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The challenge of applying chlorophyll(Chl) in aqueous media has been a significant obstacle to the diversified development of Chl a-related industries. This study presents the first report on the true-solution-scale utilization of Chl in aqueous media through the construction of chlorophyll a-phycocyanin (Chls-PC) composite nanoparticles. This study determined the optimal conditions for Chls-PC preparation: a composite ratio of 1:25, a solvent ratio of 1:4, and a stirring time of 1 h. Fluorescence spectroscopy, transmission electron microscope, and confocal microscopy confirmed Chl a and PC aggregation. Surface hydrophobicity and contact angle measurements showed that Chls-PC water solubility was similar to PC and much higher than Chl. Infrared spectroscopy, quantum chemical calculations, X-ray photoelectron spectroscopy, and molecular dynamics simulations elucidated the water solubilization mechanism of Chls-PC both experimentally and theoretically. This research provides theoretical guidance for the development and production of water-based products using Chl as a raw material.
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Self-fermented oyster homogenates were prepared to investigate core microbes and their correlations with flavor formation mechanisms. Five bacterial and four fungal genera were identified. Correlation analysis showed that Saccharomyces cerevisiae, Kazachstania, and L. pentosus were core species for the flavor of fermented products. Four core microbes were selected for inoculation into homogenates. Twelve key aroma compounds with odor activity values >1 were identified by gas chromatography-mass spectrometry. L. plantarum and S. cerevisiae were beneficial for producing key aroma compounds such as 1-octen-3-ol, (E,Z)-2,6-nonadienal, and heptanal. Fermentation with four microbes resulted in significant increases in contents of Asp, Glu, Lys, inosine monophosphate, and guanosine monophosphate, which provided freshness and sweetness. Fermentation with four microbes resulted in high digestibility, antioxidant abilities, and zinc contents. This study has elucidated the mechanism of flavor formation by microbial action and provides a reference for targeted flavor control in fermented oyster products.
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Bacterias , Crassostrea , Fermentación , Aromatizantes , Gusto , Animales , Crassostrea/microbiología , Crassostrea/metabolismo , Crassostrea/química , Aromatizantes/metabolismo , Aromatizantes/química , Bacterias/metabolismo , Bacterias/clasificación , Bacterias/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Odorantes/análisis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Hongos/metabolismo , Hongos/clasificación , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/análisis , Mariscos/análisis , Mariscos/microbiologíaRESUMEN
Fishy odor in aquatic products has a significant impact on the purchasing decisions of consumers. The production of aquatic products is a complex process involving culture, processing, transportation, and storage, which contribute to decreases in flavor and quality. This review systematically summarizes the fishy odor composition, identification methods, generation mechanism, and elimination methods of fishy odor compounds from their origin and formation to their elimination. Fishy odor compounds include aldehydes (hexanal, heptanal, and nonanal), alcohols (1-octen-3-ol), sulfur-containing compounds (dimethyl sulfide), and amines (trimethylamine). The mechanism of action of various factors affecting fishy odor is revealed, including environmental factors, enzymatic reactions, lipid oxidation, protein degradation, and microbial metabolism. Furthermore, the control and removal of fishy odor are briefly summarized and discussed, including masking, elimination, and conversion. This study provides a theoretical basis from source to elimination for achieving targeted regulation of the flavor of aquatic products, promoting industrial innovation and upgrading.
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Aldehídos , OdorantesRESUMEN
Microalgae polysaccharides (MAPS) have emerged as novel prebiotics, but their direct effects on intestinal epithelial barrier are largely unknown. Here, MAPS isolated from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 were characterized as mainly branched heteropolysaccharides, and were bioavailable to Caco-2 cells based on fluorescein isothiocyanate labeling and flow cytometry analysis. These MAPS were equally effective to scavenge hydroxyl and superoxide radicals in vitro and to attenuate the H2O2-, dextran sodium sulfate-, tumor necrosis factor α-, and interleukin 1ß-induced burst of intracellular reactive oxygen species and mitochondrial superoxide radicals, interleukin-8 production, cyclooxygenase-2 and inducible nitric oxide synthase expression, and/or tight junction disruption in polarized Caco-2 cells. MAPS and a positive drug Mesalazine were intragastrically administered to C57BL/6 mice daily for 7 d during and after 4-d dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed equivalent anti-colitis efficacies of MAPS and Mesalazine, and based on biochemical analysis of colonic tight junction proteins, goblet cells, mucin 2 and trefoil factor 3 transcription, and colonic and peripheral pro-inflammatory cytokines, MAPS alleviated dextran sodium sulfate-induced intestinal epithelial barrier dysfunction, and their activities were even superior than Mesalazine. Overall, MAPS confer direct antioxidant and anti-inflammatory protection to intestinal epithelial barrier function.
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Chlorella , Colitis , Microalgas , Humanos , Animales , Ratones , Antioxidantes/metabolismo , Dextranos/farmacología , Células CACO-2 , Mesalamina/farmacología , Peróxido de Hidrógeno/metabolismo , Superóxidos/metabolismo , Ratones Endogámicos C57BL , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/patología , Células Epiteliales , Antiinflamatorios/uso terapéutico , Sulfato de Dextran/toxicidad , Mucosa Intestinal/metabolismo , Modelos Animales de EnfermedadRESUMEN
Chlorophyll (Chl) is the most abundant pigment in photosynthetic plants, but it is prone to degradation during processing and storage, limiting its usage in the food industry. This study developed a technique for increasing Chl photostability by light-induced Chl/phycocyanin (PC) triple synergistic aggregation. Under continuous illumination settings, the results revealed that the Chl retention increased to 406% compared to the control. A model of Chl/PC complexes was constructed using multiligand molecular docking, and the aggregation mechanism was investigated by quantum chemistry, which demonstrated that PC could provide an ideal central hydrophobic cavity for Chl aggregates and thus further enhance the aggregation of Chl on the basis of Chl/PC complexes. The core driver of the improved photostability of Chl is photoexcitation-induced Chl aggregates. This study enriches our understanding of the interaction mechanism between PC and Chl, and we hope that this study can provide broader ideas for the development of natural pigment products.
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Clorofila , Ficocianina , Clorofila/metabolismo , Ficocianina/metabolismo , Clorofila A , Simulación del Acoplamiento Molecular , FotosíntesisRESUMEN
Synechococcus sp. PCC 7002 has the potential to be used as a new resource of food owing to its nutritional and functional benefits. However, little information has been published regarding the safety of Synechococcus sp. PCC 7002 biomass (SynB). The present study assessed genotoxicity, acute and subchronic toxicity of SynB for the first time. SynB did not show any genotoxicity based on the Ames test, mammalian erythrocyte micronucleus test, and mouse primary spermatocyte chromosome aberration test. SynB administered by gavage in mice at a dose of 10 g per kg body weight did not induce death or toxicity based on the acute toxicity study, indicating the median lethal dose value of SynB was over 10 g per kg body weight. In the 90-day subchronic toxicity study, no treatment-related mortality or clinical sign was noted with SynB at doses of 5 and 10 g per kg body weight in mice, and there was no adverse effect of SynB on food consumption, organ coefficients, blood biochemistry, urinalysis and histopathology. The Non Observed Adverse Effect Level for SynB in female and male mice was not less than 10 g per kg body weight per day based on subchronic toxicity. These results support the safe use of SynB as a new food raw material.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Synechococcus , Femenino , Masculino , Animales , Ratones , Biomasa , Daño del ADN , Peso Corporal , MamíferosRESUMEN
Novel food matrices (such as microalgae, plants, fungi, and microbial proteins) with high protein content and biological value, good amino acid profile, and functionality have been explored. Phycocyanin and active polysaccharides extracted from Spirulina platensis are used as food additives, treatment of colitis, as well as obesity prevention. However, most of the remaining Spirulina platensis residues are mainly used as fish feed at present. 3D food printing is one of the promising development techniques used in the food industry. The aim of this study was to develop a novel 3D printing material of Spirulina platensis residues with shear thinning characteristics, high viscosity and rapid recovery. The effects of moisture content and pretreatment method on the rheological properties of Spirulina platensis residues were clarified. Scanning electron microscopy was used to observe the microstructure and texture profile analysis was used to determine the texture characteristics of Spirulina platensis residues, rheology was used to determine the key 3D printing factors such as viscosity and modulus of Spirulina platensis residues. More importantly, the printing process could be realized under ambient conditions. The development of microalgae residue ink promoted the high-value and comprehensive utilization of microalgae, and also broadened the application of microalgae in the food field.
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Tinta , Microalgas , Animales , Polisacáridos , Impresión Tridimensional , ReologíaRESUMEN
Nanostructured iron(III) compounds are promising food fortificants with desirable iron bioavailability and food compatibility. Here, gum arabic (GA) solubilized 252 mg of iron(III) per g at neutral pH in the form of GA-stabilized ferric oxyhydroxide nanoparticles (GA-FeONPs) with Z-average size of 142.7 ± 5.9 nm and ζ-potential of -20.50 ± 1.25 mV. Calcein-fluorescence-quenching assay revealed well-absorbed iron from GA-FeONPs by polarized Caco-2 cells due to efficient macropinocytic internalization and asialoglycoprotein receptor-mediated specific endocytosis facilitated by the polypeptide and arabinogalactan fractions of GA, respectively, with endocytosed GA-FeONPs being in part basolaterally transcytosed and in another part degraded into cellular labile iron pool. GA-FeONPs showed good colloidal stability under varied pH, gastrointestinal, thermal processing, and spray/freeze drying conditions and displayed remarkably weaker pro-oxidant activity than FeSO4 in glyceryl trilinoleate emulsion (P < 0.05). Oral pharmacokinetics unveiled desirable iron bioavailability of GA-FeONPs relative to FeSO4, i.e., 124.27 ± 5.91% in aqueous solution and 161.64 ± 5.01% in milk. Overall, GA-FeONPs are a promising novel iron fortificant with food-compatible, efficient, and targeted intestinal iron delivery and sustained iron-release properties.
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Acacia , Nanopartículas , Humanos , Compuestos Férricos , Hierro , Goma Arábiga , Células CACO-2RESUMEN
The formation of advanced glycation end products (AGEs), including Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), in a fish myofibrillar protein and glucose (MPG) model system at 80 °C and 98 °C for up to 45 min of heating were investigated. The characterization of protein structures, including their particle size, ζ-potential, total sulfhydryl (T-SH), surface hydrophobicity (H0), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR), were also analyzed. It was found that the covalent binding of glucose and myofibrillar protein at 98 °C promoted protein aggregation when compared with the fish myofibrillar protein (MP) heated alone, and this aggregation was associated with the formation of disulfide bonds between myofibrillar proteins. Furthermore, the rapid increase of CEL level with the initial heating at 98 °C was related to the unfolding of fish myofibrillar protein caused by thermal treatment. Finally, correlation analysis indicated that the formation of CEL and CML had a significantly negative correlation with T-SH content (r = -0.68 and r = -0.86, p ≤ 0.011) and particle size (r = -0.87 and r = -0.67, p ≤ 0.012), but was weakly correlated with α-Helix, ß-Sheet and H0 (r2 ≤ 0.28, p > 0.05) during thermal treatment. Overall, these findings provide new insights into the formation of AGEs in fish products based on changes of protein structure.
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The biofilm clustered with putrefying microorganisms and seafood pathogens could cover the surface of aquatic products that pose a risk to cross-contaminating food products or even human health. Fighting biofilms triggers synchronous communication associated with microbial consortia to regulate their developmental processes, and the enhancement of the quorum sensing system in Lactiplantibacillus plantarum can serve as an updated starting point for antibiofilm-forming strategies. Our results showed that the exogenous 25 mM L-cysteine induced a significant strengthening in the AI-2/LuxS system of Lactiplantibacillus plantarum SS-128 along with a stronger bacteriostatic ability, resulting in an effective inhibition of biofilms formed by the simplified microbial consortia constructed by Vibrio parahaemolyticus and Shewanella putrefaciens grown on shrimp and squid surfaces. The accumulation of AI-2 allowed the suppression of the expression of biofilm-related genes in V. parahaemolyticus under the premise of L. plantarum SS-128 treatment, contributing to the inhibition effect. In addition, strengthening the AI-2/LuxS system is also conducive to eliminating preexisting biofilms by L. plantarum SS-128. This study suggests that the enhancement of the AI-2/LuxS system of lactic acid bacteria enables the regulation of interspecific communication within biofilms to be a viable tool to efficiently reduce and eradicate potentially harmful biofilms from aquatic product sources, opening new horizons for combating biofilms.
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Proteínas Bacterianas , Percepción de Quorum , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Biopelículas , Alimentos Marinos , Lactonas/metabolismo , Homoserina/metabolismoRESUMEN
To investigate the differences of volatile and non-volatile metabolites between oyster enzymatic hydrolysates and boiling concentrates, molecular sensory analysis and untargeted metabolomics were employed. "Grassy," "fruity," "oily/fatty," "fishy," and "metallic" were identified as sensory attributes used to evaluate different processed oyster homogenates. Sixty-nine and 42 volatiles were identified by gas chromatography-ion mobility spectrometry and gas chromatography-mass spectrometry, respectively. Pentanal, 1-penten-3-ol, hexanal, (E)-2-pentenal, heptanal, (E)-2-hexenal, 4-octanone, (E)-4-heptenal, 3-octanone, octanal, nonanal, 1-octen-3-ol, benzaldehyde, (E)-2-nonenal, and (E, Z)-2,6-nonadienal were detected as the key odorants (OAV > 1) after enzymatic hydrolysis. Hexanal, (E)-4-heptenal, and (E)-2-pentenal were significantly associated with off-odor, and 177 differential metabolites were classified. Aspartate, glutamine, alanine, and arginine were the key precursors affecting the flavor profile. Linking sensory descriptors to volatile and nonvolatile components of different processed oyster homogenates will provide information for the process and quality improvement of oyster products.
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The effect of oral processing on flavor release and change in composition of steamed sturgeon meat was investigated. Oral processing caused changes in the concentrations of taste compounds including amino acids, 5'-nucleotides, organic acids, and Na+. Sensory omics demonstrated that the concentrations of 12 volatile compounds increased significantly (p < 0.05) during the initial stage of oral processing. There is no significant difference in microstructure, texture, and particle size of meat bolus. The top fifteen differential lipids which including eight phospholipids in all processed samples significantly (p < 0.05) correlated with the flavor release. A total of 589 differential proteins were detected in three samples with different chewing times (0, 12, and 30 s). Analysis of the correlations between odorants and 19 differential proteins was performed. Enriched pathways including fatty acid degradation, valine, leucine and isoleucine degradation, glycine, serine and threonine metabolism, and arachidonic acid metabolism were associated with flavor release during oral processing. This study aimed to investigate potential links between flavor release and biological processes during oral processing from a proteomics perspective.
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New insights revealing the flavor formation of unrinsed mixed sturgeon surimi with chicken breast were evaluated. Seventy-two volatile compounds were identified by gas chromatography-ion mobility spectrometry among the 11 surimi sample groups. The addition of 40% chicken breast caused changes in the concentrations of amino acids, 5'-nucleotides, and organic acids. Sensory attributes of balsamic, waxy, green, fresh, fatty, citrus, and aldehydic were marked when corelated with 125 volatiles identified by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. A total of 357 different lipids were identified through UPLC-Q-Orbitrap. Analysis of the correlations between flavor-active compounds and 16 different lipids revealed that various pathways, including the degradation of triglycerides, the biosynthesis of phosphatidylcholine, and the biosynthesis of lysine, serine, and methionine, were associated with flavor formation. This study provides a theoretical basis for the development of sturgeon processing industry and surimi products from the perspective of lipid changes.
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To investigate the effects of high hydrostatic pressure (HHP) on the conformation and IgG binding capacity, tropomyosin (TM) from Pacific oysters was subjected to high pressures of 300, 450 or 600 MPa. The results showed that the α-helix of TM with HHP-induced was decreased, while ß-turn, ß-sheet (predominantly) and random coil were increased. The surface hydrophobicity and sulfhydryl group content of TM were increased, while the fluorescence/UV intensity were decreased after HHP treatment. Atomic force microscopy (AFM) result exhibited that the morphology of TM was changed at 600 MPa and formed fibrous structures. The IgG binding capacity of TM and digested TM was markedly reduced when the pressure was increased, especially at 600 MPa. Overall, this study indicated that HHP-induced conformational changes in TM contributed to the reduction in IgG binding capacity. These findings suggested that HHP may be a promising non-thermal technology for producing hypoallergenic oyster products.
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Crassostrea , Tropomiosina , Animales , Presión Hidrostática , Tropomiosina/química , Estructura Secundaria de Proteína , Inmunoglobulina GRESUMEN
This study compared the effects of pan-fried with low (LPF), high (HPF) amounts of oil and deep-fried (DF) on the profiles of advanced glycation end products (AGEs) in sturgeon patties. The surface color of the pan-fried patties, regardless of the amounts of oil used, visually presented more brown than deep-fried ones with higher internal temperature at the frying course of 3-9 min. Compared to LPF and HPF, DF significantly accelerated the furosine development for 6-9 min of frying, dynamically increased the accumulation of CML (Nε-carboxymethyl-lysine) and CEL (Nε-carboxyethyl-lysine) for up to 9 min of frying, and the level of CML in DF than LPF, HPF for 9 min of frying were increased by 209.6 % and 149.9 %, respectively. The oil level employed for pan-fried insignificantly influenced the formation of furosine and CML in patties. The principal component analysis further confirmed that DF patties had a greater influence on the formation of AGEs. The AGEs formation was positively associated with the temperature and amino groups, while remarkably negative correlation with moisture content. Therefore, pan-fried within 6 min of frying was recommended for the domestic cooking of sturgeon patties based on the potential formation of AGEs.
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Peces , Productos Finales de Glicación Avanzada , Animales , Culinaria , Análisis de Componente PrincipalRESUMEN
Lactic acid bacteria (LAB) attenuate dextran sulfate sodium (DSS)-induced colitis in mice by restoring gut flora homeostasis and modulating the immune response. Because synchronous behavior can be controlled by autoinducer-2 (AI-2)/LuxS-mediated quorum sensing, the Caco-2 cell model and DSS-induced model in C57BL/6 mice were used to explore the unknown effects of these communications involving AI-2 among various intestinal symbiotic species. The results of the cell viability and lactate dehydrogenase leakage assays indicated that the tested strains (the wild-type strains and AI-2-deficient mutants) were characterized by equal cytoprotection from hydrogen peroxide-induced injury independently of AI-2. The results of the assays of multiple indicators and proinflammatory cytokines characteristic for the symptoms of colitis in mice showed that oral administration of AI-2-deficient mutants for 7 days was more effective in ameliorating inflammation than the treatment with the wild-type strains. The treatment with AI-2-deficient mutants enriched potential probiotics (e.g., Lactobacillaceae) and controlled the proliferation of potentially harmful bacteria (e.g., Helicobacteraceae) to achieve the transformation of intestinal flora. These mutants regulated short-chain fatty acids and the intestinal epithelial barrier, thereby promoting the maintenance of relatively favorable intestinal homeostasis. These results demonstrated that the AI-2-deficient mutants provided a more pronounced ameliorative effect on colitis in a mouse model, suggesting that the background of the LAB effect is associated with the alterations in colonic flora induced by AI-2.
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Herein, the microstructure and mechanical properties of hydrogels consisting of unrinsed sturgeon surimi (URSS) and plant-derived polysaccharides such as κ-carrageenan (KC), konjac gum (KG), xanthan gum (XG), guar gum (GG) and sodium alginate (SA), were studied by texture analysis, rheological measurement and scanning electron microscopy (SEM). Rheological results showed that the apparent viscosity, storage modulus (G') and loss modulus (Gâ³) of URSS increased by addition of KC, KG, GG and SA. The gel strength of resultant surimi products fabricated with KG/URSS mixture was significantly higher than that of other groups. KG could significantly improve the hardness (44.14 ± 1.14 N), chewiness (160.34 ± 8.33 mJ) and cohesiveness (0.56 ± 0.02) of the unrinsed surimi gel. Adding SA and KC had no significant effect on the textural characteristics of printed gels. However, an apparent decrease in the relevant mechanical properties of printed hydrogels was observed when XG and GG were added into surimi. SEM indicated that the incorporation of KG and KC could further integrate the gel structure of URSS as compared to hindering the cross-linking of surimi protein by XG and GG, which were in accordance with gel strength and water-holding capacity. These results provided useful information to regulate the 3D printing performance in functionalized surimi-based material.
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Quorum sensing of lactic acid bacteria, mediated by the Autoinducer-2 (AI-2)/LuxS system, positively regulates antibacterial activity, which is an effective strategy for aquatic product preservation. This study revealed that AI-2/LuxS system regulates the nutritional competitiveness of LAB by facilitating membrane transport systems in preservation of vacuum-packaged refrigerated shrimp (VPRS), using Lactiplantibacillus plantarum SS-128 wild-type and the luxS mutant strain. In VPRS preservation, organisms that cause spoilage and total volatile basic nitrogen were significantly lower in the VPRS inoculated with L. plantarum SS-128 than those inoculated with the luxS mutant strain (L. plantarum ΔluxS/SS-128) (p < 0.05). Simulations in vitro using diluted shrimp juice showed the growth inhibitory effects of wild-type strain SS-128 on the main VPRS spoilage microorganism Shewanella baltica. This could potentially be attributed to more efficient nutrient utilization, presumably mediated by AI-2/LuxS system, as revealed by interaction analysis. In support of this, in vitro nutritional competition test showed that L. plantarum SS-128 was more competitive for nutrients when cocultured with S. baltica under conditions of limited nutrient availability. Subsequently, an integrated analysis of transcriptomic and metabolomic revealed that AI-2/LuxS enables a continuous expansion of L. plantarum SS-128 by balancing energy expenditure followed by enhancing membrane transport systems, which is the main driven forces for it to occupy a favourable niche quickly. Our results showed that the AI-2/LuxS system may regulate the nutritional competitiveness of lactic acid bacteria and may be a regulatory strategy for biological preservation of aquatic products.
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Liasas de Carbono-Azufre , Lactobacillales , Antibacterianos , Proteínas Bacterianas , Homoserina/análogos & derivados , Lactonas , Nitrógeno , NutrientesRESUMEN
Milt is an underutilized fish processing by-product containing valuable nutrients for human health. Here, a gastrointestinal hydrolysate of degreased yellowtail (Seriola quinqueradiata) milt contained 70.6% arginine-rich protein, 20% nucleic acids, 7.1% minerals and 2.3% carbohydrates. Yellowtail milt hydrolysates (YMH) effectively attenuated the H2O2-induced burst of intracellular reactive oxygen species, plasma membrane impairment, loss of cell viability, interleukin 8 production and the expression of claudin-4 and occludin in Caco-2 cells with its protein fraction playing a greater antioxidant role than its nucleic acid fraction. YMH also significantly counteracted the tumor necrosis factor α- and interleukin 1ß-stimulated interleukin 8 production and cyclooxygenase-2 and inducible nitric oxide synthase expression in Caco-2 cells and inhibited the production of nitric oxide and proinflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells depending on its protein fraction, rather than its nucleic acid fraction. YMH and a positive drug 5-aminosalicylic acid were intragastrically administered to C57BL/6 mice daily for 7 days during and after 4-day dextran sodium sulphate exposure. Based on clinical signs, colon histopathology and biochemical analysis of colonic tight junction proteins, mucus compositions and goblet cells, YMH ameliorated mouse colitis symptoms and intestinal epithelial barrier dysfunction more effectively than 5-aminosalicylic acid. According to myeloperoxidase activity, proinflammatory cytokines and NF-κB, YMH and 5-aminosalicylic acid exerted equivalent inhibitory effects on colonic and systemic inflammation. Overall, YMH have considerable antioxidant and anti-inflammatory efficacies to maintain gut health.