Discrimination of three commercial tuna species through species-specific peptides: From high-resolution mass spectrometry discovery to MRM validation.

The risk of tuna adulteration is high driven by economic benefits. The authenticity of tuna is required to protect both consumers and tuna stocks. Given this, the study is designed to identify species-specific peptides for distinguishing three commercial tropical tuna species. The peptides derived from trypsin digestion were separated and detected using ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) in data-dependent acquisition (DDA) mode. Venn analysis showed that there were differences in peptide composition among the three tested tuna species. The biological specificity screening through the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI BLAST) revealed that 93 peptides could serve as potential species-specific peptides. Finally, the detection specificity of species-specific peptides of raw meats and processed products was carried out by multiple reaction monitoring (MRM) mode based on a Q-Trap mass spectrometer. The results showed that three, one and two peptides of Katsuwonus pelamis, Thunnus obesus and Thunnus albacores, respectively could serve as species-specific peptides.

Assessment of total mercury content in tissues of Mullus argentinae in Rio de Janeiro, Brazil / Avaliação de mercúrio total em tecido comestível de Mullus argetinae coletados no Rio de Janeiro, Brasil

This study has as objective to determine total mercury (Total Hg) levels by atomic absorption spectrophotometry in 134 individuals edible part of Mullus argentinae, in two different fishing areas and two seasons in Rio de Janeiro State. Also, proximate composition was performed. Total Hg results in wet weight basis ranged from 0.0867 to 0.7476 µg.g-1 in muscle; 0.0023 to 0,1034 µg.g-1 in flippers; and 0.0177 to 0.1849 µg.g-1 in skin. Mean evaluated moisture was 73.39%; protein was 18.76%; lipid concentration of 5.36%; carbohydrates of 2.35%; and ashes were 0.85%.Results showed that Total Hg contents was lower than accepted limits established by regulatory organization. Higher averages were observed in muscle (0.2441 µg.g-1) when compared with skin (0.2386 µg.g-1) and flippers (0.0195 µg.g-1). In general, samples collected on summer showed higher values of total Hg when comparing to winter. Regarding beach areas there was no significant difference (p>0.05). We can conclude that this specie should be cautious consumed because of total Hg bioaccumulation characteristics, although neither levels were above limits established.

O objetivo deste estudo foi determinar o teor de mercúrio no tecido comestível de Mullus argentinae, conhecido como peixe trilha, espécie amplamente consumida no Rio de Janeiro, Brasil. Foi determinado o teor de mercúrio total (Hg total) por espectrofotometria de absorção atômica em 134 amostras, coletados em duas áreas e estações climáticas diferentes. Além disso, foi avaliada a composição centesimal das amostras. Os resultados de Hg total em peso úmido variaram de 0,0867 a 0,7476 µg.g-1 no músculo; 0,0023 a 0,1034 µg.g-1 nas nadadeiras; e 0,0177 a 0,1849 µg.g-1 na pele. Os valores médios da composição centesimal foram de 73,30% de umidade, 18,76% de proteína, 5,36% de lipídios, 2,35% de carboidratos e 0,85% de matéria mineral. Os resultados das 134 amostras analisadas demostraram que os teores de Hg Total apresentam concentração inferior aos limites aceitos pelos órgãos reguladores. As maiores médias foram observadas no músculo (0,2441 µg.g-1) quando comparadas à pele (0,2386 µg.g-1) e nadadeiras (0,0195 µg.g-1). Em geral, as amostras coletadas no verão apresentaram maiores valores de Hg total em relação ao inverno. Em relação aos locais de coleta não houve diferença significativa (p> 0,05). Podemos concluir que esta espécie deve ser consumida com cautela devido às características de bioacumulação do Hg total, apesar das médias apresentadas estarem abaixo dos limites estabelecidos pela legislação.

Reduction in flavor-intense components in fish protein hydrolysates by membrane filtration.

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. PRACTICAL APPLICATION: Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with >90% protein and peptide content, which is suitable for inclusion in foods.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle.

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices.

Elaboração de salsicha de peixe pargo Pagrus pagrus de tamanho reduzido proveniente da pesca de arrasto com baixo valor comercial / Development of fish sausage using low commercial value red porgy Pagrus pagrus

O objetivo deste trabalho foi desenvolver salsicha utilizando o pargo Pagrus pagrus de baixo valor comercial capturado na modalidade de pesca de arrasto e classificado como na categoria "mistura" por ter tamanho reduzido para o mercado varejista e realizar análises físico-químicas, microbiológicas, toxicológicas e sensoriais. A salsicha foi elaborada na planta piloto da Embrapa Agroindústria de Alimentos utilizando 50% de surimi e 50% de filé. As análises físico-químicas, microbiológicas e toxicológicas foram realizadas com métodos oficiais. O teste de aceitação foi realizado com 87 provadores que avaliaram o produto utilizando escala hedônica de sete pontos e também arguidos quanto à intenção de compra do produto. O produto foi considerado aceito quando 70% dos provadores atribuíram nota ≥ 4. Os resultados da composição centesimal foram: umidade 71,22%, proteínas 15,34%, lipídios totais 5,55%, carboidratos 6,11%, cinzas 1,78% e o valor energético 135,75 kcal. As avaliações microbiológicas e toxicológicas mostraram que os produtos apresentaram qualidade satisfatória conforme a legislação. A salsicha foi aceita por 82,7% dos provadores e o aspecto global do produto atingiu média de 4,93 (±1,69). Em relação à intenção de compra foi atribuída a maior porcentagem para talvez sim/talvez não (32%), seguido de provavelmente compraria (24%), e 28,1% informaram que provavelmente ou decididamente não comprariam. Com base nos resultados obtidos, conclui-se que o peixe de baixo valor agregado da categoria "mistura" resultou em produto embutido viável para o uso na tecnologia do pescado.

The aim of this work was to develop sausage using the low commercial value red porgy (Pagrus pagrus) caught in the "fish mix" category in the trawl fishery and to perform physicochemical, microbiological, toxicological and sensorial analysis. The sausage was prepared using 50% of surimi and 50% of fillet in Embrapa Food Agroindustry Pilot Plant. The physicochemical, microbiological, toxicological analyses were performed with official methods. The sensorial analyses were performed with 87 testers who evaluated the product using a hedonic scale of seven (7) points and were also accused of buying the product. The product was considered accepted when 70% of the tasters assigned a score ≥ 4. One sausage formulation was tested using 50% surimi. The results of the centesimal composition were: moisture 71.22%, proteins 15.34%, total lipids 5.55%, carbohydrates 6.11%, ash 1.78% and energy value 135.75 kcal. The parameters of microbiological and toxicological are within the current legislation for breaded products. The sausage was accepted obtaining 82.7% and the overall impression of the product reached a mean of 4.93 (± 1.69). Regarding intention to purchase for sausage, the highest percentage was attributed to maybe / perhaps not (32%), followed by likely to buy (24%), and 28.1% reported that they probably or decidedly would not buy. Based on the results obtained, it was concluded that the low commercial value fish of the "fish mix" category resulted in a viable product available for use in fish technology.

Mass spectrometric determination of disulfide bonds and free cysteine in grass carp IgM isoforms.

Disulfide bonds are fundamental in establishing Ig structure and maintaining Ig biological function. Here, we analysed disulfide bonds and free cysteine in three grass carp IgM isoforms (monomeric, dimeric/trimeric, and tetrameric IgM) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results revealed that Cys574 residue status at the C-terminal tail differed substantially in monomeric IgM in comparison with polymeric IgM, Cys574 was found as free thiol in monomeric IgM, while it formed disulfide linkages in dimeric/trimeric and tetrameric IgM. Five intra-chain disulfide bonds in the CH1~CH4 and CL1 domains, as well as one H-H and one H-L inter-chain disulfide linkages, were also observed and shown identical connectivity in monomeric, dimeric/trimeric, and tetrameric IgM. These findings represent the first experimental assignments of disulfide linkages of grass carp IgM and reveal that grass carp IgM isoform formation is due to alternative disulfide bonds connecting the Cys574 residue at the C-terminal tail.

[Detection of seven kinds of aquatic product allergens in meat products and seasoning by liquid chromatography-tandem mass spectrometry].

A liquid chromatography-tandem mass spectrometry method for the identification of marker peptides of aquatic product allergens and quantitative detection of multiple allergens in meat products and seasonings was developed. The samples were prepared by protein extraction, protein purification, and trypsin hydrolysis. The proteins and peptides were identified using ProteinPilot by the data analysis of the ion spectrum of polypeptide fragments using ultra-performance liquid chromatography-quadrupole/electrostatic orbitrap high-resolution mass spectrometry (UPLC-Q/Exactive-HRMS). The identification of 30 species-specific marker peptides in Penaeus vannamei, Eriocheir, Scylla serrata, Thunnus thynnus, and Atlantic salmon by comparison of the basic local alignment search tool (BLAST) with the UniProt database was achieved. The verification and multiple reaction monitoring (MRM) quantitative studies of these marker peptides were performed using a triple quadrupole mass spectrometry (UPLC-QqQ-MS) system. The proposed method showed a good linear relationship in the range of 5-250 mg/kg. The limits of quantitation and observed recoveries were in the range of 2-3.5 mg/kg and 88.7%-110.2%, respectively. This method presents various advantages such as good repeatability and high throughput, suitability for rapid screening, and quantitative analysis of seven aquatic allergens in meat products and seasonings.

Development and In-House Validation of an LC-MS and LC-MS/MS Assay for the Determination of Food Fraud for Different Fish Species.

Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISA- or DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC-MS and LC-MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC-electrospray-MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius (Pangasianodon hypophthalmus), salmon (Salmo salar), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), sole (Solea solea), lemon sole (Glyptocephalus cynoglossus), halibut (Reinhardtius hypoglossoides), red salmon (Oncorhynchus nerka), and great scallop (Pecten jacobaeus)] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC-MS and LC-MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future.

Adipose tissue contributes to hepatic pro-inflammatory response when dietary fish oil is replaced by vegetable oil in large yellow croaker (Larimichthys crocea): An ex vivo study.

The shortage of fish oil (FO) leads to the extensive use of vegetable oil (VO) in marine fish diets. High replacement percentage of dietary FO by VO induced pro-inflammatory response of adipose tissue (AT) and liver tissue (LT) in large yellow croaker (Larimichthys crocea). Mammalian studies showed that the secretion of cytokines by AT affected the immune response of LT. To investigate whether or not the inflammation response of LT is related to AT in large yellow croaker, LT and AT cells from fish fed FO diet (FOL and FOA) and VO diet (VOL and VOA) were co-cultured in a trans-well system, which resulted in an assembly of the two cells types sharing the culture medium but being separated by the membrane of the insert. Co-culture of FOL and FOA was selected as the control group (FOL-FOA). Results indicated that, when compared with the control group, the expression of pro-inflammatory genes (toll like receptors [TLRs], tumour necrosis factor α [TNFα], interleukin 1ß [IL1ß], suppressor of cytokine signalling 3 [SOCS3] and cyclooxygenase 2 [COX2]) in FOL was significantly increased in the co-culture group of FOL and VOA (FOL-VOA), while the expression of anti-inflammatory genes (arginase I [ArgI] and transforming growth factor ß1 [TGFß1]) in FOL was significantly depressed. On the contrary, a significantly depressed expression of pro-inflammatory genes (TLRs, TNFα, IL1ß and COX2) and increased expression of anti-inflammatory genes (interleukin 10 [IL10]) in VOL was observed in the co-culture group of VOL and FOA (VOL-FOA) when compared with the co-culture group of VOL and VOA (VOL-VOA). The change of immune-related gene expressions in LT cells was attributed to nuclear factor κB (NF-κB) signalling since the expression of the p65 protein was observed to show a similar trend to the expression of pro-inflammatory genes. It is speculated that dietary VO increased the secretion of cytokines, which induced pro-inflammatory response in LT cells. These ex vivo results indicate that AT plays a vital role in LT pro-inflammatory response in fish fed VO diet.

Skin Mucus Protein Profile, Immune Parameters, Immune-Related Gene Expression, and Growth Performance of Rainbow Trout (Oncorhynchus mykiss) Fed White Button Mushroom (Agaricus bisporus) Powder.

An 8-week feeding trial was performed to assess the effects of dietary white button mushroom (Agaricus bisporus) powder (WBMP) on the mucosal immunity and growth of rainbow trout (Oncorhynchus mykiss). Trout (n = 192; weight 13.76 ± 1.17 g) were stocked in 12 cages (65 × 65 × 65 cm) placed in 4 raceways with a flow-through water system. Trout were fed a basal diet (control group) or a basal diet supplemented with 0.5%, 1%, or 2% WBMP for 8 weeks. Evaluation of total protein levels and lysozyme activity in skin mucus revealed noticeable increases in trout fed 1% or 2% WBMP (P < 0.05), but no significant difference was observed with 0.5% WBMP administration (P > 0.05). The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed alterations in the protein profile of skin mucus following dietary administration of WBMP. Molecular studies showed a noticeable increase (P < 0.05) in tumor necrosis factor-α messenger RNA in the intestine of WBMP-fed trout, regardless of the inclusion level. Also, fish receiving the 1% or 2% WBMP treatments had a remarkable increase in interleukin (IL)-1ß expression compared with the control group (P < 0.05). In a similar way, intestinal IL-8 expression was upregulated with the 1% and 2% WBMP treatments (P < 0.05). However, no significant difference was found between the control group and the 0.5% WBMP treatment group in the case of IL-8 gene expression (P > 0.05). Furthermore, after 8 weeks of WBMP feeding, no improvement was seen in the growth parameters of trout compared with those fish fed the nonsupplemented diet (P > 0.05). These results hint at the potential immunomodulatory effects of dietary WBMP.

Comparison of proteomic profiles in the ovary of Sterlet sturgeon (Acipenser ruthenus) during vitellogenic stages.

One of the challenges of sturgeon aquaculture is that sturgeon takes an extended amount of time to reach sexual maturity. The pattern of the protein expression in relation to the late maturity of sturgeon can help to better understand changes in sexual maturity. 17ß-estradiol (E2), testosterone (T) and vitellogenin (Vtg) levels were examined at all stages of sexual maturation in Sterlet sturgeon (Acipenser ruthenus). Two-dimensional gel electrophoresis and mass spectrometry analysis were used to show the pattern of the ovarian proteins. The T levels increased from the previtellogenic to the postvitellogenic stages (P < 0.05) and Vtg showed a decremental pattern in pre- and postvitellogenic, and atresia (not significantly). The analysis showed 900 protein spots, 19 of which were successfully identified and had significant differences between the previtellogenic and the vitellogenic groups (P < 0.05). Among the identified proteins, 40% involved in cell defense (heat shock protein, Glutathione peroxidase, natural killer enhancing factor, peroxiredoxin-2), 30% in transcription and translation (constitutive photomorphogenesis 9 and Ybx2), 20% in metabolism and energy production (triose-phosphate isomerase (TPI)) and 10% in transport (glycolipid transfer protein). In the vitellogenic stage, the proteins were related to metabolism and energy production (TPI, ES1, creatin kinase, enolase, nucleoside diphosphate kinase, 50%), cell defense (thioredoxin and dislophid isomerase, 20%) and transport (fatty acid binding protein, 10%). Our findings show changes in protein expression pattern from cell defense to metabolism during egg development.

Characterization of protein hydrolysates from blue whiting (Micromesistius poutassou) and their application in beverage fortification.

Enzymatic hydrolysis of fish proteins has been employed as a principle method for converting under-utilised fish into valuable products for the pharmaceutical and health food industries. In this study, six commercial enzymes were tested for their ability to make fish protein hydrolysate powders from whole blue whiting. The chemical and functional properties of these powders were compared. The powders all had high solubility (>80%) across a wide pH range in water and their solubility improved further within a vitamin-tea beverage matrix (>85%). Varying degrees of anti-oxidant activities were recorded for the powders using three model systems (DPPH, ferrous chelating and reducing power). This study demonstrates that commercial enzymes are useful for the extraction and alteration of fish protein from a low value source to produce highly digestible, low molecular weight peptide powders that could be used as a fortifying health ingredient, especially in beverages.

Novel bioactive peptides from enzymatic hydrolysate of Sardinelle (Sardinella aurita) muscle proteins hydrolysed by Bacillus subtilis A26 proteases.

Sardinelle protein hydrolysate (SPH), prepared by treatment with Bacillus subtilis A26 proteases, was found to exhibit antibacterial, antioxidant and ACE-inhibitory activities. SPH, with a degree of hydrolysis of 4%, was fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (F1-F5). F2, which exhibited the highest antibacterial and ACE-inhibitory activities, and F4, which exhibited the highest antibacterial and antioxidant activities, were further fractionated by reverse phase-high performance liquid chromatography (RP-HPLC) and then analysed using nano-ESI-LC-MS/MS to identify the sequences of peptides. Eight peptides were identified in the sub-fraction F2-A, nine peptides in the sub-fraction F4-B, and 45 peptides in F4-C. Identified peptides were found to share sequences with previously described bioactive peptides based on Biopep database. The results of this study suggest that SPH is a good source of natural bioactive peptides. Hence, it can be used as a potential ingredient in nutraceutical field.

Digital gene expression analysis of Takifugu rubripes brain after acute hypoxia exposure using next-generation sequencing.

The adverse effects of hypoxia are confined to biochemical, physiological, developmental and behavioral processes, especially injury of the brain. In this study, a subset of genes in the brain of Takifugu rubripes were analyzed using digital gene expression (DGE) profiles and next-generation sequencing after acute hypoxia. Among 32 differentially expressed genes, 29 were up-regulated and 3 were down-regulated following hypoxia exposure. Using Gene Ontology analysis, it was found that transcription and translation, metabolism, and the stress response were affected by exposure to hypoxia. KEGG analysis revealed that the neuroactive ligand-receptor interaction pathway was significantly enriched in hypoxia-exposed T. rubripes. To further confirm the differential expression of genes, quantitative real-time PCR was performed to test six candidate genes, with the following five genes exhibiting the same expression patterns as the sequencing results: Proto-oncogene c-fos, Kruppel-like factor 2, immediate early response 2, proopiomelanocortin A and rhodopsin. This work is the first to identify and annotate genes in T. rubripes affected by hypoxia stress. This investigation provides data for understanding the molecular mechanism of fish adaptation to hypoxia and provides a reference for rationally setting dissolved oxygen levels in aquaculture.

Hidrolisado proteico de resíduo de sardinha como atrativo alimentar para juvenis de jundiá / Sardine waste protein hydrolisate as feeding stimulant for silver catfish juveniles

O objetivo deste estudo foi avaliar a utilização do hidrolisado proteico de resíduo de sardinha como atrativo na alimentação do Rhamdia quelen. No experimento 1, foram utilizados os seguintes atrativos alimentares: 1. extrato aquoso de músculo de tilápia-do-Nilo (controle positivo); 2. hidrolisado proteico de resíduo de sardinha com baixo grau de hidrólise (GH); 3. hidrolisado proteico de resíduo de sardinha com alto GH; 4. hidrolisado proteico de resíduo de sardinha com alto GH diluído (10% da concentração) e 5. controle usando somente água destilada. Após jejum de 48 horas, o comportamento foi registrado em vídeo por um período basal de dois minutos e por mais 18 minutos após a inoculação do atrativo. O delineamento foi inteiramente ao acaso, com três tratamentos e 20 repetições. O experimento 2 foi realizado para avaliar a capacidade do hidrolisado proteico de estimular a ingestão de alimento em juvenis de jundiá. Para isso, foram confeccionados pellets de ágar contendo ou não hidrolisado proteico de resíduo de sardinha. Os peixes foram avaliados individualmente e tiveram um período de adaptação de sete dias. Os resultados foram analisados por meio do teste de proporção de Goodman (1964). A inoculação dos hidrolisados com alto e baixo GH aumentou o tempo de movimentação dos barbilhões. O hidrolisado com alto GH diluído proporcionou os mesmos resultados que o hidrolisado com baixo GH , mas as médias não diferiram das obtidas para a água destilada (controle negativo) e do extrato de músculo. O incremento na movimentação de um lado para outro do aquário foi maior (P<0,05) para os hidrolisados com alto e baixo GH. No experimento 2, a proporção de peixes que ingeriu os pellets contendo hidrolisado proteico de resíduo de sardinha com alto GH foi maior (P<0,05) em relação aos que ingeriram os pellets contendo água destilada. O hidrolisado proteico foi eficiente para estimular o comportamento associado à alimentação em juvenis de Rhamdia quelen.(AU)

The aim of this study was to evaluate the use of the sardine waste hydrolysate as a feeding stimulant for Rhamdia quelen juveniles. In experiment 1 the following feeding stimulants were evaluated: 1. Aqueous extract of Nile tilapia muscle; 2. sardine waste protein hydrolysate with a low degree of hydrolysis (DH); 3. Sardine waste protein hydrolysate with high GH; 4. sardine waste protein hydrolysate with high GH diluted (10% concentration) and 5. control using only distilled water. The fish were evaluated individually. After 48 hours fasting, the behavior was videotaped for a baseline period of 2 minutes, and for another 18 minutes after attractive inoculation. The design was completely randomized with three treatments and twenty repetitions. Experiment 2 was conducted to evaluate the effect of the sardine waste protein hydrolysate on the food intake of silver catfish. For this purpose agar pellets were produced containing or not sardine waste protein hydrolysate. The fish were evaluated individually and had an adjustment period of 7 days. The results were analyzed using the Goodman test (1964). Inoculation of the sardine waste protein hydrolysate with high and low GH increased the barbel movement time. The sardine waste protein hydrolyzate diluted with high GH yielded the same results as the hydrolysate with low GH, but did not differ from the average obtained for distilled water (negative control) and muscle extract. The increase in moving side to side in the aquarium was higher (P<0.05) for sardine waste protein hydrolysate with high and low GH. In experiment 2 the proportion of fish that ingested the pellets containing sardine waste protein hydrolysate was higher (P<0.05) than the proportion of fish that ingested the pellets containing distilled water. The sardine waste protein hydrolysate was efficient to stimulate the feeding associated behavior in Rhamdia quelen juveniles.(AU)

Antioxidant activity measurement and potential antioxidant peptides exploration from hydrolysates of novel continuous microwave-assisted enzymolysis of the Scomberomorus niphonius protein.

A novel continuous microwave-assisted enzymatic digestion (cMAED) method is proposed for the digestion of protein from Scomberomorus niphonius to obtain potential antioxidant peptides. In this study, bromelain was found to have a high capacity for the digestion of the Scomberomorus niphonius protein. The following cMAED conditions were investigated: protease species, microwave power, temperature, bromelain content, acidity of the substrate solution, and incubation time. At 400W, 40°C, 1500U·g-1 bromelain, 20% substrate concentration, pH 6.0 and 5min incubation, the degree of hydrolysis and total antioxidant activity of the hydrolysates were 15.86% and 131.49µg·mL-1, respectively. The peptide analyses showed that eight of the potential antioxidant peptide sequences, which ranged from 502.32 to 1080.55Da with 4-10 amino acid residues, had features typical of well-known antioxidant proteins. Thus, the new cMAED method can be useful to obtain potential antioxidant peptides from protein sources, such as Scomberomorus niphonius.

European Carp (Cyprinus carpio L.) Protein-Derived Ex Vivo Digests and In Vitro Hydrolysates Differ in the ACE I Inhibitory Activity and Composition of Released ACE Inhibitory Peptides.

Carp muscle tissue is a valuable source of biologically active constituents known to positively influence human health. In this study, carp protein digests/hydrolysates generated by human/ porcine digestive enzymes were analysed for their angiotensin I-converting enzyme inhibitory (ACEi) activity. The ex vivo digests and in vitro hydrolysates were used in a screening for ACEi peptides as well. Carp proteins were hydrolysed more rapidly by human gastrointestinal juices than by porcine enzymes. Sarcoplasmic protein fractions were digested/hydrolysed more easily than the myofibrillar ones. The inhibitory concentrations at 50% (IC50) for ex vivo digested sarcoplasmic and myofibrillar protein fractions were established at 1.50 and 1.04 mg/mL, respectively. While, for in vitro hydrolysed sarcoplasmic and myofibrillar protein fractions, the IC50 values were calculated as 2.57 and 1.12 mg/mL, respectively. The digested/hydrolysed samples of carp sarcoplasmic and myofibrillar protein fractions were separated by RP-HPLC-MS. Amino acid sequences were identified with the use of the LC-MS/MS method coupled with in silico systematic screening for ACEi peptides. ACEi peptides with IVY, IY, VY, ALPHA and VKAGF sequences were found in the carp digests/hydrolysates. In the ex vivo carp digest, an ACEi peptide TVY was also detected, while the ACEi peptide IW was identified in in vitro hydrolysate. Our study showed that different ACEi effects of carp digests and hydrolysates were generated with the use of human gastrointestinal and porcine enzymes.

Cloning HSP70 and HSP90 genes of kaluga (Huso dauricus) and the effects of temperature and salinity stress on their gene expression.

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. KP050541) and HSP90 (GenBank accession no. KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98-99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P < 0.05).We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of kaluga under different combinations of temperature and salinity stress, including temperatures of 4,10, 25, and 28 °C at 0 ppt salinity, and salinities of 10, 20, 30, and 40 ppt at 16 °C, where 16 °C at 0 ppt (parts per thousand) served as the control. We found that levels of mRNA expression of both HSP70 and HSP90 were highest at 4 °C in the muscle, gill, and liver and changed little with salinity stress. These results increase understanding of the mechanisms of stress response of cold freshwater fish.

Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.

N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.