus) Sold in EU Retailers

Sardina pilchardus and Engraulis encrasicolus are considered the principal target species for commercial fishing in Europe and are widely consumed as semipreserved products. Although they are considered shelf-stable products, if treatment is not correctly applied, their consumption may represent a public health risk in regard to anisakiasis and allergic reactions. Little is known about the prevalence of Anisakis spp. in ripened products. This study aimed to evaluate the presence of Anisakis spp. larvae in deboned, in-oil anchovy and sardine fillets marketed in the EU to assess the influence of processing techniques on the prevalence of larvae. Ninety semipreserved anchovy and sardine products deriving from the Mediterranean Sea or Atlantic Ocean were collected from different EU retailers and examined using chloropeptic digestion to evaluate the presence of larvae and identify them. Thirty nonviable Anisakid larvae—A. pegreffii (30%) and A. simplex (70%)—were found. The frequency of larvae was higher in anchovies (28.8%). The low frequency of parasites found proved that processing technologies can influence the presence of larvae in final products, but it is important that visual inspection is performed only by trained people. The sources of raw materials should be considered in the production flow chart

A Study on the Antimicrobial and Antibiofilm Peptide 1018-K6 as Potential Alternative to Antibiotics against Food-Pathogen Salmonella enterica

Antimicrobial resistance has become one of the major global public health concerns, and it is indispensable to search for alternatives to conventional antibiotics. Recently, antimicrobial peptides have received great attention because of their broad-spectrum antimicrobial activity at relatively low concentrations, even against pathogens such as Salmonella enterica, which is responsible for most food-borne illnesses. This work aimed at evaluating the antimicrobial and antibiofilm activity of the innate defense peptide, named 1018-K6, against S. enterica. A total of 42 strains, belonging to three different subspecies and 32 serotypes, were included in this study. The antibiotic resistance profile of all the strains and the cytotoxic effects of 1018-K6 on mammalian fibroblast cells were also investigated. Results revealed that MIC (minimum inhibitory concentrations) and MBC (minimum bactericidal concentrations) values were in the ranges of 8–64 μg/mL and 16–128 μg/mL, respectively, although most strains (97%) showed MICs between 16 and 32 μg/mL. Moreover, sub-inhibitory concentrations of 1018-K6 strongly reduced the biofilm formation in several S. enterica strains, whatever the initial inoculum size. Our results demonstrated that 1018-K6 is able to control and manage S. enterica growth with a large potential for applications in the fields of active packaging and water disinfectantsThis research was funded by: Ministero dello Sviluppo Economico “Packaging Bioattivi e bIOsanitizzanti: Sviluppo di stratEgie iNnovaTIve ed ecososteNibili pEr L’Industria Alimentare—(BIO-SENTINEL) project, grant number F/200092/01-03/X45. Fondo per la Crescita Sostenibile—Sportello “Agrifood” PON I&C 2014-2020; Regione Campania-“Sviluppo di una tecnologia Intelligente contro spoilage ed agenti patogeni: dal peptide antimicrobico ad un PACKaging innovativo nella filiera ittica del Mediterraneo (iPACK-Med)project, grant number PO FEAMP 2014/2020, MISURA 1.26 “INNOVAZIONE” DD n. 266/2019-n. B68D19001380009; Ministero della Salute-“Attività battericida ed anti-biofilm di nano-sistemi ibridi coniugati con peptidi antimicrobici: una nuova strategia per la formulazione di bio-sanitizzanti contro ceppi patogeni resistenti”- Ricerca Corrente 2018 project, grant number IZS ME 06/18 RCS

Real-world Performance of a New Strategy for Off-Label Use of Guselkumab in Moderate to Severe Psoriasis: Super-Responder Patients as the Epitome of Efficacy and Optimisation

Background Guselkumab is a drug used to treat moderate to severe plaque psoriasis. However, real-life clinical data on its of-label use are limited, especially regarding the optimal drug dosage regimen for diferent patient profles. Objective The main objective of this real-world, single-centre, retrospective study was to identify the of-label guselkumab dosing regimen used in clinical practice. The study also aimed to evaluate the drug's efcacy, safety, and survival, as well as the proportion of super-responders (SR) based on a newly proposed defnition. Methods The study included 69 patients who started treatment with guselkumab between March 2019 and July 2021. Patients were followed up until April 2022, during which time their efcacy, safety, persistence, and use of guselkumab were recorded. Patients were aged ≥ 18 years and had moderate to severe plaque psoriasis. Results The mean disease duration was 18.6 years, and 59% of patients had received at least one biologic treatment before guselkumab with a mean of 1.3 biologics per patient. The initial absolute Psoriasis Area and Severity Index (PASI) was 10.1 and decreased to 2.1 between Week 11–20 without signifcant changes in the PASI value throughout the 90 weeks of followup. The cumulative probability of drug survival was 93.5% at Week 52. No diferences were found in terms of efcacy and survival associated with the of-label drug dosage regimens compared to the doses described in the Summary of Product Characteristics (SmPC). The greatest adjustments in the drug administration regimen were achieved in the subgroups of bionaïve and SR patients, with a reduction in the number of administrations by 40% and 47% compared to the regimen described in the SmPC. Super-response to guselkumab was mainly associated with patients naïve to previous biologic treatment. Conclusion The study demonstrated that of-label use of guselkumab was safe and efective in real-life clinical practice. The fndings suggest that adjustments to the drug administration regimen may be necessary to optimise its use in diferent patient profles, especially in SR and bio-naïve patients. Further studies are needed to confrm these fnding

Effects of Different Levels of Inclusion of Apulo-Calabrese Pig Meat on Microbiological, Physicochemical and Rheological Parameters of Salami during Ripening

This study focused on the characterization of salami produced with meat from different pig breeds. The aim consisted in evaluating the added value of the inclusion of Apulo-Calabrese meat in the production of salami, which was characterized by production until the end of maturation (1, 30, 60, and 120 days). The experimental design involved three types of salami, two of which were produced by partial inclusion of 50 and 75% of the Italian breed pork meat (S50 and S75, respectively). Physicochemical (pH, aw, fatty acid analysis, and malondialdehyde concentration), rheological parameters (texture analyses and color measurement), and bacterial biodiversity were evaluated. Results showed that the partial inclusion of Apulo-Calabrese meat influences the fatty acid profile of final products, which were characterized by a higher percentage of monounsaturated fatty acids compared to traditional salami; however, due to the high content of unsaturated fatty acids, S50 and S75 showed higher values of secondary lipid oxidation up to the 120th day. The linoleic and palmitic acid content significantly affected hardness and brightness. Overall, the ripening process was able to control the microbiological profile and the S50 formulation appeared as a suitable choice that could satisfy consumers for nutritional expectations and sensory profiles

Innovative food safety and hygiene management systems in the agri- food industry

Food sector contributes significantly to the financial statements of the European Union (EU), despite the economic losses caused by the contamination of the foods, which are exposed to various hazards along the food chain. The microbiological contamination plays an important role in the food spoilage and foodborne diseases, causing public health and economic damage. Indeed, in the EU, around 90 million tons of food are wasted annually, with associated burden of microbial spoilage estimated to affect more than a quarter of the total weight. Furthermore, the European Food Safety Authority has attributed to pathogenic bacteria the role of mail culprit agents of human diseases linked to the ingestion of contaminated food. These microorganisms are also partly responsible of the spread of the antimicrobial resistance phenomenon, which has become one of the major global public health concerns. In this context, the general aim of the thesis was to search for molecules capable of replacing the chemical additives and conventional antibiotics, and develop an “end-product” with antimicrobial activity ready to be put on the food market. Ideally, the novel preservatives should have broad spectrum of action, be non-toxic, be active a low concentration, no affect organoleptic properties of food, and have a good ratio cost-effectiveness. Plants and their extracts, and antimicrobial peptides (AMPs) are gaining attention due to their naturalness, high bioavailability, and innumerable antimicrobial properties. In Chapter 1, Loranthus europaeus, well‐known medicinal plant, was chosen to evaluate its potential antimicrobial action. To this aim, different protocols were performed to selectively extract protein compounds, from L. europaeus yellow fruits, and evaluate the antimicrobial activity against four phytopathogenic fungi (Aspergillus niger, Alternaria spp., Penicillium spp., Botrytis cinerea) and a number of foodborne bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus strains, Salmonella Typhimurium and Escherichia coli) by using serial dilutions and colony formation assays. Results evidenced no antifungal activity but a notable bactericidal efficiency of a crude protein extract against two foodborne pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 0.2 and 0.5 mg/mL, being S. aureus strains the most susceptible bacteria. Moreover, a strong bactericidal activity against S. aureus M7 was observed by two partially purified protein fractions of about 600 and 60 kDa molecular mass in native conditions. Therefore, these plant protein extracts could be used as natural alternative preventives to control food poisoning diseases and preserve foodstuff avoiding health hazards of chemically antimicrobial applications. Chapter 2 aimed at evaluating the antimicrobial and antibiofilm activity of the innate defense peptide, named 1018-K6, against Salmonella enterica. A total of 42 strains, belonging to three different subspecies and 32 serotypes, were included in this study. The antibiotic resistance profile of all the strains and the cytotoxic effects of 1018-K6 on mammalian fibroblast cells were also investigated. Results revealed that MIC (minimum inhibitory concentrations) and MBC (minimum bactericidal concentrations) values were in the ranges of 8–64 μg/mL and 16–128 μg/mL, respectively, although most strains (97%) showed MICs between 16 and 32 μg/mL. Moreover, sub- inhibitory concentrations of 1018-K6 strongly reduced the biofilm formation in several S. enterica strains, whatever the initial inoculum size. The results showed that 1018-K6 was able to control and manage S. enterica growth with a large potential for applications in the fields of active packaging and disinfectants. To this purpose, the goal of Chapter 3 was to investigate the effects of a food contact surface of polyethylene terephthalate (PET) functionalized with a previously characterized antimicrobial peptide mitochondrial-targeted peptide 1 (MTP1), in reducing the microbial population related to spoilage and in extending the shelf- life of different types of fresh foods such as ricotta cheese and buffalo meat. Modified polymers were characterized concerning the procedure of plasma-activation by water contact angle measurements and Fourier transform infrared spectroscopy measurements in attenuated total reflection mode (ATR-FTIR). Results showed that the MTP1-PETs provided a strong antimicrobial effect for spoilage microorganisms with no cytotoxicity on human cell line. Finally, the activated polymers revealed high storage stability and good reusability. Chapter 4 aimed to evaluate the efficacy of packaging in polyethylene terephthalate (PET) functionalized with the antimicrobial peptide 1018-K6 to extend the shelf life of fish burgers of dolphinfish (Coryphaena hippurus). At 1, 3, 5 and 7 days, microbiological analyses, pH, aw (activity water) and the sensory analyses were carried out. Sensory results showed that samples packed with functionalized packaging were considered acceptable two days longer than the control group. Microbiological data underlined an evident antimicrobial activity of the active packaging at 5 days against coliforms, Enterobacteriaceae, Pseudomonas spp., E. coli and fecal streptococci, with a reduction of about 1 Log (CFU/g) compared to the control group. The advantage to use natural antimicrobial compounds is also due to their poor link with the common bacterial resistance than conventional drugs. To this purpose, a better comprehension of the mechanisms of action of the antimicrobial peptide is essential for promoting their use in the food industry. Although antimicrobial activity of 1018-K6 towards both Gram-positive and Gram-negative bacteria responsible for foodborne diseases has been extensively demonstrated, the mechanism of action at the molecular level is still unclear. In Chapter 5, mechanistic studies (binding saturation assay, and fluorescence methodologies) were carried out using model membranes and bacterial membranes extracted from pathogenic bacteria isolated in various food matrices. Results reveal that the antimicrobial activity of 1018-K6 is connectable to its affinity for bacterial membranes. The study highlights differences in peptide-lipid interaction and partitioning between mimetic membranes of bacteria, suggesting that peptides have to establish more deeper interactions for binding to Gram- positive membrane that could be favored with arginine (Arg) residues. Moreover, the peptide ability to interact with bacterial mimetic liposomes was confirmed by the formation of vesicular aggregates at ratios of lipid to peptide lower than 80. In conclusion, it is worth pointing out the higher affinity of 1018-K6 for bacterial membranes than those of the respective multilaminar vesicles (MLVs). Thus far, the knowledge gathered on the mechanism of action of this peptide shows a high specificity against anionic liposomes and provides valuable information on the time of action. By calculating that the peptide needs only about 30 minutes or 2 hours of contact to enter in the membranes of Gram- negative and Gram-positive, respectively, the prospect of using 1018-K6 in the formulation of active packaging becomes more concrete. The antimicrobial packaging is offering a viable solution to tackle the economic and safety issues above mentioned, by extending the shelf-life and improving the quality and safety of fresh products. Overall, this thesis provided valuable information to develop alternative antimicrobial packaging for enhancing and extending the microbial quality and safety of perishable foods during storage by using natural compounds

Mitochondrial Analysis of Sparidae Species to Detect a New DNA Barcoding Marker for Dentex gibbosus to Utilize against Fraud

Dentex gibbosus (Pink dentex) is a fish species of increasing economic interest in the Mediterranean Sea that is consumed both whole and processed. The growing value of this sparid in European markets is responsible for its substitution with fraudulent species. The distinctive morphologic feature of D. gibbosus is the conspicuous hump on the forehead in the older and larger specimens. However, the head is regularly convex in young individuals, requiring high skills and competencies for correct identification. Authentication becomes even more challenging in the case of prepared and processed products. Therefore, the molecular characterization of Pink dentex plays a crucial role in preventing commercial fraud with species substitution. This paper proposes a comparative mitogenome analysis between 19 sparid species of commercial interest as a tool to accurately design species-specific primers targeting a fragment of the NAD2 gene for the identification of D. gibbosus. We successfully detected Pink dentex DNA both using endpoint and real-time PCR. The findings showed the high specificity of the designed primers, demonstrating this a suitable, fast, and cost-effective method that could be used for the unambiguous identification of Pink dentex. This innovative approach for sparid authentication is expected to contribute to seafood traceability, public health assurance, integrity, and the credibility of the seafood industry

ANALISI COMPARATIVA DEL MITOGENOMA COMPLETO DI ALCUNE SPECIE DI SPARIDI E VALUTAZIONE DI UN NUOVO MARKER MOLECOLARE PER PAGELLUS ERYTHRINUS

Il Pagello fragolino (Pagellus erythrinus) è una delle specie di Sparidi più pescate e commercializzate nel Mar Mediterraneo e nell’Oceano Atlantico. Tale specie è molto apprezzata nei mercati Europei ed è spesso soggetta a frodi per sostituzione di specie. Le sostituzioni avvengono soprattutto mediante utilizzo di altri Sparidi con differente pregio commerciale. Le sequenze di DNA mitocondriale (mt) maggiormente utilizzate per l'identificazione di specie ittiche sono il citocromo b-CYTB, la citocromo c ossidasi I-COI, i geni che codificano per le subunità ribosomiali 16S e 12S. Ricerche recenti hanno dimostrato che lo studio dell'intero mtDNA consente di identificare nuovi marcatori molecolari specie-specifici e quindi più efficaci. In particolare, il gene NAD5 ha un'elevata capacità discriminatoria per le specie appartenenti alla famiglia Sparidae. Tuttavia, l'utilizzo di tutti questi markers molecolari richiede diversi steps analitici, come l'amplificazione del frammento target ed il sequenziamento. Quest’ultima fase rappresenta un momento critico dell’identificazione di specie, poiché richiede tempi lunghi. Inoltre, tale analisi deve essere effettuata in laboratori altamente specializzati, poiché la lettura errata di pochi nucleotidi può falsare la corretta identificazione della specie. Scopo di questo studio è stato quello di analizzare e confrontare l'intera sequenza di mtDNA di alcune specie di Sparidi al fine di identificare un marcatore molecolare utile per riconoscere la specie P. erythrinus, evitando la fase di sequenziamento. In questo studio sono stati confrontati i mitogenomi di n°13 Sparidi. L’allineamento degli mtDNA è stato condotto mediante l’utilizzo del software UGENE. L'algoritmo Hamming Distance è stato utilizzato per valutare la percentuale di diseguaglianza genetica tra le specie ed i geni. La p-genetic distance è stata valutata utilizzando il Maximum Composite Likelihood model. La variabilità della sequenza nucleotidica è stata determinata allineando le sequenze gene-by-gene utilizzando MEGA 6.0. I primers sono stati disegnati a mano in seguito ad allineamento multiplo delle sequenze complete di mtDNA mediante il software BioEdit Sequence Alignment Editor. L'efficienza dei primers per l'identificazione di P. erythrinus è stata testata mediante PCR end-point. Sono stati testati n°10 campioni di P. erythrinus con differente provenienza geografica e n°30 campioni di filetti appartenenti ad altre specie ittiche. I risultati della Hamming Distance analysis, della valutazione della p-genetic distance e dell’analisi di variabilità della sequenza nucleotidica hanno consentito di identificare il gene NAD2 come potenziale marcatore molecolare per gli Sparidi. La reazione PCR ha confermato la capacità di discriminazione del gene NAD2. In particolare, l'amplificazione di un frammento selezionato del gene NAD2 è stata possibile solo per la specie P. erythrinus. Tale risultato può consentire di verificare la specie senza necessità di sequenziamento, riducendo tempi e costi delle analisi ed aumentando l’efficienza dei risultati. La valutazione di presenza/assenza della specie P. erythrinus può essere ottenuta in poche ore di lavoro di laboratorio. Ulteriori indagini sono in corso al fine di identificare primers specie-specifici anche per altre specie appartenenti alla famiglia degli Sparidi. In accordo con il Regolamento (UE) 1379/2013, questo studio contribuisce alla tracciabilità molecolare dei prodotti della pesca

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide–membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium2GN, Escherichia coli3GN, and Staphylococcus aureus3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics

Effect of Breeding Techniques and Prolonged Post Dry Aging Maturation Process on Biomolecule Levels in Raw Buffalo Meat

Recently, several concerns have been expressed on red meat quality and consumption. The aims of this study were to evaluate the influence of different breeding techniques and a prolonged post dry aging (PDA) maturation process on biomolecules level in raw buffalo meat. In the first experiment, two groups of animals were maintained with different space availability (15 vs. 10 m2/animal) for 90 days and biomolecules content was evaluated. In experiment 2, two diets (with or without ryegrass green forage) were used to assess the concentration of these biomolecules. Finally, in experiment 3, the meat of the animals that showed the highest content of biomolecules was chosen to assess the influence of the PDA maturation process. Buffaloes reared at 15 m2 showed a significantly (p < 0.05) higher content of the considered biomolecules compared with their counterparts. Similarly, buffaloes fed green forage showed higher content of biomolecules (p < 0.05) compared with the control group. The meat of the animals bred at 15 m2 and fed green forage showed a significant (p < 0.01) increase of biomolecules content during the PDA maturation process up to 60 days without influence microbiological profile in terms of total aerobic bacterial counts, yeasts, and molds. In conclusion, breeding techniques and PDA maturation system could enhance biomolecules levels in terms of quality, without affect health standards