Cell-Cultured Fish Meat via Scale-Up Expansion of Carassius auratus Skeletal Muscle Cells Using Edible Porous Microcarriers and Quality Evaluation.

Emerging technologies for cell-cultured fish meat as an environmentally friendly protein source for humans still have many obstacles, including large-scale production of high-quality cells, differentiation and bioassembly of cellular material, and improvement of the quality of meat products. Here, we used edible porous microcarriers as scaffolds to support scalable skeletal muscle cell expansion to prepare centimeter-scale cell-cultured fish (CCM) of Carassius auratus for the first time. The quality of CCM was assessed by analyzing the texture, nutrition, flavor, and safety. The results indicated that CCM demonstrated a softer texture than natural fish due to a high moisture content. CCM contained higher protein and lower fat contents, with no significant difference in energy from natural golden crucian carp meat (NGM). CCM had better digestible properties, and 17 volatile components were identified in CCM, ten cocontained compared to NGM. ELISA quantified penicillin, streptomycin, vitamin D, and insulin residues as risk factors in CCM. In conclusion, we utilized edible porous microcarriers to scale-up the expansion of Carassius auratus skeletal muscle cells and bioassembled high-quality CCM of Carassius auratus for the first time, which represents a state-of-the-art protocol applicable to different fish species and even to other economic animals and provides a theoretical basis for scaling up cell-cultured meat production.

TLR21 is involved in the NF-κB and IFN-ß pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Zn(II) and Cu(II) Coordination Enhances the Antimicrobial Activity of Piscidin 3, but Not That of Piscidins 1 and 2.

Piscidins, antimicrobial peptides isolated from fish, are potent against a variety of human pathogens; they show minimum inhibitory concentration values comparable to those of commercially used antimicrobials. Piscidins 1 and 2 are generally more effective than piscidin 3 when applied alone; the contrary is observed for their metal complexes: Zn(II) and Cu(II) coordination does not enhance the efficacy of piscidins 1 and 2, while a moderate enhancement is observed for piscidin 3. All three piscidins bind Cu(II) in a so-called albumin-like binding mode, while for Zn(II) complexes, two coordination modes are observed: piscidins 1 and 2 bind Zn(II) by imidazole nitrogens from His4, His11, and His17 side chains; piscidin 3 coordinates Zn(II) by His3, His4, and His11 imidazole nitrogens and additionally supports the interaction, formed by carbonyl oxygen from His4. Most likely, the high antimicrobial activity of piscidin complexes is due to neither the stability of their complexes nor the change in their secondary structure. Copper(II) complexes with piscidins 1 and 2 can form hydroxyl radicals, which could be responsible for the antimicrobial membrane damaging activity of these complexes. Clearly, a different mechanism (most likely an intercellular targeted one) is observed for piscidin 3 metal complexes; in most cases, the coordination of Cu(II) and Zn(II) enhances the antimicrobial potency of piscidin 3, showing that not only piscidin 3 alone but also its metal complexes have a different mode of action than piscidins 1 and 2.

A Comparative Study of the Stability, Transport, and Structure-Activity Relationship of Round Scad Derived Peptides with Antineuroinflammatory Ability.

Our previous study identified round scad neuroprotective peptides with different characteristics. However, the intrinsic relationship between their structure and bioactivity, as well as their bioavailability, remains unclear. The aim of this study is to elucidate the bioavailability of these peptides and their structure-activity relationship against neuroinflammation. Results showed that the SR and WCP peptides were resistant to gastrointestinal digestion. Additionally, peptides SR, WCP, and WCPF could transport Caco-2 monolayers as intact peptides. The permeability coefficients (Papp) of SR, WCP, and WCPF in Caco-2 monolayer were (1.53 ± 0.01) × 10-5, (2.12 ± 0.01) × 10-5, and (8.86 ± 0.03) × 10-7 cm/s, respectively. Peptides SR, WCP, and WCPF, as promising inhibitors of JAK2 and STAT3, could attenuate the levels of pro-inflammatory cytokines and regulate the NFκB and JAK2/STAT3 signaling pathway in LPS-treated BV-2 cells. WCPF exerted the highest anti-inflammatory activity. Moreover, bioinformatics, molecular docking, and quantum chemistry studies indicated that the bioactivity of SR was attributed to Arg, whereas those of WCP and WCPF were attributed to Trp. This study supports the application of round-scad peptides and deepens the understanding of the structure-activity relationship of neuroprotective peptides.

Microfluidic Chip Coupled with MALDI-TOF MS for Multitarget Detection of Allergens in Crucian Carp (Carassius auratus).

The goal of the present study was to establish a rapid, simple method for simultaneous allergy testing of sera from multiple fish-allergic patients. Sera from fish-allergic patients were pooled and used for capturing allergens in fish muscle of crucian carp (Carassius auratus), which was studied as a fish model. Sarcoplasmic proteins of crucian carp (Carassius auratus) were extracted for the analysis of allergens. Anti-human IgE antibody-functionalized magnetic beads were utilized to collect IgE antibodies from human pooled sera. The isolation of allergenic proteins was immunomagnetically performed in microfluidic channels, and the elution of the captured allergenic proteins was done with 5% (v/v) acetic acid aqueous solution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting were used for the analysis of tryptic digests of eluted proteins. Ten potential allergenic proteins were identified from crucian carp (Carassius auratus). The present protocol provides a rapid, efficient, and simple method for simultaneous detection of multiple allergens, based on multitargeted antibodies from pooled sera of allergic patients. The constructed multiple antibody-modified MBs can be applied for the deallergenicity of food matrices. The efficiency of allergen detection can be greatly improved, with promising application in allergen discovery and filtration for other muscle-based foods.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Generation, Characterisation and Identification of Bioactive Peptides from Mesopelagic Fish Protein Hydrolysates Using In Silico and In Vitro Approaches.

This study generated bioactive hydrolysates using the enzyme Alcalase and autolysis from mesopelagic fish, including Maurolicus muelleri and Benthosema glaciale. Generated hydrolysates were investigated for their bioactivities using in vitro bioassays, and bioactive peptides were identified using mass spectrometry in active hydrolysates with cyclooxygenase, dipeptidyl peptidase IV and antioxidant activities. In silico analysis was employed to rank identified peptide sequences in terms of overall bioactivity using programmes including Peptide Ranker, PrepAIP, Umami-MRNN and AntiDMPpred. Seven peptides predicted to have anti-inflammatory, anti-type 2 diabetes or Umami potential using in silico strategies were chemically synthesised, and their anti-inflammatory activities were confirmed using in vitro bioassays with COX-1 and COX-2 enzymes. The peptide QCPLHRPWAL inhibited COX-1 and COX-2 by 82.90% (+/-0.54) and 53.84%, respectively, and had a selectivity index greater than 10. This peptide warrants further research as a novel anti-inflammatory/pain relief peptide. Other peptides with DPP-IV inhibitory and Umami flavours were identified. These offer potential for use as functional foods or topical agents to prevent pain and inflammation.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate.

Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.

Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

A cytokine-like factor 1 homolog acts as a macrophage chemoattractant in grass carp.

Cytokine-like factor 1 (CYTL1) is a small cytokine and has diverse biological functions in mammals. However, whether CYTL1 exists in lower vertebrates is not clear. In this study, we identified cytl homologs in fish and characterized the immune functions in a teleost species, grass carp (Ctenopharyngodon idella). Fish CYTL1 homologs share conserved molecular features with their mammalian counterparts, including 6 cysteine residues in the mature peptide, genomic organization and synteny. Gene expression analysis revealed that cytl1 was constitutively expressed in tissues of grass carp, with the highest expression detected in the heart. Upon infection with Aeromonas hydrophila (A. hydrophila), cytl1 was downregulated in the hindgut, head kidney, skin, and spleen. In the primary head kidney leukocytes (HKLs), stimulation with inactivated A. hydrophila, LPS, poly(I:C), IL-22, IFN-a or IFN-γrel resulted in downregulation of cytl1 expression. Recombinant grass carp CYTL1 protein produced in the HEK293-F cells was potent to induce il-10 expression, but had little effect on the expression of il-1ß and il-6. In vivo experiments revealed that CYTL1 was effective to recruit macrophages to the muscle injected with cytl expression plasmids. Taken together, our results indicate that CYTL1 is a potent chemokine for recruitment of macrophages in fish.

Genome-wide identification, characterization and expression profiling of TLR family genes in Chromileptesaltivelis.

Toll-like receptors (TLRs) represent a prominent category of pattern recognition receptors that have been extensively investigated for their pivotal role in combating pathogen incursions. Despite this, there has been a notable absence of comprehensive identification and exploration of the immune response associated with the TLR family genes in C. altivelis. This study successfully identified and named fourteen genes as Catlr1-1, Catlr1-2, Catlr2-1, Catlr2-2, Catlr3, Catlr5, Catlr7, Catlr8, Catlr9, Catlr13-1, Catlr13-2, Catlr18, Catlr21, and Catlr22. A series of bioinformatic analysis were performed, encompassing analysis of protein properties, examination of gene structures, evolutionary assessments, and prediction of protein tertiary structures. The expression patterns of Catlr genes were analyzed in five immune tissues: liver, spleen, kidney, gill, and intestine, in both healthy and bacterial stimulated-fish. The results showed that different tissue and different genes showed differed expression patterns after V. harveyi infection, indicating the involvement of all Catlr members in mounting immune responses following infection in various tissues. Additionally, histological evaluations of immune tissues unveiled varying levels of damage. In conclusion, this investigation into the TLR gene family offers novel information that contribute to a more profound comprehension of the immune response mechanisms in C. altivelis.

A review of inhibition mechanisms of surimi protein hydrolysis by different exogenous additives and their application in improving surimi gel quality.

It is well known that aquatic products such as fish and shellfish, when stored for a long period of time under inappropriate conditions, can suffer from muscle softening. This phenomenon is mainly caused by endogenous proteases, which are activated during heating and accelerates the degradation of myofibrillar proteins, directly leading to weaker gels and poorer water retention capacity. This paper reviews the changes in fish proteins during storage after death and the factors affecting protein hydrolysis. A brief overview of the extraction of protease inhibitors, polysaccharides and proteins is given, as well as their mechanism of inhibition of protein hydrolysis in surimi and the current status of their application to improve the properties of surimi.

Changes in protein and volatile flavor compounds of low-salt wet-marinated fermented Golden Pomfret during processing.

In this experiment, the changes in protein hydrolysis, protein oxidation, and flavor of low-salt wet-marinated fermented golden pomfret were studied during processing. During processing, a decrease in sulfhydryl content (P < 0.05), a significant increase in protein surface hydrophobicity (P < 0.05), a significant increase in carbonyl content and TCA-soluble peptide (P < 0.05), an increase in TVB-N and amino acid nitrogen (P < 0.05), and a significant increase in the content of free amino acids (P < 0.05), indicating that proteins were gradually oxidized and degraded to small molecules and flavor precursors under the action of bacterial reduction pretreatment, deodorization, marination and fermentation processes, small molecules and aroma precursors was generated by gradual oxidative decomposition. In the course of processing, a total of 113 volatile flavor compounds were identified using GC-MS analysis, while OPLS-DA analysis and VIP value determination led to the identification of 10 characteristic flavor compounds. The results showed that an abundance of flavor compounds was generated during the processing, thereby imparting a more pronounced taste profile to the low-salt wet-marinated fermented golden carp. The results showed that a large number of flavor substances were generated during the processing to give a richer flavor to low-salt wet-marinated fermented golden pomfret that could provide data and theoretical support for the subsequent processing industry of golden pomfret.

Characteristics and stabilization of Pickering emulsions constructed using myosin from bighead carp (Aristichthys nobilis).

Myosin from bighead carp (Aristichthys nobilis) as a main type of fish protein possesses a good emulsifying ability. However, whether bighead carp myosin (BCM) could construct stable Pickering emulsions is still unclear. Therefore, myosin particles and Pickering emulsions stabilized by bighead carp myosin (BCMPEs) were analyzed. The surface structure of BCM particles at 0.6 mol/L NaCl treatment was uniform and compact with a contact angle of 86.4 ± 2.7°, exhibiting the potential ability to construct O/W Pickering emulsions. The size and flocculation index (FI) of BCMPEs decreased with the increase in BCM concentrations of 1%-4% (w/v). Reversely, the size of BCMPEs increased with the increase in oil-water ratios. BCM particles could uniformly distribute at the oil-water interface to stabilize BCMPEs at a BCM concentration of 4% (w/v) and an oil-water ratio of 6:4 (v/v). This study could help explore fish proteins to construct Pickering emulsions for the deep processing of fish products.

Genome-wide identification and characterization of large yellow croaker (Larimichthys crocea) suppressors of cytokine signaling (SOCS) in immune response to Pseudomonas plecoglossicida infection and acute hypoxia stress.

The suppressor of cytokine signaling (SOCS) gene family is a group of genes involved in the negative regulation of cytokine signal transduction. The members of this family play a crucial role in regulating immune and inflammatory processes. However, comprehensive investigations of these genes have not yet been conducted in the economically significant fish large yellow croaker (Larimichthys crocea). In this study, a total of 13 SOCS genes (LcSOCS1a, LcSOCS1b, LcSOCS2, LcSOCS3a, LcSOCS3b, LcSOCS4, LcSOCS5a, LcSOCS5b, LcSOCS6, LcSOCS7a, LcSOCS7b, LcCISHa and LcCISHb) were identified and analyzed in L. crocea. The phylogenetic tree revealed a high conservation of SOCS genes in evolution, and the gene structure and motif analysis indicated a high similarity in the structure of LcSOCSs in the same subfamily. In addition, the expression patterns of LcSOCSs showed that LcSOCS1b was significantly down-regulated in all time under acute hypoxia stress, but it was markedly up-regulated throughout the entire process after P. plecoglossicida infection, revealing its different immune effects to two stresses. Besides, LcSOCS2a, LcSOCS6 and LcSOCS7a only participated in acute hypoxic stress, while LcSOCS5a was more sensitive to P. plecoglossicida infection. In summary, these results indicated that SOCS genes were involved in stress responses to both biological and non-biological stimuli, setting the foundation for deeper study on the functions of SOCS genes.

Genome-wide identification, characterization, molecular evolution and expression profiling analysis of scavenger receptors in black rockfish (Sebastes schlegelii).

The scavenger receptors (SRs) gene family is considered as the membrane-associated pattern recognition receptors that plays important roles in the immune responses of organisms. However, there is currently limited research on the systematic identification of the SRs gene family in teleost and their role in the innate immunity of S. schegelii. In this study, we identified and annotated 15 SRs genes in S. schegelii. Through phylogenetic analysis, analysis of conserved domains, gene structure, and motif composition, we found that SRs gene family within different classes were relatively conserved. Additionally, we used qRT-PCR to analyze the expression patterns of SRs genes in immune-related tissues from healthy and Acinetobacter johnsonii-infected S. schegelii. The results showed that SRs genes exhibited different tissue expression patterns and the expression of SRs genes significantly changed after A. johnsonii infection. These results provided a valuable basis for further understanding of the functions of SRs in the innate immune response of S. schegelii.

Serum proteinogram of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) as a new useful approach for detecting loss of haemostasis.

Proteinograms, a semiquantitative analytical method that separates proteins into multiple bands, have not been explored in teleosts for diagnostic or prognostic purposes. This study aimed to establish reference values for proteinograms in the serum of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), two important farmed fish species in the Mediterranean region. Serum proteins were studied using SDS-PAGE, electropherogram, and HPLC-mass spectrometry. SDS-PAGE analysis revealed four major bands of proteins around 11, 25, 70, and 100 kDa in the serum of gilthead seabream and European sea bass. Electropherogram results showed that a protein with a molecular weight of 76.8 kDa was the most abundant protein in the serum of gilthead seabream, while a peak of 75.5 kDa was the most abundant in European sea bass. HPLC-mass spectrometry detected 87 proteins and 119 proteins in the serum of gilthead seabream and European sea bass, respectively, including α1-globulins, α2-globulins, ß-globulins, and γ-globulins. Notably, the albumin sequence was not detected in either of the two species. These results help to characterize the serum protein profile and to establish reference proteinograms for these two fish species. They also provide a basis for the development of novel approaches for the rapid detection of loss of haemostasis due to stress, health disorders or disease in farmed fish.

The immune function of heme oxygenase-1 (HO-1) from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-ß), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1ß, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia.

Investigation into Potential Allergenicity and Digestion-Resistant Linear Epitopes of Fish Skin Gelatin in Cell-Cultured Meat Scaffolds.

As a key component of cell-cultured fish, fish skin gelatin (FSG)-based cell scaffold provides support structures for cell growth, proliferation, and differentiation. However, there are potential allergenicity risks contained in FSG-based scaffolds. In this study, 3D edible scaffolds were prepared by phase separation method and showed a contact angle of less than 90°, which indicated that the scaffolds were favorable for cell adhesion. Besides, the swelling ratio was greater than 200%, implying a great potential to support cell growth. The sequence homology analysis indicated that FSG was prone to cross-reaction with collagen analogues. Additionally, a food allergic model was constructed and represented that mice gavaged with cod FSG exhibited higher levels of specific antibodies, mast cell degranulation, vascular permeability, and intestinal barrier impairment than those gavaged with pangasius and tilapias FSG. Its higher allergenicity might be attributed to a higher number of digestion-resistant linear epitopes. Moreover, the higher hydrolysis degree linked to the exposure of linear epitopes to promote the combination with IgE, which was also responsible for maintaining the higher allergenicity of cod FSG. This study clarifies allergenic risks in cell-cultured fish and further study will focus on the allergenicity reduction of FSG-based cell scaffolds.

Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.