A peptidomic approach for the identification of antioxidant and ACE-inhibitory peptides in sardinelle protein hydrolysates fermented by Bacillus subtilis A26 and Bacillus amyloliquefaciens An6

Antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities of sardinelle (Sardinella aurita) protein hydrolysates (SPHs) obtained by fermentation with Bacillus subtilis A26 (SPH-A26) and Bacillus amyloliquefaciens An6 (SPH-An6) were investigated. Both hydrolysates showed dose-dependent antioxidant activities evaluated using various in vitro antioxidant assays. Further, they were found to exhibit ACE-inhibitory activity. Peptides from SPH-A26 and SPH-An6 were analyzed by nESI-LC–MS/MS and approximately 800 peptides were identified. Identified peptides derived mainly from myosin (43% and 31% in SPH-An6 and SPH-A26, respectively). Several peptides identified in both hydrolysates were found to share sequences with previously identified antioxidant and ACE-inhibitory peptides based on Biopep database. Some of these peptides were selected for synthesis and their biological activities were evaluated. Among the synthesized peptides, NVPVYEGY and ITALAPSTM were found to be the most effective ACE-inhibitors with IC50 values of 0.21 and 0.23 mM, respectively. On the other hand, NVPVYEGY, which exhibited the highest ACE-inhibitory activity, showed the highest reducing power and peroxyl radical scavenging activities, followed by SLEAQAEKY and GTEDELDKY. The results of this study suggest that fermented sardinelle protein hydrolysates are a good source of natural antioxidant peptides and could have the potential to act as hypotensive nutraceutical ingredients.This work was funded by grant AGL2014-57367-R from MINECO and FEDER funds as well as the Ministry of Higher Education and Scientific Research-Tunisia and the Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138). JAEDOC-CSIC postdoctoral contract of L.M. cofunded by the European Social Found is also acknowledged. LC-MS/MS analysis was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.Peer reviewe

Effect of ultrasound pretreatment and Maillard reaction on structure and antioxidant properties of ultrafiltrated smooth-hound viscera proteins-sucrose conjugates

The effect of ultrasound (US) pre-treatment on the evolution of Maillard reaction (MR), induced between low molecular weight (LMW) peptides and sucrose, was studied. LMW peptides (<1 kDa) were obtained by the ultrafiltration of smooth hound viscera protein hydrolysates, produced by Neutrase, Esperase and Purafect. MR was induced by heating the LMW peptides in the presence of sucrose for 2 h at 90 °C, without or with US pre-treatment. During the reaction, a marked decrease in pH values, coupled to the increase in colour of the Maillard reaction products (MRPs), were recorded. In addition, after sonication, the glycation degree was significantly enhanced in Esperase-derived peptides/sucrose conjugates (p < 0.05). Moreover, results showed that thermal heating, particularly after US treatment, reduced the bitter taste and enhanced the antioxidant capacities of the resulting conjugates. Hence, it could be concluded that US leads to efficient mixing of sugar-protein solution and efficient heat/mass transfer, contributing to increase the MR rate.This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.Peer reviewe

Peptidomic analysis of bioactive peptides in zebra blenny (Salaria basilisca) muscle protein hydrolysate exhibiting antimicrobial activity obtained by fermentation with Bacillus mojavensis A21

The present study investigates the antibacterial activity of zebra blenny (Salaria basilisca) protein hydrolysates obtained by fermentation with a proteolytic bacterium, Bacillus mojavensis A21. The fermentative zebra blenny protein hydrolysate (FZPH), with a degree of hydrolysis (DH) of 17.35%, was fractionated by size exclusion chromatography on a Sephadex G-25 into six major fractions (F1-F6). Fraction F2, which exhibited antibacterial activity against several Gram-positive and Gram-negative bacteria, was further fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Fractions A and B from RP-HPLC exhibiting the highest antibacterial activity, were analysed using nano ESI-LC–MS/MS to identify the sequences of the peptides. A total of 28 and 41 peptides, containing from 8 to 31 amino acid residues, were identified in sub-fractions A and B, respectively. Further, identified bioactive peptides sharing sequences with previously identified peptides were reported. The results of this study suggest that FZPH is a good source of natural antimicrobial peptides and therefore, they could serve as a beneficial ingredient for nutraceuticals.This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.Peer reviewe

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.This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. Grant AGL2014-57367-R from MINECO (Spain) and FEDER funds and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged. LC-MS/MS analysis was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.Peer reviewe

Hypolipidemic, antiobesity and cardioprotective effects of sardinelle meat flour and its hydrolysates in high-fat and fructose diet fed Wistar rats

Aims: The present study aims to evaluate the antiobesity, hypolipidemic and cardioprotective effects of fermented sardinelle (Sardinella aurita) protein hydrolysates (FSPHs) produced with two proteolytic bacteria, Bacillus subtilis A26 (FSPH-A26) and Bacillus amyloliquefaciens An6 (FSPH-An6). Main methods: Wistar rats were fed during 10 weeks a standard laboratory diet, a high caloric diet (HCD) and a HCD coupled with the oral administration of sardinelle meat flour (SMF) or FSPHs. Key findings: HCD caused hyperlipidemia and increased body weight (BW). The daily oral administration of FSPHs or SMF reduced the total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-c) serum levels, and increased the level of high-density lipoprotein cholesterol (HDL-c). Nevertheless, FSPHs were found to be more efficient than SMF. FSPHs also lowered hepatic TC and TG content and decreased the pancreatic lipase activity. Further, the administration of FSPHs or SMF decreased the BW gain, the food intake and the relative epididymal adipose tissue weight. FSPHs exhibited a potent cardioprotective effect against heart attack, which was demonstrated by returning atherogenic indexes to their normal levels and the conservation of standard histological structure of the heart and aorta. Significance: The overall results indicate that FSPHs contained bioactive peptides which significantly attenuated hyperlipidemia, and might reduce the risk of cardiovascular disease (CVD) in rats fed HCD.This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. AGL2014-57367-R from MINECO and FEDER funds, and JAEDOC-CSIC post-doctoral contract of L.M. cofounded by the European Social Found are acknowledged. MALDI-TOF analysis was carried out by in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform.Peer reviewe

Combined biocatalytic conversion of smooth hound viscera: Protein hydrolysates elaboration and assessment of their antioxidant, anti-ACE and antibacterial activities

The aim of this study was to investigate the antioxidant, anti-ACE and antibacterial activities of smooth hound viscera hydrolysates (SHVHs). Viscera, as by-product, were hydrolyzed with crude alkaline protease extract from the same species, three commercial proteases and a combination endogenous and exogenous preparations. Hydrolysates showed different degrees of hydrolysis and the digestion with endogenous enzymes in combination with commercial proteases was found to enhance protein hydrolysis. The resulting SHVHs had high protein content (70.89%–89.06%) and they were mainly constituted by Gly, Glu and Gln, while Tau was the major free amino acid. In addition, they contained high amounts of UMP, Uridine, GMP and Guanosine, while undigested proteins were mainly rich in IMP and Xanthine. Furthermore, SHVHs showed different molecular mass distribution and RP-HPLC profiles proving their molecular mass and hydrophilic/hydrophobic peptide heterogeneity. All the SHVHs exhibited antioxidant activity, in terms of radical-scavenging activity, reducing power, metal chelating activity, β-carotene protection, lipid peroxidation inhibition and DNA breakage assay, and it was found that a medium degree of hydrolysis was appropriate to obtain hydrolysates with good antioxidant capacity. In addition, the SHVHs antioxidant activity was improved after simulated gastrointestinal digestion. Besides their antibacterial effect against several Gram-positive and Gram-negative bacteria, all the hydrolysates showed varying degrees of ACE inhibitory activities and the highest one was achieved by the Purafect hydrolysate (IC50 = 75 μg/mL). The overall data suggested that the SHVHs could be used as potential source of natural antioxidant, antimicrobial and anti-ACE peptides to formulate functional foods.Funded by the Ministry of Higher Education and Scientific Research, Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.Peer reviewe

Beneficial effects of fermented sardinelle protein hydrolysates on hypercaloric diet induced hyperglycemia, oxidative stress and deterioration of kidney function in wistar rats

This study investigated the potential effects of fermented sardinelle protein hydrolysates (FSPHs) obtained by two proteolytic bacteria, Bacillus subtilis A26 (FSPH-A26) and Bacillus amyloliquefaciens An6 (FSPH-An6), on hypercaloric diet (HCD) induced hyperglycemia and oxidative stress in rats. Effects of FSPHs on blood glucose level, glucose tolerance, α-amylase activity and hepatic glycogen content were investigated, as well as their effect on the oxidative stress state. Biochemical findings revealed that, while undigested sardinelle proteins did not exhibit hypoglycemic activity, oral administration of FSPHs to HCD-fed rats reduced significantly α-amylase activity as well as glycemia and hepatic glycogen levels. Further, the treatment with FSPHs improved the redox status by decreasing the levels of lipid peroxidation products and increasing the activities of the antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) and the level of glutathione in the liver and kidneys, as compared to those of HCD-fed rats. FSPHs were also found to exert significant protective effects on liver and kidney functions, evidenced by a marked decrease in alkaline phosphatase activity and a modulation of creatinine and uric acid contents. These results indicated the beneficial effect of FSPHs on the prevention from hyperglycemia and oxidative stress.This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.Peer reviewe