Conjugation of (-)-epigallocatechin-3-gallate and protein isolate from large yellow croaker (Pseudosciaena crocea) roe: improvement of antioxidant activity and structural characteristics.

BACKGROUND: Large yellow croaker (Pseudosciaena crocea) roe is the main by-product in the processing of large yellow croaker. Previous studies have found that its protein isolates are composed of vitellogenin, as well as vitellogenin B and C, having good functional properties. (-)-Epigallocatechin-3-gallate (EGCG) is a natural antioxidant component that can be combined with protein to improve antioxidant activity and structural characteristics of protein. RESULTS: EGCG was bound with the P. crocea roe protein isolate (pcRPI) by the free radical method to prepare the conjugate. The formation of pcRPI-EGCG conjugates was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography, which showed that the calculated weight-average molar masses of native-pcRPI and pcRPI-EGCG conjugates were 86.9 and 215.3 kDa, respectively. The results of fluorescence, ultraviolet, circular and infrared spectra indicated that the conjugation of EGCG with native-pcRPI changed the secondary and tertiary structure of native-pcRPI. The pcRPI-EGCG conjugates exhibited higher thermal stability than native-pcRPI. The radical scavenging and reducing power of native-pcRPI were increased by 2.0-2.5- and 1.4-fold, respectively, after the EGCG-grafting reaction. CONCLUSION: These results indicate that the binding of pcRPI and EGCG effectively improved the antioxidant properties and structural characteristics of the pcRPI. © 2021 Society of Chemical Industry.

Molecular characterization and expression analysis of toll-like receptor 1 from large yellow croaker (Pseudosciaena crocea).

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP) and induce antimicrobial immune responses. Here we report the molecular cloning and characterization of a TLR1 homologue from the large yellow croaker (LycTLR1). The complete cDNA of LycTLR1 is 3487 nucleotides long, encoding a protein of 802 amino acids. The deduced LycTLR1 has a typical TLR domain architecture including 4 leucine-rich repeats (LRRs) (residues 42-491), one C-terminal LRR domain (residues 527-583) at the extracellular region and a TIR domain (residues 646-791) in the cytoplasmic region. Homology comparison shows that LycTLR1 has 76.8%-47.6% amino acid identity to known fish TLR1. Genomic analysis revealed that LycTLR1 consisted of only one exon in the coding region, which is conserved among other TLR1 from different mammalian species and fish analyzed to date, except the zebrafish. The mRNA of LycTLR1 was constitutively expressed in spleen, head kidney, blood, liver, heart, gills, intestine, brains and muscle, with the highest levels in spleen and blood. Upon stimulation with LPS, the LycTLR1 expression obviously increased in the anterior kidney cells of large yellow croaker, suggesting a role for LycTLR1 in the immune response to LPS.

Vaccination efficiency of surface antigens and killed whole cell of Pseudomonas putida in large yellow croaker (Pseudosciaena crocea).

Large yellow croaker (Pseudosciaena crocea), a major marine fish aquacultured in the southeastern coastal region of China, has become endangered by the pathogen Pseudomonas putida in recent years. P. putida infections occur in low water temperatures when fish reduce food intake, thus oral antibiotic administration is not practical. Therefore, vaccination may be the only method to prevent the infection. In the present study, main surface antigens of P. putida, including lipopolysaccharide (LPS), outer membrane proteins (OMP), extracellular biofilm polysaccharide (EPS), and formalin-killed cell (FKC) bacterin, were prepared and the fish vaccinated. On post-immunization day 28, serum antibody titers, phagocytic responses of leukocytes, and lysozyme activities of the fish were evaluated. The efficiency of vaccination was tested by artificial challenge via intraperitoneal injection of live bacteria on post-immunization day 28 and 35, respectively. The results showed that although significant humoral and innate immune responses were elicited in all vaccination groups, the challenge produced similar poor protection in both tests, with a relative percent survival (RPS) of 0-40%. Although the EPS group showed a complete lack of protection, LPS reached the highest RPS value (40%), suggesting that LPS may be involved in protection immunity against the pathogen. Further analysis of the ultra-structures of tissues from infected fish via TEM revealed macrophage survival and intracellular replication ability of the pathogen. New strategies for development might put more emphasis on efficient clearance of intracellular bacteria. The present study is the first to report vaccination against the fish pathogen P. putida and the first investigation of intracellular survival of this pathogen in host macrophages.

Molecular characterization and expression analysis of TLR 7 and TLR 8 homologs in large yellow croaker (Pseudosciaena crocea).

The two toll-like receptor (TLR) genes, LycTLR7 and LycTLR8, were cloned from large yellow croaker (Pseudosciaena crocea), an economically important marine fish in China. The full-length cDNAs of LycTLR7 and LycTLR8 are 3544 and 3593 bp, with an open reading frame (ORF) of 3165 and 3093 bp, encoding 1053 and 1030 amino acids, respectively. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in the LycTLR7 and LycTLR8, with 17 and 14 LRRs, and with a TIR domain, respectively. It is also noted that an LRR N-terminal domain (LRR-NT, residues 24-60) is present in the LycTLR7 but not in the LycTLR8. Both LycTLR7 and LycTLR8 contain a conserved extracellular CxRCxxxxxPCxxC motif, which was found in TLR7/TLR8 of other species and required for stimulus-induced signal transduction. Homology comparison shows that LycTLR7 has 79%, 71.9%, 65.9% and 65.8% identity to fugu, rainbow trout, carp and catfish TLR7, while LycTLR8 has 67.1%, 60.7%, 60.6%, 52.4%, and 51.5% identity to fugu TLR8, rainbow trout TLR8a1, rainbow trout TLR8a2, catfish TLR8-2, and catfish TLR8-1, respectively. Subcellular localization analysis revealed that both LycTLR7 and LycTLR8 are located in the endoplasmic reticulum of epithelioma papulosum cyprini (EPC) cells, which is similar to TLR7/TLR8 in mammals. The two TLRs were constitutively expressed in all tissues tested, especially in immune-related tissues such as spleen, head kidney and gills. An increased expression of LycTLR7 and LycTLR8 was observed in head kidney and spleen of large yellow croakers stimulated by poly (I:C), a viral mimic. In head kidney, their mRNA expression was up-regulated more than 10 times compared to the controls at 12 h after poly (I:C) stimulation. These results suggested that LycTLR7 and LycTLR8 may play a role in the defense against viral infection like their mammalian homologs.

Formation and characteristics of curcumin-loaded binary gels formed from large yellow croaker (Pseudosciaena crocea) roe protein isolate and gellan gum.

The aim of this study was to investigate the effect of gellan gum (GG) on the cold gelation of large yellow croaker roe protein isolate (pcRPI). The water-holding ability and storage modulus of the pcRPI-GG binary gels increased with the GG concentration, where the storage modulus of the pcRPI-0.2% GG gel was approximately 30.7 times that of the pure pcRPI gel. Compare to the other binary gels, pcRPI-0.2% GG gels exhibited a lower lacunarity and higher junction density, with a denser, more aggregated microstructure. Consequently, curcumin was embedded in pcRPI-0.2% GG gels, and simulated gastrointestinal digestion test results showed that GG addition effectively protected and slowed curcumin release in the gastrointestinal environment. These findings may contribute to elucidating the interaction of pcRPI with GG and demonstrate the potential of binary gels for the embedding and delivery of active substances.

Exploring the Effect of Dehydration on Water Migrating Property and Protein Changes of Large Yellow Croaker (Pseudosciaena crocea) during Frozen Storage.

This study aimed to explore the effect of dehydration on the water migrating property and protein changes of large yellow croaker during frozen storage. A freeze-dryer was used to accelerate experiments, which was isolated from oxygen and excluded the effects of protein oxidation. After dehydration time (3, 9, 18, and 30 h) for both fast- and slow-freezing samples, the results showed that the ice sublimation of samples containing small ice crystals was faster than that of samples containing large ice crystals in the early stages of dehydration, but in the latest stage, there was an opposite trend. The results indicated that dehydration reduced the water freedom degrees and water-protein interaction. At the same time, dehydration had a significant effect on protein secondary and tertiary structures. The significant increase in surface hydrophobicity and particle size indicated that dehydration exacerbated myofibrillar protein aggregation. The ΔH1 values (from 1.275 to 0.834 J/g for slow-freezing group and from 1.129 to 0.855 J/g for fast-freezing group) decreased gradually as the dehydration time extended, indicating the decrease in protein thermal stability. Additionally, significant protein degradation occurred when the water content of the sample decreased to a certain level. This study showed that ice crystal size had an important effect on the rate of ice sublimation, and the occurrence of dehydration during frozen storage accelerated the water loss and the decrease in protein stability.

Identification and functional characterization of the transcription factor coding Dp1 gene in large yellow croaker Pseudosciaena crocea.

The transcription factor Dp1, as a binding partner, often forms a dimerization complex with typical E2F to play a central role in regulating gene expression during G1/S cell cycle progression. In this study, a full-length dp1 cDNA (Pcdp1) was successfully cloned and characterized from the large yellow croaker Pseudosciaena crocea. The nucleotidic sequence of Pcdp1 is 1,427 bp long with an open reading frame (ORF) of 1,239 bp encoding a putative protein of 412 amino acids, a 5'-untranslated region of 116 bp and a 3'-untranslated region of 70 bp. Prediction of protein domains showed that PcDp1 contains a DNA-binding domain (DBD) with a DEF box, a dimerization domain and an acidic region at C terminus with transcription activity. Homology comparisons indicated that PcDp1 shared the highest sequence identity of 98.55% with Oreochromis niloticus dp1, followed by 88.72% identity with Danio rerio dp1 and a relatively low identity of 78.91-80.55% with its mammalian and amphibian counterparts. The mRNA of Pcdp1 showed ubiquitously expression in all analyzed tissues, with the highest level of expression in the body kidney. Moderate expression levels of Pcdp1 was found in several immune-related tissues including the gills, head kidney and liver, indicating that PcDp1 might play an important role in osmotic pressure regulation and immune response of the large yellow croaker. The subcellular localization of PcDp1 revealed that it is mainly distributed in the cytoplasm both in COS-7 and parenchymal cells of the spleen, head kidney and kidney tissues. Furthermore, the recombinant PcDp1 exhibited DNA-binding activity to E2F site in vitro. In conclusion, these results indicated that PcDp1 may participate in immune regulation and provide a foundation for further study of the regulatory mechanism of Dp1 in teleosts.

Simultaneous extraction by acidic and saline solutions and characteristics of the lipids and proteins from large yellow croaker (Pseudosciaena crocea) roes.

The objective of this study was to simultaneously obtain protein isolates and lipids from the dried powder of large yellow croaker (Pseudosciaena crocea) roes (pcRs) to achieve high-value utilization. Protein isolates and lipids were extracted simultaneously from pcRs by saline and acidic solutions. The purity of the protein isolates from the pcRs (pcRPIs) was greater than 70%, with vitellogenin, vitellogenin B and vitellogenin C as the main proteins. The lipids from pcRs (pcRLs) were mainly composed of triglycerides with high levels of EPA and DHA. The pcRPIs exhibited a higher surface hydrophobicity, water/oil holding capacity and emulsifying ability than those of the pcRs. Moreover, pcRPIs had a better oil holding capacity and emulsifying ability than soy protein isolate. These results suggest that protein isolates and lipids can be simultaneously extracted by saline and acidic solutions, and pcRPIs and pcRLs can be used as functional materials in the food industry.

Purification and Characterization of Protamine, the Allergen from the Milt of Large Yellow Croaker (Pseudosciaena crocea), and Its Components.

The protamine in fish milt can cause anaphylaxis in humans. To determine the allergen in the milt of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera from allergic patients. The results showed that a 12 kDa multicomponent protein was the major allergen in the milt of large yellow croaker. The multicomponent protein was purified, and physicochemical characterization showed that it was a glycoprotein, highly stable in acid-alkali conditions, and weakly retained immunoglobulin E (IgE)-binding activity at high temperatures. Separation and immunoreactivity analysis of the components of the multicomponent protein showed that it had six components, and component 5 had the strongest IgE-binding activity with patient sera. N-terminal sequencing confirmed the multicomponent protein was protamine. Following analysis of protamine from different fish by reversed-phase liquid chromatography and circular dichroism spectra, the protamines from different fish were found to have a similar secondary structure, although their components were different.

Purification, characterization and immunoreactivity of ß'-component, a major allergen from the roe of large yellow croaker (Pseudosciaena crocea).

Fish roe, a nutritious food, is favored by consumers, but has also been confirmed to be allergenic in salmonid fish. However, little information is available in other fish species. To determine the allergen in the roe of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera of allergic patients. The major allergen was purified by column chromatography methods, revealing a single band with 16 kDa and was confirmed as ß'-component (ß'-c) by mass spectrometry. The results of physicochemical characterization showed that ß'-c was a glycoprotein and was relatively stable following thermal or acid/alkali treatment. Furthermore, ß'-c was easily degraded by pepsin, but was resistant to trypsin and α-chymotrypsin. After treatment with different processing methods, including Maillard reaction (MR), ultraviolet radiation (UVR), ultrasound-heat (UH), and retorting (RT), the IgG-binding activity of ß'-c decreased obviously by MR, but decreased slightly by UVR and UH. Cross-immunoreactivity results of the allergens in the roes of different species revealed that ß'-c was a specific allergen in teleostean, and the cross-immunoreactivity between the roe of large yellow croaker and other kinds of fish roe was relatively strong.