Search

Endotoxins carried within LDL are cleared from the circulation via hepatic LDL receptor (LDLR)-mediated endocytosis. Proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces this clearance by down-regulating LDLR density on hepatocytes. In addition to hepatocytes, vascular endothelial cells also express receptor targets of PCSK9, including LDLR. Therefore, we hypothesized that PCSK9 may regulate vascular endothelial cell uptake of lipopolysaccharide (LPS) and alter the vascular endothelial cell inflammatory response.We found that LPS is internalized by human umbilical vein vascular endothelial cells (HUVECs) and LPS uptake dose-dependently increased with increasing LDL concentration. Intracellular LPS co-localized with LDL. PCSK9 and, separately, blocking antibodies against LDLR, dose-dependently decreased the vascular endothelial cell uptake of LPS and, furthermore, inhibition of endocytosis using Dynasore blocked LPS uptake. In contrast, blocking antibodies against TLR4 did not alter LPS uptake. PCSK9 decreased the LPS-induced proinflammatory response (IL-6 and IL-8 gene expression and protein secretion, and VCAM-1/ICAM-1 expression) in vascular endothelial cells. In addition, a decrease in PCSK9 and increase in LDLR, mediated by triciribine or siPCSK9, increased LPS uptake and the LPS-induced proinflammatory response. Similar results were also found in aortic vascular tissue from Pcsk9Our data suggest that, similar to PCSK9 treatment in hepatocytes, PCSK9 reduces vascular endothelial cell uptake of LPS via LDLR-mediated endocytosis. Consequently, PCSK9 decreases the LPS-induced proinflammatory response in vascular endothelial cells. These results raise the possibility that PCSK9 inhibition may have additional effects on vascular endothelial inflammation via this alternative pathway, beyond the known effects of PCSK9 inhibition on LDL lowering and hepatic endotoxin clearance.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

Lab-grown bovine meat analogues are emerging alternatives to animal sacrifices for cultured meat production. The most challenging aspect of the production process is the rapid proliferation of cells and establishment of the desired 3D structure for mass production. In this study, we developed a direct ink writing-based 3D-bioprinted meat culture platform composed of 6% (w/v) alginate and 4% (w/v) gelatin (Alg/Gel)-based hydrogel scaffolds supplemented with naturally derived protein hydrolysates (PHs; 10%) from highly nutritive plants (soybean, pigeon pea, and wheat), and some selected edible insects (beetles, crickets, and mealworms) on in vitro proliferation of bovine myosatellite cells (bMSCs) extracted from fresh meat samples. The developed bioink exhibited excellent shear-thinning behavior (

11 Páginas.-- 5 Figuras.-- 2 Tablas, We have previously reported the in vitro hypocholesterolemic, anti-inflammatory, and antioxidant effects of Alcalase-generated lupin protein hydrolysate (LPH). Given that lipoprotein deposition, oxidative stress, and inflammation are the main components of atherogenesis, we characterized the LPH composition, in silico identified LPH-peptides with activities related to atherosclerosis, and evaluated the in vivo LPH effects on atherosclerosis risk factors in a mouse model of atherosclerosis. After 15 min of Alcalase hydrolysis, peptides smaller than 8 kDa were obtained, and 259 peptides out of 278 peptides found showed biological activities related to atherosclerosis risk factors. Furthermore, LPH administration for 12 weeks reduced the plasma lipids, as well as the cardiovascular and atherogenic risk indexes. LPH also increased the total antioxidant capacity, decreased endothelial permeability, inflammatory response, and atherogenic markers. Therefore, this study describes for the first time that LPH prevents the early stages of atherosclerosis., Ministerio de Economía y Competitividad, Gobierno de España [AGL2012-40247-C02-01 and AGL2012-40247-C02-02], Consejería de Salud, Junta de Andalucía [PC-0111-2016-0111 and PEMP-0085-2020], and the Programa PAIDI from the Junta de Andalucía [CTS160]. G.S.-S. was supported by Formación Profesorado Universitario grants from the Ministerio de Educación, Cultura y Deporte, Gobierno de España [FPU16/02339]. I.C.-C. was supported by a postdoctoral fellowship from the Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía [DOC_00587/2020]. N.Á.-S. was supported by a fellowship from the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) [RD12/0043/0012 from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Gobierno de España]. B.R.-O. was supported by a grant from the Programa de Empleo Juvenil of Ministerio de Empleo y Seguridad Social, Gobierno de España [EJ-086]. A.I.Á.-L. was funded by the Consejería de Salud, Junta de Andalucía [PI-0136-2019]. I.B. was supported by the VI Plan Propio de Investigación y Transferencia of Universidad de Sevilla [VI PPIT-US].

Nattokinase (NK), a protease enzyme produced by Bacillus subtilis, has various biological effects such as lipid-lowering activity, antihypertensive, antiplatelet/anticoagulant, and neuroprotective effects. Exposure to environmental toxicants such as bisphenol A (BPA) or γ-radiation (IR) causes multi-organ toxicity through several mechanisms such as impairment of oxidative status, signaling pathways, and hepatic and neuronal functions as well as disruption of the inflammatory responses. Therefore, this study is designed to evaluate the ameliorative effect of NK against BPA- or IR-induced liver and brain damage in rats. Serum ammonia level and liver function tests were measured in addition to brain oxidative stress markers, amyloid-beta, tau protein, and neuroinflammatory mediators. Moreover, relative quantification of brain nuclear factor-erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) genes, as well as apoptotic markers in brain tissue, was carried out in addition to histopathological examination. The results showed that NK improved liver functions, impaired oxidative status, the cholinergic deficits, and minified the misfolded proteins aggregates. Furthermore, NK alleviated the neuroinflammation via modulating NF-κB/Nrf2/HO-1 pathway and glial cell activation in addition to their antiapoptotic effect. Collectively, the current results revealed the protective effect of NK against hepatic and neurotoxicity derived from BPA or IR.

Soybean dregs are one of the most important albeit underutilized byproducts in soybean processing. In this study, soluble soybean polysaccharides with lipid-lowering activity were extracted from soybean dregs and used as a wall material for embedding metformin. Metformin-soluble soybean polysaccharide nanoparticles (MET-SSPS-NPs) were prepared by electrostatic interaction. The lipid-lowering activity and possible mechanism of MET-SSPS-NPs were investigated. Western blotting was used to detect the expression levels of cell-related protein proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR)

Background: PCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9. Methods: We obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay. Results: Median plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant ( P =0.83 [95% CI, −6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P =0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (R S =−0.3634; P Conclusions: Administration of UFH does not result in a clinically meaningful effect on circulating PCSK9 among an unselected population of humans. The results cast doubt on the clinical utility of disrupting the PCSK9:HSPG interaction as a general therapeutic strategy for PCSK9 inhibition. However, the observations suggest that in selected populations, disrupting the PCSK9:HSPG interaction could still affect PCSK9 reuptake and offer a therapeutic benefit.

The current research work aims at optimization, production, purification and evaluation of fibrinolytic extracellular protease from Bacillus subtilis VITMS2 isolated from fermented milk of Vigna unguiculata. The optimal production was achieved at 4.0% inoculum, pH7.0, 30 °C with (1% w/v) sucrose, (2% w/v) soya bean meal and (2% w/v) malt extract and 10 mM of CaCl2, MgSO4, Na2HPO4 and K2HPO4. The clear cell-free supernatant was purified using conventional ammonium sulphate salt fractionation (75%), ultrafiltration, ion-exchange (DEAE Sepharose FF) and gel filtration (Sephadex G-50). The molecular mass was determined to be 29 kDa using SDS-PAGE analysis. The purified enzyme showed strong fibrinolytic activity with a specific activity of 2418.85 U/mg and has a yield of 12.01%. The enzyme was highly stable up to 60 °C and a pH range of 10.0 until 72 h of incubation. The purified enzyme showed 97.4% in vitro thrombolytic activity. The Km and Vmax values of the enzyme was determined to be 0.0114 mM and 147.8 µmol min−1 using the chromogenic substrate S-7388. IC50 of ace inhibition was assessed to be 0.06 mg/mL suggesting anti-hypertensive property of the fibrinolytic enzyme. The above-obtained ace-inhibition results was supported by in silico molecular docking studies which revealed better binding affinity of nattokinase with a HADDOCK score of − 22.0 ± 8.5 confirms affinity towards angiotensin converting enzyme.

Cholesterol is central to pancreatic β-cell physiology and alterations of its homeostasis contribute to β-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane. PCSK9 is also expressed in the pancreas and loss of function mutations of PCSK9 result in higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Aim of this study was to investigate whether PCSK9 also impacts β-cells function.Pancreas-specific Pcsk9 null mice (Pdx1Cre/Pcsk9 fl/fl) were generated and characterized for glucose tolerance, insulin release and islet morphology. Isolated Pcsk9-deficient islets and clonal β-cells (INS1E) were employed to characterize the molecular mechanisms of PCSK9 action.Pdx1Cre/Pcsk9 fl/fl mice exhibited normal blood PCSK9 and cholesterol levels but were glucose intolerant and had defective insulin secretion in vivo. Analysis of PCSK9-deficient islets revealed comparable β-cell mass and insulin content but impaired stimulated secretion. Increased proinsulin/insulin ratio, modifications of SNARE proteins expression and decreased stimulated‑calcium dynamics were detected in PCSK9-deficient β-cells. Mechanistically, pancreatic PCSK9 silencing impacts β-cell LDLR expression and cholesterol content, both in vivo and in vitro. The key role of LDLR is confirmed by the demonstration that LDLR downregulation rescued the phenotype.These findings establish pancreatic PCSK9 as a novel critical regulator of the functional maturation of the β-cell secretory pathway, via modulation of cholesterol homeostasis.

Background: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. Methods: We used Ldlr -/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr -/- mouse macrophages. Results: In Ldlr -/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1β (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr -/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. Conclusions: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

Sickle cell disease (SCD) is with associated altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 uM) or medroxyprogesterone (6.75 uM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

Low molecular weight (lt;5 kDa) peptides from mussels (

Background: MT1-MMP (membrane-type 1 matrix metalloproteinase, MMP-14) is a transmembrane-anchored protein with an extracellular proteinase domain and a cytoplasmic tail devoid of proteolytic functions but capable of mediating intracellular signaling that regulates tissue homeostasis. MT1-MMP extracellular proteolytic activity has been shown to regulate pathological remodeling in aortic aneurysm and atherosclerosis. However, the role of the nonproteolytic intracellular domain of MT1-MMP in vascular remodeling in abdominal aortic aneurysms (AAA) is unknown. Methods: We generated a mutant mouse that harbors a point mutation (Y573D) in the MT1-MMP cytoplasmic domain that abrogates the MT1-MMP signaling function without affecting its proteolytic activity. These mice and their control wild-type littermates were subjected to experimental AAA modeled by angiotensin II infusion combined with PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression and high-cholesterol feeding. Results: The mutant mice developed more severe AAA than the control mice, with concomitant generation of intraaneurysmal atherosclerotic lesions and dramatically increased macrophage infiltration and elastin degradation. Aortic lesion-associated and bone marrow–derived macrophages from the mutant mice exhibited an enhanced inflammatory state and expressed elevated levels of proinflammatory Netrin-1, a protein previously demonstrated to promote both atherosclerosis and AAA. Conclusions: Our findings show that the cytoplasmic domain of MT1-MMP safeguards from AAA and atherosclerotic plaque development through a proteolysis-independent signaling mechanism associated with Netrin-1 expression. This unexpected function of MT1-MMP unveils a novel mechanism of synchronous onset of AAA and atherogenesis and highlights its importance in the control of vascular wall homeostasis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis. Cilostazol exerts favorable cellular and metabolic effects; however, the effect of cilostazol on the expression of PCSK9 has not been previously reported. Our study aimed to investigate the potential mechanisms of action of cilostazol on the expression of PCSK9 and lipid homeostasis. We evaluated the effects of cilostazol on the expression of PCSK9 in HepG2 cells and evaluated potential molecular mechanisms by measuring signaling molecules in the liver and serum lipid profiles in high-fat diet-induced obese mice and normal chow-fed mice. Cilostazol treatment significantly induced the messenger RNA and protein expression of PCSK9 in HepG2 cells and enhanced PCSK9 promoter activity. Chromatin immunoprecipitation assays confirmed that cilostazol treatment enhanced PCSK9 transcription by binding to peroxisome proliferator-activated receptor-γ (PPARγ) via the PPARγ DNA response element. PPARγ knockdown attenuated the stimulatory effect of cilostazol on PCSK9. In vitro, cilostazol treatment increased PCSK9 expression in vehicle-treated HepG2 cells but decreased PCSK9 expression in palmitic acid-treated HepG2 cells. In vivo, cilostazol treatment increased the serum levels of PCSK9 in normal mice but significantly reduced PCSK9 levels in obese mice. The expressions of PCSK9-relevant microRNAs also showed similar results. Clinical data showed that cilostazol treatment significantly reduced serum PCSK9 levels in patients with obesity. The obesity-dependent effects of cilostazol on PCSK9 expression observed from bench to bedside demonstrates the therapeutic potential of cilostazol in clinical settings.

This study aimed to produce bioactive protein hydrolysates from undervalued fish, namely Baltic herring, and its filleting by-products. Protein hydrolysates were produced with Alcalase and Flavourzyme to achieve effective hydrolysis. The hydrolysates were evaluated for chemical composition, molecular weight distribution, antioxidant capacity, dipeptidyl-peptidase 4 (DPP4) inhibitory activity, effects on cell proliferation and surface hydrophobicity. The protein content of the hydrolysates was high, from 86% to 91% (dm), while the fat content was low, from 0.3% to 0.4% (dm). The hydrolysates showed high DPP4 inhibition activities with IC

Endothelial dysfunction plays a central role in the pathogenesis of sepsis-mediated multiple organ failure. Several clinical and experimental studies have suggested that the glycocalyx is an early target of endothelial injury during an infection. Colivelin, a synthetic derivative of the mitochondrial peptide humanin, has displayed cytoprotective effects in oxidative conditions. In the current study, we aimed to determine the potential therapeutic effects of colivelin in endothelial dysfunction and outcomes of sepsis in vivo. Male C57BL/6 mice were subjected to a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP) and were treated with vehicle or colivelin (100-200 µg/kg) intraperitoneally at 1 h after CLP. We observed that vehicle-treated mice had early elevation of plasma levels of the adhesion molecules ICAM-1 and P-selectin, the angiogenetic factor endoglin and the glycocalyx syndecan-1 at 6 h after CLP when compared to control mice, while levels of angiopoietin-2, a mediator of microvascular disintegration, and the proprotein convertase subtilisin/kexin type 9, an enzyme implicated in clearance of endotoxins, raised at 18 h after CLP. The early elevation of these endothelial and glycocalyx damage biomarkers coincided with lung histological injury and neutrophil inflammation in lung, liver, and kidneys. At transmission electron microscopy analysis, thoracic aortas of septic mice showed increased glycocalyx breakdown and shedding, and damaged mitochondria in endothelial and smooth muscle cells. Treatment with colivelin ameliorated lung architecture, reduced organ neutrophil infiltration, and attenuated plasma levels of syndecan-1, tumor necrosis factor-α, macrophage inflammatory protein-1α and interleukin-10. These therapeutic effects of colivelin were associated with amelioration of glycocalyx density and mitochondrial structure in the aorta. At molecular analysis, colivelin treatment was associated with inhibition of the signal transducer and activator of transcription 3 and activation of the AMP-activated protein kinase in the aorta and lung. In long-term outcomes studies up to 7 days, co-treatment of colivelin with antimicrobial agents significantly reduced the disease severity score when compared to treatment with antibiotics alone. In conclusion, our data support that damage of the glycocalyx is an early pathogenetic event during sepsis and that colivelin may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Oxidative stress is a major cause of cardiovascular diseases (CVDs). Antioxidant peptides have potential to improve CVDs. The aim of this study was to develop novel antioxidant peptides from silver carp muscle hydrolysates (SCHs) and investigate their gastrointestinal digestion (GID) stability, and bioavailability. Results showed that SCHs generated by Alcalase and Papain exhibited the highest free radical scavenging activity, peroxynitrite scavenging activity and low density lipoprotein (LDL) oxidation inhibitory capacity. All these antioxidant activities were enhanced in different SCHs after GID. Notably, the GID products of Papain-SCH and Alcalase-SCH possessed potent ameliorating effects on oxidative stress injury in endothelial cells. After transepithelial transport, total of nine peptides was identified from permeates of GID products of Papain-SCH and Alcalase-SCH. The VKVGNEF and MEAPPH were both novel peptides in the current study that possess the highest activities in scavenging free radicals, inhibiting LDL oxidation and alleviating oxidative stress injury in endothelial cells.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Enzymatic hydrolysis could be one of the crucial means to limit the allergenicity of allergens. The allergenicity of pea peptides was evaluated using indirect ELISA and RBL-2H3 cell assay, thereby obtaining hypoallergenic pea peptide sequences. Results indicated that pea protein-sensitized mice produced higher levels of total IgG1 and IgE antibodies than the mice in the control group (P < 0.05). Moreover, the allergenicity of hydrolysates decreased significantly after enzymolysis, and the allergenicity of ultrafiltration component F1 and purified component F1–2 was significantly lower than that of other isolated and purified components (P < 0.05). Furthermore, ADLYNPR identified from F1–2 had lower binding capacity to specific IgE and IgG1 and lower degree of cell degranulation with a higher EC50 value of 6.63 ng mL−1, which was about 36.83 times that of pea protein (P < 0.05). Based on the above results, ADLYNPR might be a potential source of hypoallergenic peptides.

Background: modern society, there is a tendency to consume products with natural origins and minimum chemical additives. This has encouraged the replacement of synthetic antioxidants for the ones obtained from natural sources, such as the antioxidants acquired from enzymatic protein hydrolysates. Objective: In this study, the process of enzymatic hydrolysis of proteins from bovine plasma, which produces hydrolysates with an Antioxidant Capacity (AC), was scaled up from 1 to 5 L. Methods: An experimental design was developed in 1 L to evaluate the effect of the Substrate concentration (So) on the time needed to reach a Degree of Hydrolysis (DH) of 20% as well as the AC. Results: The best conditions in the 1 L reactor controlled by a Titrando 842 were transferred to 5L in a BioFlo310 reactor. These conditions were achieved at a ratio of 80g/L of the substrate and 0.89 AU of Alcalase 2.4L/g of the substrate in order to obtain a level of 16.36 ± 0.21min of the 20% of DH and antioxidant capacity of 58.98 ± 1.80%. Conclusion: The results showed that DH depends significantly on So, while the antioxidant capacity only depends on the DH. Additionally, the dimensional analysis using Re as a scaling criterion allowed us to obtain the same results in the model (1 L) and the prototype (5 L).

Milk-derived protein hydrolysates have generated a great deal of interest recently due to their numerous beneficial health effects. However, there are few comparative studies on protein hydrolysates from different dairy species, their production, characterization, and bioactivity. In the present study, skimmed milk from both major and minor dairy species was hydrolyzed with alcalase, and its protein profiles were studied using tricine polyacrylamide gel electrophoresis and reverse phase-high protein liquid chromatography. The antioxidant and in vitro immunostimulatory properties were determined.Iron chelation activity was highest in hydrolysates of whey (25.00 ± 0.32 mmol LThis study comparatively evaluates the antioxidative and immunomodulatory properties of different skimmed MPHs and their potential applications as ingredients in pediatric, geriatric, and other health-promoting foods. © 2022 Society of Chemical Industry.

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) deficiency leads to lower cholesterol and is associated with reduced vascular complications in the general population. Cholesterol lowering may also have beneficial effects in sickle cell disease (SCD). The objective of this study was to determine effects of PCSK9 deficiency in a mouse model of SCD. Bone marrow transplantation (BMT) was performed from donor SCD mice to wild-type, PCSK9-deficient, and LDLR-deficient recipients to generate SCD controls (Pcsk9+/+, SCDbmt) with preserved PCSK9 status, SCD mice with deficiency of PCSK9 (Pcsk9−/−, SCDbmt), and SCD mice with deficiency of LDLR (Ldlr−/−, SCDbmt). Although cholesterol levels were lower in Pcsk9−/−, SCDbmt mice compared to Pcsk9+/+, SCDbmt mice, anemia was more severe in Pcsk9−/−, SCDbmt mice. Increased reticulocytosis, enhanced ex vivo erythrocyte sickling, and increased erythrocyte phosphatidylserine exposure was also observed. Livers, spleens, and kidneys contained increased iron in Pcsk9−/−, SCDbmt mice compared to Pcsk9+/+, SCDbmt mice consistent with greater hemolysis. SCD mice with deficiency of LDLR (Ldlr−/−, SCDbmt mice) had similar anemia as Ldlr+/+, SCDbmt mice despite higher serum cholesterol. In conclusion, deficiency of PCSK9 is associated with worsened anemia in SCD mice due to increased hemolysis. These findings may have implications for lipid-lowering strategies in patients with SCD, as well as for potential novel modifiers of anemia severity.

Shiga-toxigenic Escherichia coli (STEC) is a major bacterium responsible for disease resulting from foodborne infection, including bloody diarrhea and hemolytic uremic syndrome. STEC produces important virulence factors such as Shiga toxin (Stx) 1 and/or 2. In the STEC family, some locus of enterocyte effacement-negative STEC produce two different types of cytotoxins, namely, Stx2 and subtilase cytotoxin (SubAB). The Stx2 and SubAB cytotoxins are structurally similar and composed of one A subunit and pentamer of B subunits. The catalytically active A subunit of SubAB is a subtilase-like serine protease and specifically cleaves an endoplasmic reticulum (ER) chaperone 78-kDa glucose-regulated protein (GRP78/BiP), a monomeric ATPase that is crucial in protein folding and quality control. The B subunit binds to cell surface receptors. SubAB recognizes sialic carbohydrate-modified cell surface proteins as a receptor. After translocation into cells, SubAB is delivered to the ER, where it cleaves GRP78/BiP. SubAB-catalyzed BiP cleavage induces ER stress, which causes various cell events including inhibition of protein synthesis, suppression of nuclear factor-kappa B activation, apoptotic cell death, and stress granules formation. In this review, we describe SubAB, the SubAB receptor, and the mechanism of cell response to the toxin.

To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.

The structural and physicochemical characteristics of protein hydrolysates prepared from Chinese sturgeon through the enzymatic hydrolysis process were evaluated. Two different enzymes including papain and alcalase 2.4L were used in the hydrolysis process. The papain enzyme significantly increased the degree of hydrolysis (20.62%) and decreased the ζ-potential (12.4 ± 1.31 mV) as compared to the alcalase enzyme, which represented 15.55% and 15.53 ± 0.77 mV, respectively. Alcalase 2.4L hydrolysate exhibited smaller particle size (822.047 ± 61.26 nm) than papain hydrolysate (1425.39 ± 44.82 nm). Hydrolysis by papain and alcalase 2.4L enzymes decreased the molecular weights (MW ≤ 1,000 Da) to 98.27% and 86.84%, respectively. The surface hydrophobicity and turbidity of the hydrolysates significantly affected by enzyme type and protein concentrations. By using the X-ray diffraction analysis, the papain hydrolysate showed a higher relative crystallinity degree (30.33%) than alcalase 2.4L hydrolysate (29.40%), whereas the Fourier transform infrared spectroscopy showed more clearly peaks for the amide bands of alcalase hydrolysate. The thermal properties also affected by enzymatic hydrolysis conditions, since the melting temperatures were 159.17 and 149.58 °C, whereas the rate of mass loss was 67.04% and 62.8%, for papain and alcalase hydrolysates, respectively. PRACTICAL APPLICATION: The enzymatic hydrolysis process of proteins is employed to obtain the nutritionally and functionality important peptides that result during the preparation of fish protein hydrolysate. The objective of this study was to investigate the structural and physicochemical characteristics of protein hydrolysate prepared from Chinese sturgeon. This study showed that these characteristics were affected by enzymatic hydrolysis conditions especially enzyme type. The finding of this study may be useful in terms of providing new information on the properties of the protein hydrolysate and the structural changes resulting from controlled enzymatic hydrolysis conditions. Overall, these conditions could potentially alter the secondary structure of the protein hydrolysates or peptides and enhance their functional properties.

In this study, bovine sodium caseinate (NaCas) was hydrolyzed with four proteases, alcalase, savinase, subtilisin A, and flavourzyme. In addition to the structural changes occurred through the enzymatic hydrolysis, the solubility, oil binding capacity, zeta potential, emulsification properties, and in vitro antioxidant capacity, anti-carcinogenic and antidiabetic properties of hydrolysates were determined. FTIR combined with hierarchical cluster analysis (HCA) made in Amide I region enable to classification of the samples based on the changes of the secondary structure depending on the enzyme type and degree of fragmentation. Technological properties of NaCas were enhanced through the enzymatic hydrolysis, and those were more prominent in serine-type enzymes, regardless of the enzyme type, all hydrolysates showed high antioxidant capacities. All hydrolysates, specifically those produced by savinase and alcalase, reduced the viability of the carcinogenic Caco-2 cells in a dose-dependent manner and showed a very low level of cytotoxicity against healthy HEK-293 cells. The hydrolysis treatment made a significant contribution to the antidiabetic activity of NaCas. Particularly alcalase and savinase hydrolysates suppressed the activity of alpha- amylase and alpha- glucosidase. Therefore, the generated milk protein hydrolysates could be used in functional food developments for specific dietary purposes.

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 μM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

In this study, to improve the quality and shelf life of hamburgers, sesame meal protein hydrolysates (SPH) were produced using two enzymes of alcalase and flavourzyme and then four hamburger treatments: T1: control (10% soybean), T2: 1% SPH + soybean 9%, T3: 2% SPH + soybean 8%, and T4: 3% SPH + soybean 7% were prepared. Physicochemical properties were analyzed at the beginning of the storage period; microbial and chemical quality was evaluated at intervals of 0, 4, 8, 12, and 16 days. The results of SPH showed that alcalase enzyme can produce a SPH with a higher antioxidant properties (DPPH, FRAP, and beta-carotene-linoleic acid) (P 0.05); therefore, this SPH was used for hamburger properties. According to the results, with the addition of SPH, moisture, fat, texture firmness decreased, protein, and brightness increased (P 0.05), and all treatments had the allowable range. SPH replacement with soybean slowed down the increasing trend of oxidation and microbial spoilage (P 0.05). In general, better results were observed in T3 and T4, which had a permissible range chemical and microbial index until the end of the storage period, as well as these treatments inhibited the growth of Staphylococcus aureus and Escherichia coli. Only T3 was approved by the evaluators.

Chicken hydrolysates (CHs) have been reported to protect mice against alcoholic liver injury possibly through oxidative stress reduction. In this study, the antioxidant activity of CHs was studied. Results showed that CHs exhibited significant antioxidant activity (around 600 and 400 μM TEAC/g in ORAC and ABTS assay, respectively) and could resist simulated gastrointestinal digestion. A total of 22 peptides were identified after antioxidant activity-oriented isolation using size-exclusion chromatography and high-performance liquid chromatography. Further in silico analysis and the validation of antioxidant activity revealed that novel peptides (RWGG and YYCQ) exhibited strong antioxidant activity. The most active peptide YYCQ displayed a TEAC value of 3.54 and 4.28 μM TEAC/μM in ORAC and ABTS assay, respectively. These peptides could contribute to reduce oxidative stress and protect against alcohol-induced liver injury. However, further studies understanding the bioactivity of such peptides in vivo are necessary before further applying them as functional food ingredient., This work was supported by the National Natural Science Foundation of China (No. 32101896 and No. 31871746), Key-Area Research and Development Program of Guangdong Province (2021B0707060001), and Self-innovation Research Funding Project of Hanjiang Laboratory (HJL202010B002). Grant AGL2017-89381-R funded by MCIN/ AEI /10.13039/501100011033 by “ERDF A way of making Europe” is acknowledged and grant RYC-2016-19635 (L.M.) funded by MCIN/AEI /10.13039/501100011033 and “ESF Investing in your future” is also acknowledged.

Food-originated angiotensin-I-converting enzyme (ACE)-inhibitory peptides to preserve hypertension are widely investigated over the past decade. Our research aims to discovery novel ACE-inhibitory peptides from bovine milk by couple of complex proteases (alcalase and protease). By means of response surface methodology with the conditions of pH 9.01, 61.81 °C and 6.5% ratio of enzyme to substrate, the hydrolysis model contributes to best-performing ACE-inhibitory activity of 85.02%. Through the further purification by consequent ultrafiltration, macroporous resin and gel chromatography, fraction G

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions. ispartof: MOLECULAR METABOLISM vol:66 ispartof: location:Germany status: published

High molecular weight (Mw) collagen hydrolysates have been demonstrated to produce a higher synthesis of collagen type I mRNA. Mw determination is a key factor maximizing the effect of collagen hydrolysates on collagen type I synthesis by fibroblasts. This work aimed to achieve a high average Mw in Blue Shark Collagen Hydrolysate, studying different hydrolysis parameters by GPC-LS analysis and testing its effect on mRNA Type I collagen expression. Analysis revealed differences in blue shark collagen hydrolysates Mw depending on hydrolysis conditions. Papain leads to obtaining a significantly higher Mw hydrolysate than Alcalase at different times of hydrolysis and at different enzyme/substrate ratios. Besides, the time of the hydrolysis factor is more determinant than the enzyme/substrate ratio factor for obtaining a higher or lower hydrolysate Mw when using Papain as the enzyme. Contrary, Alcalase hydrolysates resulted in similar Mw with no significant differences between different conditions of hydrolysis assayed. Blue shark collagen hydrolysate showing the highest Mw showed neither cytotoxic nor proliferation effect on fibroblast cell culture. Besides, it exhibited an increasing effect on both mRNA expression and pro-collagen I production.

Background Nattokinase (NK) is a promising alternative in the prevention and treatment of cardiovascular diseases due to its potent fibrinolytic activity. In this study, we investigated the effect of crude nattokinase extract on the healing of acetic acid-induced oral mucositis in mice. Methods Bacillus subtilis culture media (BSCM) was isolated into the supernatant, named nattokinase crude extract (NCE), and the pellet was named Bacillus subtilis mass (BSM). An oral mucositis model was established in mice by applying 50% glacial acetic acid to the buccal mucosa. According to the treatment conditions, the mice were divided into BSCM, NCE, BSM and phosphate buffered saline (PBS) groups. The weight of the mice, oral mucositis healing score and histopathological examination were used to evaluate the treatment. Results Fibrinolytic activities of BSCM, NCE and BSM were approximately 8069, 10,800 and 80 U/ml, respectively. The weight gain of mice in the NCE group was significantly different from the PBS group after three days’ treatment (p p p Conclusions NCE could possess remarkable potential to reduce pain and promote oral mucositis healing with minimal safety concerns. In this study, we first report that NCE from the supernatant of Bacillus subtilis can promote the healing of oral mucositis, which extends the application scope of NK.

Milk protein concentrate was hydrolyzed using one-step enzymatic hydrolysis. Both the peptide profiles and antioxidant activities of the resulting extensive hydrolysates of milk protein concentrate (EMPH) were analyzed using a peptidomics approach based on liquid chromatography-tandem mass spectrometry. The results demonstrated that the degrees of hydrolysis of the 4 EMPH by Alcalase-Protamex, Alcalase-Protease A 2SD, Alcalase-Flavorzyme, and Alcalase-ProteAXH were 12.02%, 16.85%, 15.87%, and 15.77%, respectively. Using size exclusion chromatography, 99.85% of the peptides in the Alcalase-Protease A 2SD hydrolysate were shown to have a molecular weight of3 kDa. A total of 33 common peptides were identified in the EMPH by liquid chromatography-tandem mass spectrometry, 16 of which were identified as bioactive peptides using bioinformatics. The peptide profiles and the coverage of master proteins of the 4 EMPH were different. The EMPH also exhibited strong free radical scavenging capacity, as indicated by the results of the 1,1-diphenyl-2-picrylhydrazyl radical, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroxyl radical, and reducing power assays. The results of this study provided useful information on the peptide profiles and antioxidant activity of EMPH.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteins that activate other proteins in cells. Functionally, PCSK9 binds to the receptor of low-density lipoprotein particles (LDL) to regulate cholesterol metabolism and lipoprotein homeostasis in human body. PCSK9 inhibition is a novel pharmacological strategy to control hypercholesterolemia and cardiovascular diseases. Recently accumulating evidence realizes that PCSK9 possesses other roles in cells, such as regulation of tissue inflammatory response, intratumoral immune cell infiltration, and tumor progression. This review discussed the advancement of PCSK9 research on its role and underlying mechanisms in tumor development and progression. For example, PCSK9 inhibition could attenuate progression of breast cancer, glioma, colon tumor, hepatocellular cancer, prostate cancer, and lung adenocarcinoma and promote apoptosis of glioma, prostate cancer, and hepatocellular cancer cells. PCSK9 deficiency could reduce liver metastasis of B16F1 melanoma cells by lowering the circulating cholesterol levels. PCSK9 gene knockdown substantially attenuated mouse tumor growth in vivo by activation of cytotoxic T cells, although PCSK9 knockdown had no effect on morphology and growth rate of different mouse cancer cell lines in vitro. PCSK9 inhibition thus can be used to control human cancers. Future preclinical and clinical studies are warranted to define anti-tumor activity of PCSK9 inhibition.

Blood is a meat by-product rich in proteins with properties that can be improved after hydrolysis, making it a sustainable alternative for use in the generation of bioactive peptides. The objective of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides obtained from different chicken blood hydrolysates prepared using combinations of four different enzymes. Best results were observed for AP (2% Alcalase + 5% Protana Prime) and APP (2% Alcalase + 5% Protana Prime + 3% Protana UBoost) hydrolysates obtaining inhibition values of 60.55 and 53.61%, respectively, assayed at a concentration of 10 mg/mL. Free amino acids were determined to establish the impact of exopeptidase activity in the samples. A total of 79 and 12 sequences of peptides were identified by liquid chromatography and mass spectrometry in tandem (LC-MS/MS) in AP and APP samples, respectively. Nine of the identified peptides were established as potential DPP-IV inhibitory using in silico approaches and later synthesized for confirmation. Thus, peptides GPF, IGL, and GGGW showed good DPP-IV inhibitory activity with IC50 values of 0.94, 2.22, and 2.73 mM, respectively. This study confirmed the potential of peptides obtained from chicken blood hydrolysates to be used as DPP-IV inhibitors and, therefore, in the control or modulation of type 2 diabetes., This research was funded by Grant PID2020-119684RB-I00 funded by MCIN/AEI/10.13039/501100011033. Grant GRISOLIAP/2020/021 de la Consellería de Innovació, Universitats, Ciència i Societat Digital de la Generalitat Valenciana (GCA) is also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to Proteored.

19 pages, 8 figures, 4 tables.-- This is an open access article distributed under the Creative Commons Attribution License, Rising trends in fish filleting are increasing the amount of processing by-products, such as skins of turbot, a flatfish of high commercial value. In line with circular economy principles, we propose the valorization of turbot skins through a two-step process: initial gelatin extraction described for the first time in turbot, followed by hydrolysis of the remaining solids to produce collagen hydrolysates. We assayed several methods for gelatin extraction, finding differences in gelatin properties depending on chemical treatment and temperature. Of all methods, the application of NaOH, sulfuric, and citric acids at 22 °C results in the highest gel strength (177 g), storage and loss moduli, and gel stability. We found no relation between mechanical properties and content of pyrrolidine amino acids, but the best performing gelatin displays higher structural integrity, with less than 30% of the material below 100 kDa. Collagen hydrolysis was more efficient with papain than alcalase, leading to a greater reduction in Mw of the hydrolysates, which contain a higher proportion of essential amino acids than gelatin and show high in vitro anti-hypertensive activity. These results highlight the suitability of turbot skin by-products as a source of gelatin and the potential of collagen hydrolysates as a functional food and feed ingredient., This research was funded by GAIN project (EU, Horizon 2020 Framework Research and Innovation Programme under GA n. 773330), and Xunta de Galicia (Grupos de Potencial Crecimiento, IN607B 2021/11

A new preservation approach is presented in this article to prolong the lifetime of raw chicken meat and enhance its quality at 4 °C via coating with highly soluble kidney bean protein hydrolysate. The hydrolysates of the black, red, and white kidney protein (BKH, RKH, and WKH) were obtained after 30 min enzymatic hydrolysis with Alcalase (E/S ratio of 1:100, hydrolysis degree 25–29%). The different phaseolin subunits (8S) appeared in SDS-PAGE in 35–45 kD molecular weight range while vicilin appeared in the molecular weight range of 55–75 kD. The kidney bean protein hydrolysates have considerable antioxidant activity as evidenced by the DPPH-scavenging activity and β-carotine-linolenic assay, as well as antimicrobial activity evaluated by disc diffusion assay. BKH followed by RKH (800 µg/mL) significantly (p ≤ 0.05) scavenged 95, 91% of DPPH and inhibited 82–88% of linoleic oxidation. The three studied hydrolysates significantly inhibited the growth of bacteria, yeast, and fungi, where BKH was the most performing. Kidney bean protein hydrolysates could shield the chicken meat because of their amphoteric nature and many functional properties (water and oil-absorbing capacity and foaming stability). The quality of chicken meat was assessed by tracing the fluctuations in the chemical parameters (pH, met-myoglobin, lipid oxidation, and TVBN), bacterial load (total bacterial count, and psychrophilic count), color parameters and sensorial traits during cold preservation (4 °C). The hydrolysates (800 µg/g) significantly p ≤ 0.05 reduced the increment in meat pH and TVBN values, inhibited 59–70% of lipid oxidation as compared to control during 30 days of cold storage via eliminating 50% of bacterial load and maintained secured storage for 30 days. RKH and WKH significantly (p ≤ 0.05) enhanced L*, a* values, thus augmented the meat whiteness and redness, while, BKH increased b* values, declining all color parameters during meat storage. RKH and WKH (800 µg/g) (p ≤ 0.05) maintained 50–71% and 69–75% of meat color and odor, respectively, increased the meat juiciness after 30 days of cold storage. BKH, RKH and WKH can be safely incorporated into novel foods.

Background: Keratin is among the most abundant structural proteins of animal origin, however it remains broadly underutilized. Objective: Bioinformatic investigation was performed to evaluate selected keratins originating from mass-produced waste products, i.e., chicken feathers and pig hair, as potential sources of bioactive peptides. Methods: Pepsin, trypsin, chymotrypsin, papain, and subtilisin were used for in silico keratinolysis with the use of “Enzyme(s) action” and fragmentomic analysis of theoretical products was performed using “Profiles of potential biological activity” in BIOPEP-UWM database of bioactive peptides. Bioactivity probability calculation and toxicity prediction of the peptides obtained were estimated using PeptideRanker and ToxinPred tools, respectively. Results: Our results showed that the keratins are a potential source of a variety of biopeptides, including dipeptidyl peptidase IV, angiotensin converting enzyme, prolyl endopeptidase inhibitory and antioxidative. Papain and subtilisin were found to be the most appropriate enzymes for keratin hydrolysis. This study presents possible structures of keratin-derived bioactive peptides that have not been previously described. Conclusion: Our data suggest additional in vitro and in vivo studies to verify theoretical predictions and further investigate the possibility of using keratin-rich waste as a source of peptide nutraceuticals.

This study investigated peptide fractions from fish skin collagen for antibacterial activity against Escherichia coli and Salmonella strains. The collagen was hydrolyzed with six commercial proteases, including trypsin, Alcalase, Neutrase, Flavourzyme, pepsin and papain. Hydrolyzed samples obtained with trypsin and Alcalase had the largest number of small peptides (molecular weight

Background Subtilisin QK is a serine protease in the subtilisin family, and is fermented by Bacillus subtilis QK02. The fibrinolytic activity of subtilisin QK was measured by detecting low molecular weight degradation products using a spectrophotometric method developed by Japan Bio Science Laboratory Co., Ltd. Subtilisin QK powder can maintain its fibrinolytic activity for more than 24 months when it is stored at room temperature and protected from light. Our previous results showed that subtlisin QK directly degraded cross-linked fibrins in the fibrin plate assay and effectively inhibited thrombosis in the mouse thrombus model. The aim of this study was to determine the acute toxicity, potential subchronic toxicity, and safety pharmacology of subtilisin QK in Sprague–Dawley (SD) rats. Methods In the acute toxicity study, a single oral dose of 100,000 FU/kg was administered to 10 female and 10 male SD rats. In the 28-day subchronic toxicity, 60 female and 60 male SD rats were randomly assigned to four experimental groups (daily oral dose of 0, 2500, 7500 and 25,000 FU/kg). In the safety pharmacology study, 20 female and 20 male SD rats were randomly assigned to four experimental groups (single oral dose of 0, 500, 1500 and 5000 FU/kg). Results No death occurred and no adverse effects were observed in the acute toxicity study at a dose of 100,000 FU/kg. In the 28-day subchronic toxicity study, several hematological and blood biochemical parameters showed increases or decreases; however, due to the lack of a dose–response relationship, these differences were considered unrelated to treatment. In the safety pharmacology study, no adverse effects were observed on the central nervous of SD rats post-administration up to a dose of 5000 FU/kg subtilisin QK. Conclusion The results showed that oral consumption of subtilisin QK is of low toxicological concern. No adverse effects were observed at doses of 2500, 7500, and 25,000 FU/kg in the 28-day subchronic toxicity, and the no-observed-adverse-effect level (NOAEL) of subtilisin QK was 25,000 FU/kg.

For utilizing the largest source of marine proteins, Antarctic krill (Euphausia superba) proteins were defatted and hydrolyzed separately using pepsin, alcalase, papain, trypsin, and netrase, and alcalase hydrolysate (EPAH) showed the highest DPPH radical (DPPH·) and hydroxyl radical (HO·) scavenging activity among five hydrolysates. Using ultrafiltration and chromatography methods, fifteen antioxidant peptides were purified from EPAH and identified as Asn-Gln-Met (NQM), Trp-Phe-Pro-Met (WFPM), Gln-Asn-Pro-Thr (QNPT), Tyr-Met-Asn-Phe (YMNF), Ser-Gly-Pro-Ala (SGPA), Ser-Leu-Pro-Tyr (SLPY), Gln-Tyr-Pro-Pro-Met-Gln-Tyr (QYPPMQY), Glu-Tyr-Glu-Ala (EYEA), Asn-Trp-Asp-Asp-Met-Arg-Ile-Val-Ala-Val (NWDDMRIVAV), Trp-Asp-Asp-Met-Glu-Arg-Leu-Val-Met-Ile (WDDMERLVMI), Asn-Trp-Asp-Asp-Met-Glu-Pro-Ser-Phe (NWD-DMEPSF), Asn-Gly-Pro-Asp-Pro-Arg-Pro-Ser-Gln-Gln (NGPDPRPSQQ), Ala-Phe-Leu-Trp-Asn (AFLWA), Asn-Val-Pro-Asp-Met (NVPDM), and Thr-Phe-Pro-Ile-Tyr-Asp-Tyr-Pro-Gln (TFPIYDPQ), respectively, using a protein sequencer and ESI/MS. Among fifteen antioxidant peptides, SLPY, QYPPMQY and EYEA showed the highest scavenging activities on DPPH· (EC50 values of 1.18 ± 0.036, 1.547 ± 0.150, and 1.372 ± 0.274 mg/mL, respectively), HO· (EC50 values of 0.826 ± 0.027, 1.022 ± 0.058, and 0.946 ± 0.011 mg/mL, respectively), and superoxide anion radical (EC50 values of 0.789 ± 0.079, 0.913 ± 0.007, and 0.793 ± 0.056 mg/mL, respectively). Moreover, SLPY, QYPPMQY and EYEA showed strong reducing power, protective capability against H2O2-damaged plasmid DNA, and lipid peroxidation inhibition ability. Furthermore, SLPY, QYPPMQY, and EYEA had high stability under temperatures lower than 80 °C, pH values ranged from 6–8, and simulated GI digestion for 180 min. The results showed that fifteen antioxidant peptides from alcalase hydrolysate of Antarctic krill proteins, especially SLPY, QYPPMQY and EYEA, might serve as effective antioxidant agents applied in food and health products.