Integrating omics technologies for improved quality and safety of seafood products

Dborah M Powr, Ptros Taoukis, Dimitra Houhoula, Thoania Tsironi,Emmanouil Flmtakis

a Comparative Endocrinology and Integrative Biology Group, Centro de Ciencias do Mar, University of Algarve, Faro, Portugal

b International Institution of Marine Science, Shanghai Ocean University, Shanghai, China

c Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Greece

d University of West Attica, School of Food Sciences, Department of Food Science & Technology, Greece

e Agricultural University of Athens, Department of Food Science and Human Nutrition, Laboratory of Food Process Engineering, Greece

f Agricultural University of Athens, Department of Biotechnology, Laboratory of Molecular Biology, Greece

Keywords:Microbiome Proteome Fish Microalgae Seafood Quality Molecular methods

ABSTRACT An essential aspect of product quality of aquatic foods is the rapid and accurate identification of bacterial species.From this perspective omics technologies prove to be very useful in the assessment of the quality and safety of seafood products.Such technologies can identify and detect low levels of contamination by pathogenic and spoilage bacteria and can be used to study the effects caused by processing and storage of seafood products.The integration of food processing with the monitoring of the microbial characteristics using conventional microbiological assays, coupled to molecular techniques may establish the baseline for the development of quicker and more sensitive and reliable methods for seafood safety screening.The use of combined omics technologies,including metagenomics, proteomics and metabolomics, coupled to conventional quality indices such as colour,texture and flavour offer a new tool for novel processing optimization to ensure seafood quality.The aim of this brief review is to outline how omics technologies can generate novel tools for integration into seafood processing and quality control.Considering that the main aspect of the review is the improvement of safety and quality of the final product, from production to consumption, emphasis is given to microbial identification and metabolite detection, the evaluation of the allergenic capacity of fish and seafood and optimization of postharvest processing.Deployment of omics for identification of potential microalgal products of relevance to seafood quality and safety is also considered.

1.From Omics to Foodomics

Food quality and safety are critical components to maintaining a safe and economically sustainable food supply chain, and thus improve food security across the world (WHO, 2015).The globalization of the market for food has raised interest in issues linked to food authenticity and safety, especially in the case of imported food products.Consumers are currently sceptical about processed foods and the potential alterations that occur to food during production at an industrial level and during distribution (Tsironi et al., 2021).Consumer awareness regarding food quality and safety is continuously increasing, resulting in exhaustive labelling, and reporting of details in accordance with current legislation and regulatory restrictions (Ferri et al., 2015).

In line with the current trend for quality monitoring using more sophisticated and biology-based techniques alongside traditional analysis methods, considerable research is focussed on the identification of novel markers that can potentially be used for quality assessment and microbiological spoilage fingerprints using novel omics technologies.Omics tools (i.e.genomics, transcriptomics, proteomics and metabolomics) have been developing with the recent technology revolution.The increased use of omics in life sciences has promoted a rapid advance in applications and their validation and recent significant advances in technologies and the reduction in cost and complexity of analysis have brought them into the mainstream (Den Besten et al., 2018; Ferri et al.,2015).Part of this revolution has been the development of robust tools to gain insight into microbial communities along the food supply chain and their implications for human and animal health (Cook & Nightingale, 2018; Davies, 2010).The termFoodomicshas been defined as “a discipline that studies the Food and Nutrition domains through the application of omics technologies” (Cifuentes, 2009; Ellis, 2019).

The starting point for expanding these omics techniques, to provide robust descriptors of the target system under investigation, leverages concepts from the central biological dogma: (1) DNA replication, (2)transcription to mRNA, and (3) translation to protein.The development of molecular fingerprints and patterns associated with foodstuffs may provide a more in-depth insight into how the application of conventional and novel food processing technologies’ affect microorganisms, product structure, colour and texture and enzymatic alterations all of which are related to food quality (Cook & Nightingale, 2018).Omics technologies prove to be very useful in the assessment of the quality of perishable food products, through the identification and detection of pathogenic and spoilage bacteria and the evaluation of the effects caused by processing and storage on food proteins, carbohydrates, and fats in different food products (Carrera et al., 2013; Ferri et al., 2015).

Global food demand is rising and further expanding land-based food production is fraught with environmental and health concerns.As seafood is nutritionally diverse and avoids several of the environmental burdens of terrestrial food production, it is uniquely positioned to contribute to both food provision and future global food and nutrition security (Costello et al., 2020).The seafood industry and consequently the challenge of feeding the world population, has experienced unprecedented breakthroughs through blue biotechnology (Vieira et al.,2020).The demand for seafood products is growing significantly while the wild capture resources have become limited.Currently, aquaculture provides more than 50% of all the aquatic food consumed and this percentage keeps rising (FAO, 2016).To meet the current and future global demand for high-quality, sustainable aquatic food products, the world needs to transition quickly towards more diversified production systems and species.Simultaneously, the search for technological innovations needs to be intensified since they can boost food production and quality while protecting the environment.High value, niche species and raw materials based on aquatic organisms have great potential to improve sector productivity, since they can expand the scope and dimension of the sector without competing with the existing well-established production models.In addition, novel foods are being developed and produced, utilizing low-trophic marine sources such as microalgae, as alternative raw materials.New product development and utilization of novel protein sources from aquatic organisms for human consumption, raises the need to develop improved, more accurate analytical techniques and to establish sensitive and cost-effective screening methods, to evaluate the quality of aquatic food products at all stages of the supply chain (Costello et al., 2020; Draaisma et al.,2013).

The aim of this article is to provide an overview of how omics technologies can be integrated into fish and microalgae quality monitoring by identifying and validating novel markers for quality assessment and microbiological spoilage fingerprints.

2.Omics for microbial identification and metabolite detection in fish and seafood

An essential aspect of fish and seafood quality is the rapid and accurate identification of bacterial species (Gram & Dalgaard, 2002).Up until the last decade, the evaluation of fish and seafood microbiota was predominantly carried out by phenotypic tests (morphological,biochemical) on the isolated microorganisms, using specific non-selective or selective growth media.This means that the conventional methods for the determination of microbiota in a specific food product only targeted species when the potential bacterial contamination was already defined or expected in advance.The reason for the limitations in the scope of microbiota detected with the conventional methods is that the discriminatory analytical methods for their identification must be specifically tailored.Currently, the conventional phenotypic tests for microbial identification have been replaced by or accompanied with (when viable cells need to be enumerated) genotypic bacterial identification methods.These tests include DNA-based methods, such as analysis of 16S-23S rDNA intergenic spacer region polymorphism and examination of amplified-fragment length polymorphism (Laursen et al., 2005; Woo et al., 2008).

Recently, novel omics technologies, such as Next Generation Sequencing (NGS), have shown the advantage of DNA analysis methods,combining sequencing and quantification of DNA within a relatively short period of time (Nogueira & Botelho, 2021).NGS techniques undoubtedly represent a step change in the way microbiologists address microbial ecology and diversity in foods (Cocolin et al., 2018).Table 1 summarises examples of applications of omics techniques for quality evaluation of fish and seafood products.Tsironi, Lougovois, et al.(2019)assessed the microbial ecology of fish, such as Atlantic salmon (Salmo salar), albacore tuna (Thunus alalunga), European anchovy (Engraulis encrasicolus), chub mackerel (Scomber japonicus), Atlantic mackerel,(Scomber scombrus), European pilchard (Sardina pilchardus), grey mullet(Mugil cephalus), European hake (Merluccius merluccius), gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), picarel(Spicara smaris), comber (Serranus cabrilla), dentex (Dentex macrophthalmus) and striped red mullet (Mullus surmuletus), displayed in the Greek market within the spring and summer of 2016, using NGS and correlated the species represented in the microbiota with the production of histamine in the case of scombroid fish species.The main pathogenic bacterial species identified in the tested fish samples includedVibriospp.,Clostridiumspp.,Staphylococcus,FlavobacteriumandJanthinobacterium, which are representative of native freshwater habitats and contaminants arising from sources such as sewage, wild animals, livestock, and feed.The initial spoilage microbiota of fish consisted of several psychrotrophic Gram-negative bacteria, mainlyPseudomonas,Acinetobacter,Moraxella,Shewanella,Psychrobacter,Lactobacillus,BrochothrixandPhotobacterium.The results of the study showed the potential and the usefulness of NGS for the determination of microbiota associated with food-borne diseases and spoilage in fish products.The study by Tsironi, Lougovois, et al.(2019) was the first systematic evaluation of the microbiota of the edible part of fish in a public market using NGS.However, further research is needed into the effect of storage conditions on the profile of the microbiota of fish and seafood and bacteria - bacteria and food - bacteria interactions during storage.This would enable a better understanding of the spoilage mechanisms of fish and seafood under different processing, packaging, and storage conditions.Tsironi, Lougovois et al., (2019) also showed that histamine formation in scombroid fish was correlated with the concentration and number of bacteria, (as determined by a combination of molecular and conventional culture-based techniques) and to a lesser extent with the genus of the identified microorganisms.Low concentrations of histamine in certain fish species were attributed to either a low free-histidine level in the fish flesh or low microbial counts of microorganisms such asMorganella morganii,Raoultellaspp.andPhotobacterium phosphoreum.The results available to date suggest that the NGS approach may enable the selection of appropriate biomarkers for preventing foodborne poisoning outbreaks, and thus contribute to reduce fish product recalls and improve consumer confidence (Kumar et al., 2020).

The total cultivable microbiota of ice-stored European sea bass(Dicentrarchus labrax) has been reported using 16S rRNA gene sequence analysis (Syropoulou et al., 2020).High Resolution Melting (HRM)analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles.The ten groups consisted of representatives ofPsychrobacter glacincola,Ps.alimentarius,Ps.cryohalolentis,Ps.maritimus,Ps.fozii,Pseudomonassp.,Paeniglutamicibactersp.,Carnobacteriumsp.,Leucobacter aridicolisandBacillus thuringiensis.Based on this approach,Ps.cryohalolentiswas foundto be the most dominant phylotype at the beginning of the sea bass shelf life compared to other bacterial species.The abundance of this bacterium decreased throughout storage, whilePs.glacincolaincreased and dominated when sensory minimums of acceptability and rejection were attained.

Table 1 Examples of omics tools for quality evaluation of aquatic products.

Table 1 (continued)

The 16S rRNA gene sequencing analysis was used by Vogel et al.(2005) for the identification of the dominant species in iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time.More than 500 H2S-producing strains were isolated, which were identified asShewanellaspecies by phenotypic tests.DifferentShewanellaspecies were present on newly caught fish.During the warm summer months, the mesophilic human pathogenicS.algaedominated the H2S-producing bacterial population.After iced storage, a shift in theShewanellaspecies was reported and most of the H2S-producing strains were identified asS.baltica.

The “volatilome”, and more specifically detected microbial metabolites, are proposed as a promising tool for spoilage monitoring of modified atmosphere packed sea bass fillets and Atlantic salmon slices(Kritikos et al., 2020).In the case of thevolatilome, spoilage was monitored by determining the metabolites produced by the main spoilage microorganisms, and an acceptable correlation was observed with microbial growth.According to the aforementioned study, further research is needed to define product-specific volatilomes.Consequently, there is a need to establish potential volatile spoilage markers, that are quantifiable and species specific under specified storage conditions.An alternative approach could be the exploration of the overall volatilome through multivariate data analysis coupled to the development of a database with the volatile spoilage fingerprint of each fish species under specific storage conditions.A GC-MS metabolomic approach has been proposed by Mallouchos et al.(2010) as an efficient tool to monitor quality loss of gilthead sea bream during storage on ice.The results of the study indicated a set of hydrophilic metabolites linked directly to the storage time of the fish that could be used as potential markers of freshness and spoilage.With the application of multivariate data analysis, the gilthead seabream samples were successfully classified into freshness grades, and the K-value was predicted using a PLS-R model.

3.Omics for the assessment of allergenic hazards in fish and seafood

Food allergens are proteins or glycoproteins (mainly 10–80 kDa)generally resistant to digestive enzymes in the gastrointestinal tract and to thermal processing.Due to the complexity of food matrices, it is very difficult to standardize crude food allergen extracts which may vary in allergen content, and this is a cause of the limited sensitivity of conventional diagnostic procedures for allergen detection (Andjelkovi´c et al., 2017).For seafood products, allergens that provoke a major allergenic response are β-parvalbumin, tropomyosin and arginine kinase, and these proteins are frequently the cause of immunological and clinical responses (L´opez-Pedrouso et al., 2020; Ruethers et al., 2018).Food legislation requires detailed declarations of potential allergens in food products and therefore an increased capability to analyze for the presence of food allergens.Currently, antibody-based methods are mainly utilized to quantify allergens.However, these methods have several disadvantages.(Koeberl et al., 2014).Recently, mathematical models have been built based on genomic, transcriptomic and proteomic data to predict or discriminate disease phenotypes, and to describe the biomolecular networks behind allergies (Tang et al., 2020).

Several proteomics approaches, involving the high-throughput analysis of the proteins of a specific biological sample, have been recently reported as tools to provide the characterization of some of the principal issues associated with fish farming conditions (Carrera et al.,2013 and 2018; L´opez-Pedrouso et al., 2020).Proteomics is a promising approach to address some of the main challenges in aquaculture, such as dietary management, fish welfare, the stress response, food safety and antibiotic resistance.Several publications suggest that the composition of fish feeds influences the nutritional value and the proteome of fish muscle (Carrera et al., 2020; Raposo de Magalh˜aes et al., 2018).Efforts have been made to discover protein markers for such quality traits (Un Nissa et al., 2021).Currently, innovative fast targeted proteomics workflows have demonstrated their usefulness for the rapid detection of fish allergens, parasites and microorganisms in aquaculture (Carrera et al., 2020; Gamage et al., 2022; L´opez-Pedrouso et al., 2020).Another approach has been reported by Abdelmoteleb et al.(2021), where potential proteins from three novel food sources (Chlorella variabilis,Galdieria sulphuraria, andFusariumstrain flavolapis) were predicted from genomic sequences and were evaluated for potential risks of allergic cross-reactivity by comparing the predicted amino acid sequences against allergens in the www.AllergenOnline.org database.Based on this study, changes in the allergen databases or methods of identifying matches for risk evaluation of new food sources were strongly recommended.

2D gel electrophoresis has been also introduced by Tomm et al.(2013) for the identification of species-specific allergens, together with individual sensitization, as a means to investigate the immunoglobulin(Ig) E-reactive proteins that may be allergens in Nile perch (Lates niloticus).

Shotgun proteomics has been successfully applied for the molecular characterization of β-cell epitopes for parvalbumin in several fish species, such as white seabream, hake, salmon, sole, yellowfin tuna and mackerel (Carrera et al., 2019).Mass spectrometry (MS) methods have been introduced and used for the evaluation of food allergenicity.Koeberl et al.(2014) described 46 allergens from 11 different food sources, which were characterized using different MS techniques, based on relevant literature.Apart from this study, the quantification of allergens using MS has been not extensively reported.When MS analysis included multiple reaction monitoring, low limits of quantification for multiple allergens were reported (Zhang et al., 2012).MS followed by SDS-PAGE and subsequent immunoblotting with antibodies detected 4 fish allergens (i.e.parvalbumin, tropomyosin, aldolase and collagen) in cod, flounder, hake, herring, mackerel, tuna, trout and salmon (Ruethers et al., 2019).LC-MS/MS has also been reported as an adequate proteomic approach for the determination of superoxide dismutase, troponin C, aldolase А and thioredoxin h in macroalgaeUlvasp.(Polikovsky et al.,2019).These novel techniques have been reported as high-throughput methods, appropriate for the investigation and identification of new isoforms of allergens in different seafood matrixes (Irizar et al., 2021).

4.Omics for fish processing optimization

Progress toward a more sustainable, more secure, safer food system cannot be envisaged without novel food processing technologies.On the other hand, the food industry is aware of how important perceived freshness/naturalness is for consumers, seeking high quality food, with enhanced nutritional value and an extended shelf life while at the same time retaining fresh-like sensory characteristics (Siegrist & Hartmann,2020).In order to manage the perceived naturalness of food, quality improvement and shelf-life extension, two parallel actions need to be implemented, i.e.design and application of innovative, minimal processing technologies of seafood and development of novel analytical techniques for effective and rapid food quality monitoring during processing.The integration of food processing procedures with the monitoring of the microbial characteristics using conventional microbiological assays, coupled to molecular techniques may establish the baseline for the development of quicker, predictive and more sensitive methods for seafood quality and safety screening (Bagge-Ravn et al., 2003).

Omics data may be integrated with processing data and the results of conventional analysis (eg.microbial plate counts, physico-chemical parameters etc) to generate novel targeted tools for quality monitoring of fish along the supply chain.More specifically, the development and application of proteomics analytical tools and resources may result in more efficient and tailored approaches of quality monitoring for fish and seafood.Within the scope of the ERA-NET COFASP project SUSHIFISH(https://www.sushifish.eu, 2016-2019) was the evaluation of the effect of high pressure (HP) processing on fish flesh using microbiological,physicochemical and sensory indices coupled to omics technologies,with the aim of defining optimal processing conditions for the tested fish products.The target fish product was the European sea bass (Dicentrarchus labrax).In this systematic study, omics technologies proved to be very useful in the assessment of the quality of fish, by the identification and detection of pathogenic and spoilage bacteria and the study of the effects on proteins in seafood products caused by processing and storage.Based on the data reported by Tsironi, Anjos, et al.(2019) and Anjos et al.(2019), it was concluded that the metagenomics and proteomics results corroborated and explained the reduced microbial growth and the physico-chemical and organoleptic degradation of the stored fillets.The conclusions of the study were based on integration of the hierarchical clustering of samples according to their microbiome composition and proteomic data obtained from Sequential Windowed data independent Acquisition of the Total High-resolution-Mass Spectra(SWATH-MS).Overall, the results revealed the complementarity and value of allying and streamlining conventional and omics approaches.The potential of omics for the generation of new monitoring tools for fish processing and quality was demonstrated for fresh and for high pressure treated European seabass fillets stored at 2◦C.The obtained data can be used in the future for the optimization of HP processing of fish and the selection of appropriate HP conditions for quality improvement and shelf-life extension of whole and filleted fish.Future experiments may be directed at the identification of markers correlated with efficacy of HPP and product quality.

The MCSA-RISE ICHTHYS project (https://www.ichthys-eu.org,2020-2025) aims to integrate omics technologies into fish and seafood quality monitoring by identifying and validating novel markers for quality assessment and microbiological spoilage fingerprints.The tools generated will form the basis of sensitive and cost-effective screening and contribute to the development of a new generation of smart packaging systems.

Increased data analysis skills through machine learning and implementation of Artificial intelligence (AI) technology may enable the large-scale evaluation of microbial composition, gene content, gene function, functional activity and metabolites and result in effective correlations.The huge amount of data generated through a multi-omics approach will contribute to extend knowledge and understanding of complex food processing technologies and lead to refinement and improvement of such technologies.Optimized protocols for bacterial inactivation linked to minimal protein modifications to deliver minimally processed foods that retain their “natural” character but have an extended shelf-life is the long-term goal (Ferrocino & Cocolin, 2017).Such advances can contribute to reduce the problem of food waste while securing the supply of good quality and tasty food for all.

5.Omics for identifying molecules of interest for seafood processing and preservation from microalgae

The diversity of marine species (conservative estimates >1 × 106(Bouchet, 2006), and the physical and chemical extremes of marine environments makes the probability of finding unique and useful bioactive molecules very high.Interest in marine microalgae from this perspective is high particularly since they are very abundant and grow rapidly as shown by their contribution of up to 50% of all aquatic productivity (Raven & Falkowski, 1999).Marine microalgae are of interest for biofuel production and as potential feeds due to their high protein content (40–70% dry weight), although technological barriers still need to be overcome.Microalgae protein hydrolysates are being mined for bioactive molecules with health giving properties or for treatment and management of chronic disease (Draaisma et al., 2013; Ejike et al.,2017).It is this context and their multi-functional characteristics that make them interesting for seafood processing.Ideas for their use include bioactive molecules to control microbial growth or to function as antioxidants in processed foods.Furthermore, products from microalgae are being studied as environmentally friendly alternatives to plastic packaging.

Specific microalgae proteins already on the market for human consumption include a prebiotic TetraSOD® (Mantec´on et al., 2019) that contains 3 enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) that are strong antioxidants.The target enzymes collectively, described as primary antioxidants, are produced by all aerobic organisms and provide defense against free radicals and harmful inflammatory reactions (Case, 2017; Ighodaro et al., 2018).Such characteristics highlight the potential of secondary streams coming from microalgae products (e.g.extraction of natural pigments, carotenoids, feeds or biofuels) as a source of natural anti-oxidants for fresh or processed seafood.

The need for novel antimicrobial compounds, both for health and food applications has dramatically increased during recent years.To this end, it is well established that both marine and freshwater microalgae species can provide a largely unexploited source for novel antimicrobial compounds (Falaise et al., 2016).Both eukaryotic and prokaryotic microalgae can produce a wide range of bioactive compounds with a range of applications such as antimicrobials and food preservatives,including fatty acids, carbohydrates, cyclophanes, alkaloids, indoles,phenols, terpenes and other pigments, peptides, etc (Rojas et al., 2020;Senhorinho et al., 2015).Unsaturated fatty acids are among the best characterised microalgae derived antimicrobial compounds, including chlorellin, a mixture of bioactive fatty acids and carbohydrates produced byChlorellasp., which in pioneering work was shown to inhibit the growth of both gram-positive and -negative bacteria (Pratt et al.,1944).In addition, high nutritional value PUFAs including eicosapentaenoic (EPA) and hexadecatrienoic acids etc., produced by many microalgae species includingNannochloropsissp.andPhaeodactylum tricornutum,Scenedesmus intermediusandHaematococcus pluvialisare known to be potent antimicrobial compounds (Smith et al., 2010).In addition, microalgae derived extracts are also known to possess both antifungal and antiviral activities (Ahmadi et al., 2015; Dewi et al.,2018, pp.235–261; Rechter et al., 2006).

6.Future prospects

The future contribution of food from the sea to the global food supply depends on a range of ecological, economic, policy and technological factors.It has been estimated, based on demand shifts and supply scenarios, that edible food from the sea could increase by 21–44 million tons by 2050, a 36–74% increase compared to current yields.Whether these production potentials will be realized and will be sustainable depends on factors, such as policy reforms, technological innovation, and the extent of future shifts in demand (Costello et al., 2020).Technological advances in terms of quality evaluation of “new” foods, is a prerequisite for the production of safe, innovative food products with improved quality and nutritional value.The detection of contaminating non-cultivable bacteria in aquatic food products remains a challenge, as these microorganisms may play a significant role in the safety and spoilage process of seafood.Omics technologies open the way for new innovative monitoring tools that will be essential for the development of next generation risk assessment approaches (den Besten et al., 2018).Current technologies based on digital information systems, such as web platforms and smartphone apps, will be crucial for the adoption and widespread implementation of such molecular labelling (Ferri et al.,2015).Future developments of molecular microbiological tools should focus on further optimization of universal primer sets and tools towards the discrimination between positive results and actual pathogen contamination.Since most of the omics tools discussed in this article have mainly been developed and/or validated at laboratory scale or on model systems, it is necessary to test them in a relevant environment, i.e.on real food and at an industrial scale.

7.Conclusions

Current and emerging innovative omics approaches can reveal patterns of response that cannot be detected by classical methods and have the potential to speed-up and ultimately uncover new and powerful methods to control hazards in food and feed.They have the potential to bring much more insight than the usual snapshot of the farm-to-fork contamination process analysis.The combination of a range of novel tools allowing alternative approaches for food quality and safety monitoring may pave the way towards the creation of “molecular labelling” of food translated into an easily understood output for a diversity of stakeholders, such as producers, distributors and consumers.

CRediT authorship contribution statement

Deborah M Power:Conceptualization, Funding acquisition, Project administration, Resources, Writing – original draft, Writing – review &editing.Petros Taoukis:Writing – review & editing.Dimitra Houhoula:Writing – review & editing.Theofania Tsironi:Conceptualization, Funding acquisition, Project administration, Resources, Writing –original draft, Writing – review & editing.Emmanouil Flemetakis:Project administration, Resources, Writing – original draft, Writing –review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

Research was conducted for the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant agreement 872217 (Project acronym: ICHTHYS) and under the Section 2 PRIMA call 2019 project (Project acronym: FRUALGAE).