Effect of Physicochemical Characteristics and Storage Atmosphere on Microbiological Stability and Shelf-Life of Minimally Processed European Sea Bass (Dicentrarchus labrax) Fillets.

The aim of the present work was to evaluate the effect of various hurdles such as aw and pH as well as the storage atmosphere on the microbiological and sensory changes of minimally processed (lightly brined or marinated with acetic or citric acid) European sea bass (Dicentrarchus labrax) fillets. The results indicated that the shelf-life of brined fillets stored aerobically was 6 d while that of vacuum and MAP-stored was 12 d, since a reduced growth rate of spoilage bacteria was recorded. The physicochemical characteristics such as aw and water phase salt (WPS) were not considerably changed, while the oxygen levels into the packages ensure the microbiological safety of the product. The fillets marinated with acetic acid exhibited a longer shelf-life at 30 and 40 d under aerobic and reduced oxygen conditions, respectively, while the products marinated with citric acid were at 25 and 35 d respectively. A low pH resulted in reduced or even limited microbial levels, especially for the fillets marinated with acetic acid; something that ensures microbiological safety as well. The low or limited microbial levels in conjugation with the sensory attributes indicated that spoilage may be due to other mechanisms such as autolysis rather than microbial activity. Overall, the present work highlights the potential for further research and development of minimally processed, microbiologically safe and stable with extended shelf-life value added seafood to satisfy the corresponding consumer demands.

Microbiota profile of filleted gilthead seabream (Sparus aurata) during storage at various conditions by 16S rRNA metabarcoding analysis.

The aim of the present work was to study the microbiota profile of gilthead seabream (Sparus aurata) fillets stored either aerobically or under Modified Atmosphere Packaging (MAP) conditions at 0, 4, 8 and 12 °C, via 16S rRNA metabarcoding sequencing. Throughout storage, sensory assessment was also applied to estimate fillets' end of shelf-life. Results indicated that storage conditions strongly influenced the shelf-life of the fillets, since the sensorial attributes of air-stored samples deteriorated earlier than that of MAP-stored fillets, while higher temperatures also contributed to a more rapid products' end of shelf-life. Metataxonomic analysis indicated that Pseudomonas was by far the dominant genus at the end of fillet's shelf-life, in the vast majority of treatments, even though a sporadic but noteworthy presence of other genera (e.g, Shewanella, Carnobacterium, Brochothrix etc.) at the middle stages of MAP-stored fillets is also worth mentioning. On the other hand, a completely different profile as well as a more abundant bacterial diversity was observed at the end of shelf-life of MAP-stored fillets at 12 °C, in which Serratia was the most dominant bacterium, followed by Kluyvera, Hafnia, Rahnella and Raoultella, while Pseudomonas was detected in traces. The findings of the present work are very important, providing useful information about the spoilage status of gilthead seabream fillets during several storage conditions, triggering in parallel the need for further studies to enrich the current knowledge and help stakeholders develop innovative strategies that delay the growth of key spoiler players and consequently, retard spoilage course.

Growth and Volatile Organic Compound Production of Pseudomonas Fish Spoiler Strains on Fish Juice Agar Model Substrate at Different Temperatures.

Microbial spoilage is the main cause of quality deterioration in seafood. Several strains of psychotropic Pseudomonas have been found to dominate in such products, producing a plethora of volatile organic compounds (VOC). Herein, we investigated the growth of and VOC production by seven strains of Pseudomonas associated with spoiled fish after inoculation as single and mixed cultures on model fish substrate and storage at 0, 4 and 8 °C. The results indicated a strain-dependent VOC profile that was also affected by the storage temperature. Hierarchical cluster analysis (HCA) successfully grouped the strains based on VOC profile at each studied temperature, while some potential Chemical Spoilage Indices (CSI) were revealed. The findings of the present work will contribute to the understanding of the metabolic activity of particular strains of Pseudomonas and to reveal any potential CSI for rapid evaluation of fish spoilage/freshness status.

Bacterial Communities and Antibiotic Resistance of Potential Pathogens Involved in Food Safety and Public Health in Fish and Water of Lake Karla, Thessaly, Greece.

Bacterial communities, microbial populations, and antibiotic resistance of potential pathogens in the water and fish (Cyprinus carpio, flesh and gut) from different areas (A1, A2 and A3-A1 was linked with river water, A2 with cattle activity, and A3 with waters of a spring after heavy rains) of Lake Karla (Thessaly, Central Greece) were investigated. The isolated bacteria were identified using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and were tested for resistance in 21 antibiotics. The microbiota composition of fish flesh was also studied using 16S amplicon-based sequencing Serratia fonticola and several species of Aeromonas (e.g., Aeromonas salmonicida, Aeromonas bestiarium, Aeromonas veronii, etc.) exhibited the highest abundances in all studied samples, while the microbiota profile between the three studied areas was similar, according to the culture-dependent analysis. Of them, S. fonticola was found to be resistant in the majority of the antibiotics for the water and fish (gut and flesh), mainly of the areas A1 and A2. Regarding 16S metabarcoding, the presence of Serratia and Aeromonas at genus level was confirmed, but they found at very lower abundances than those reported using the culture-dependent analysis. Finally, the TVC and the rest of the studied microbiological parameters were found at acceptable levels (4 log cfu/mL or cfu/g and 2-4 log cfu/mL or cfu/g, extremely low levels of E. coli/coliforms) in both water and fish flesh. Based on our findings, the water of Lake Karla would be used for activities such as irrigation, recreation and fishing, however, the development and implementation of a quality management tool for Lake Karla, to ensure environmental hygiene and prevention of zoonosis during the whole year, is imperative.

Microbiota Succession of Whole and Filleted European Sea Bass (Dicentrarchus labrax) during Storage under Aerobic and MAP Conditions via 16S rRNA Gene High-Throughput Sequencing Approach.

In the present work, the profiles of bacterial communities of whole and filleted European sea bass (Dicentrarchus labrax), during several storage temperatures (0, 4, 8 and 12 °C) under aerobic and Modified Atmosphere Packaging (MAP) conditions, were examined via the 16S rRNA High-Throughput Sequencing (HTS) approach. Sensorial attributes were also assessed to determine products' shelf-life. Results indicated that shelf-life was strongly dependent on handling, as well as on temperature and atmosphere conditions. HTS revealed the undisputed dominance of Pseudomonas from the very beginning and throughout storage period in the majority of treatments. However, a slightly different microbiota profile was recorded in MAP-stored fillets at the middle stages of storage, which mainly referred to the sporadic appearance of some bacteria (e.g., Carnobacterium, Shewanella, etc.) that followed the dominance of Pseudomonas. It is noticeable that a major difference was observed at the end of shelf-life of MAP-stored fillets at 12 °C, where the dominant microbiota was constituted by the genus Serratia, while the relative abundance of Pseudomonas and Brochothrix was more limited. Furthermore, at the same temperature under aerobic storage of both whole and filleted fish, Pseudomonas almost co-existed with Acinetobacter, while the presence of both Erwinia and Serratia in whole fish was noteworthy. Overall, the present study provides useful information regarding the storage fate and spoilage status of whole and filleted European sea bass, suggesting that different handling and storage conditions influence the shelf-life of sea bass by favoring or delaying the dominance of Specific Spoilage Organisms (SSOs), affecting in parallel to some extent the formation of their consortium that is responsible for products' sensorial deterioration. Such findings enrich the current knowledge and should be used as a benchmark to develop specific strategies aiming to delay spoilage and thus increase the products' added value.

Volatile Organic Compounds and 16S Metabarcoding in Ice-Stored Red Seabream Pagrus major.

The profiles of bacterial communities and volatile organic compounds (VOCs) of farmed red seabream (Pagrus major) from two batches during ice storage were studied using 16S metabarcoding (culture independent approach) and headspace Solid Phase Micro-Extraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS) analysis, respectively. Sensory attributes and microbiological parameters were also evaluated. At Day 12 (shelf-life for both batches based on sensory evaluation), using classical microbiological analysis, Total Viable Counts (TVC) were found at the levels of 7-8 log cfu/g, and Pseudomonas and/or H2S producing bacteria dominated. On the other hand, the culture independent 16S metabarcoding analysis showed that Psychrobacter were the most abundant bacteria in fish tissue from batch 1, while Pseudomonas and Psychrobacter (at lower abundance) were the most abundant in fish from batch 2. Differences were also observed in VOC profiles between the two batches. However, combining the VOC results of the two batches, 15 compounds were found to present a similar trend during fish storage. Of them, 2-methylbutanal, 3-methylbutanal, 3-methyl-1-butanol, ethanol, 2,4 octadiene (2 isomers), ethyl lactate, acetaldehyde and (E)-2-penten-1-ol could be used as potential spoilage markers of red seabream because they increased during storage, mainly due to Psychrobacter and/or Pseudomonas activity and/or chemical activity (e.g., oxidation). Additionally, VOCs such as propanoic acid, nonanoic acid, decanoic acid, 1-propanol, 3,4-hexanediol and hexane decreased gradually with time, so they could be proposed as freshness markers of red seabream. Such information will be used to develop intelligent approaches for the rapid evaluation of spoilage course in red seabream during ice storage.

Fermented table olives from Cyprus: Microbiota profile of three varieties from different regions through metabarcoding sequencing.

The knowledge about the microbial diversity of different olives varieties from diverse regions in the Mediterranean basin is limited. This work aimed to determine the microbial diversity of three different fermented olive varieties, collected from different regions in Cyprus, via Next Generation Sequencing (NGS) analysis. Olives were spontaneously fermented for 120 days, microbial DNA was extracted from the final products, and subjected to 16S rRNA gene and ITS1 loci metabarcoding analysis for the determination of bacterial and fungal communities, respectively. Results revealed that the bacterial profile of the studied varieties was similar, while no noteworthy differences were observed in olives from different regions. The bacterial profile was dominated by the co-existence of Lactobacillus and Streptococcus, while the genera Lactococcus and Salinivibrio and the family Leuconostocaceae were also present in increased relative abundances. Regarding fungal communities, the analysis indicated discrimination among the different varieties, especially in Kalamata ones. The most abundant fungi were mainly the genera Aspergillus, Botryosphaeria, Meyerozyma, and Zygosaccharomyces for Cypriot olives, the genera Botryosphaeria, Saccharomyces, Geosmithia, and Wickeromyces for Kalamata variety, while the dominant fungi in the Picual variety were mainly members of the genera Candida, Penicillium, Saccharomyces, Hanseniospora and Botryosphaeria. Potential microbial biomarkers that distinguish the three varieties are also proposed. Moreover, interaction networks analysis identified interactions among the key taxa of the communities. Overall, the present work provides useful information and sheds light on an understudied field, such as the comparison of microbiota profiles of different varieties from several regions in Cyprus. The study enriches our knowledge and highlights the similarities and the main differences between those aspects, booming in parallel the need for further works on this frontier, in the attempt to determine potentially olives' microbial terroir in Cyprus. Our work should be used as a benchmark for future works in this direction.

Spoilage Investigation of Chill Stored Meagre (Argyrosomus regius) Using Modern Microbiological and Analytical Techniques.

Spoilage status of whole and filleted chill-stored meagre caught in January and July was evaluated using sensory, microbiological, 16S metabarcoding and Volatile Organic Compounds (VOCs) analysis. Based on the sensory analysis, shelf-life was 15 and 12 days for the whole fish taken in January and July, respectively, while 7 days for fish fillets of both months. For the whole fish, Total Viable Counts (TVC) at the beginning of storage was 2.90 and 4.73 log cfu/g for fish caught in January and July respectively, while it was found about 3 log cfu/g in fish fillets of both months. The 16S metabarcoding analysis showed different profiles between the two seasons throughout the storage. Pseudomonas (47%) and Psychrobacter (42.5%) dominated in whole meagre of January, while Pseudomonas (66.6%) and Shewanella (10.5%) dominated in fish of July, at the end of shelf-life. Regarding the fillets, Pseudomonas clearly dominated at the end of shelf-life for both months. The volatile profile of meagre was predominated by alcohols and carbonyl compounds. After univariate and multivariate testing, we observed one group of compounds (trimethylamine, 3-methylbutanoic acid, 3-methyl-1-butanol) positively correlating with time of storage and another group with a declining trend (such as heptanal and octanal). Furthermore, the volatile profile seemed to be affected by the fish culturing season. Our findings provide insights into the spoilage mechanism and give information that helps stakeholders to supply meagre products of a high-quality level in national and international commerce.

Cyprus Sausages' Bacterial Community Identification Through Metataxonomic Sequencing: Evaluation of the Impact of Different DNA Extraction Protocols on the Sausages' Microbial Diversity Representation.

Cyprus traditional sausages from the Troodos mountainous region of Pitsilia gained the protected geographical indication (PGI) designation from the European Committee (EU 2020/C 203/06). Still, we lack authentication protocols for the distinction of "Pitsilia" from industrially produced Cyprus sausages. Microbial activity is an essential contributor to traditional sausages' sensorial characteristics, but whether the microbial patterns might be associated with the area of production is unclear. In the present research, we applied high-throughput sequencing (HTS) to provide a linkage between the area of production and Cyprus sausages' bacterial diversity. To strengthen our findings, we used three different DNA extraction commercial kits: (i) the DNeasy PowerFood Microbial Kit (QIAGEN); (ii) the NucleoSpin Food Kit (MACHEREY-NAGEL); and (iii) the blackPREP Food DNA I Kit (Analytik Jena), in which we applied three different microbial cell wall lysis modifications. The modifications included heat treatment, bead beating, and enzymatic treatment. Results regarding metagenomic sequencing were evaluated in terms of number of reads, alpha diversity indexes, and taxonomic composition. The efficacy of each method of DNA isolation was assessed quantitatively based on the extracted DNA yield and the obtained copy number of (a) the 16S rRNA gene, (b) the internal transcribed spacer (ITS) region, and (c) three Gram-positive bacteria that belong to the genera Latilactobacillus (formerly Lactobacillus), Bacillus, and Enterococcus via absolute quantification using qPCR. Compared with some examined industrial sausages, Pitsilia sausages had significantly higher bacterial alpha diversity (Shannon and Simpson indexes). Principal coordinates analysis separated the total bacterial community composition (beta diversity) of the three Pitsilia sausages from the industrial sausages, with the exception of one industrial sausage produced in Pitsilia, according to the manufacturer. Although the eight sausages shared the abundant bacterial taxa based on 16S rDNA HTS, we observed differences associated with bacterial diversity representation and specific genera. The findings indicate that the microbial communities may be used as an additional tool for identifying of the authenticity of Cypriot sausages.

Current Status, Recent Advances, and Main Challenges on Table Olive Fermentation: The Present Meets the Future.

Table olives are among the most well-known fermented foods, being a vital part of the Mediterranean pyramid diet. They constitute a noteworthy economic factor for the producing countries since both their production and consumption are exponentially increasing year by year, worldwide. Despite its significance, olive's processing is still craft based, not changed since antiquity, leading to the production of an unstable final product with potential risk concerns, especially related to deterioration. However, based on industrial needs and market demands for reproducible, safe, and healthy products, the modernization of olive fermentation processing is the most important challenge of the current decade. In this sense, the reduction of sodium content and more importantly the use of suitable starter cultures, exhibiting both technological and potential probiotic features, to drive the process may extremely contribute to this need. Prior, to achieve in this effort, the full understanding of table olive microbial ecology during fermentation, including an in-depth determination of microbiota presence and/or dominance and its functionality (genes responsible for metabolite production) that shape the sensorial characteristics of the final product, is a pre-requisite. The advent of meta-omics technology could provide a thorough study of this complex ecosystem, opening in parallel new insights in the field, such as the concept of microbial terroir. Herein, we provide an updated overview in the field of olive fermentation, pointing out some important challenges/perspectives that could be the key to the olive sector's advancement and modernization.

Evolution of Bacterial Communities, Physicochemical Changes and Sensorial Attributes of Natural Whole and Cracked Picual Table Olives During Spontaneous and Inoculated Fermentation.

Table olives are one of the most well-known traditionally fermented products, and their global consumption is exponentially increasing. In direct brining, table olives are produced spontaneously, without any debittering pre-treatment. Up to date, fermentation process remains empirical and inconstant, as it is affected by the physicochemical attributes of the fruit, tree and fruit management of pro and post-harvest. In the present study, whole and cracked Picual table olives were fermented at industrial scale for 120 days, using three distinct methods (natural fermentation, inoculation with lactic acid bacteria (LAB) at a 7 or a 10% NaCl concentration). Microbial, physicochemical and sensorial alterations monitored during the whole process, and several differences were observed between treatments. Results indicated that in all treatments, the dominant microflora were LAB. Yeasts also detected in noteworthy populations, especially in non-inoculated samples. However, LAB population was significantly higher in inoculated compared to non-inoculated samples. Microbial profiles identified by metagenomic approach showed meaningful differences between spontaneous and inoculated treatments. As a result, the profound dominance of starter culture had a severe effect on olives fermentation, resulting in lower pH and higher acidification, which was mainly caused by the higher levels of lactic acid produced. Furthermore, the elimination of Enterobacteriaceae was shortened, even at lower salt concentration. Although no effect observed concerning the quantitated organoleptic parameters such as color and texture, significantly higher levels in terms of antioxidant capacity were recorded in inoculated samples. At the same time, the degradation time of oleuropein was shortened, leading to the production of higher levels of hydroxytyrosol. Based on this evidence, the establishment of starter culture driven Picual olives fermentation is strongly recommended. It is crucial to mention that the inoculated treatment with reducing sodium content was highly appreciated by the sensory panel, enhancing the hypothesis that the production of Picual table olives at reduced NaCl levels is achievable.

Benefits of the Use of Lactic Acid Bacteria Starter in Green Cracked Cypriot Table Olives Fermentation.

Table olives are one of the most established Mediterranean vegetables, having an exponential increase consumption year by year. In the natural-style processing, olives are produced by spontaneous fermentation, without any chemical debittering. This natural fermentation process remains empirical and variable since it is strongly influenced by physicochemical parameters and microorganism presence in olive drupes. In the present work, Cypriot green cracked table olives were processed directly in brine (natural olives), using three distinct methods: spontaneous fermentation, inoculation with lactic acid bacteria at a 7% or a 10% NaCl concentration. Sensory, physicochemical, and microbiological alterations were monitored at intervals, and major differences were detected across treatments. Results indicated that the predominant microorganisms in the inoculated treatments were lactic acid bacteria, while yeasts predominated in control. As a consequence, starter culture contributed to a crucial effect on olives fermentation, leading to faster acidification and lower pH. This was attributed to a successful lactic acid fermentation, contrasting the acetic and alcoholic fermentation observed in control. Furthermore, it was established that inhibition of enterobacteria growth was achieved in a shorter period and at a significantly lower salt concentration, compared to the spontaneous fermentation. Even though no significant variances were detected in terms of the total phenolic content and antioxidant capacity, the degradation of oleuropein was achieved faster in inoculated treatments, thus, producing higher levels of hydroxytyrosol. Notably, the reduction of salt concentration, in combination with the use of starter, accented novel organoleptic characteristics in the final product, as confirmed from a sensory panel; hence, it becomes obvious that the production of Cypriot table olives at reduced NaCl levels is feasible.