Proteolytic Hydrolysis Research Articles

Keratin-based pet food produced through proteolytic hydrolysis of chicken feather: Characterisation and palatability study

Valorisation of poultry co-products can fulfil the protein demand of the pet food sector thereby supporting a circular bioeconomy. In this investigation, keratin was produced through an optimised reduction (0.8 % sodium sulphite, for 90 min) and hydrolysis (using papain for 120 min) with a yield of 56.87 %. FTIR analysis, in vitro digestibility, SEM imaging, and amino acid profiling of the derived keratin were done and it was subsequently used for pet formulations. Results revealed significant alteration in secondary protein structure post-hydrolysis with in vitro digestibility of 90 %. Higher levels (>100 % increase) of histidine, arginine, lysine, and methionine were observed in the hydrolysate compared to conventional feather meal. Pet food was formulated with 20 % protein replacement that showed maximum palatability in single and two-bowl tests for 2–4 year old Labrador dogs. The developed pet food exhibited desirable colour, water activity (0.5), moisture (4.7 %), and relatively lower TBARS value (0.8 mg/kg) than the critical value.

Assessment of Potential Probiotic Properties and Biotechnological Activities of Lactobacillus Strains Isolated from Traditional Algerian Fermented Wheat ELHAMOUM

Background: The objective of the current study was to explore the in vitro probiotic and technological characteristics of lactic acid bacteria (LAB) isolated from Algerian fermented wheat. Methods: Three LAB strains were selected and identified using 16S rRNA gene sequencing, in vitro assessments of probiotic properties included resistance to simulated gastric and intestinal conditions, auto-aggregation and antimicrobial susceptibility against common pathogens. Furthermore, the strains were evaluated for their biotechnological properties. Results: The results of 16S rRNA gene sequencing showed that two of them were Levilactobacillus brevis (LAn91 and LAn110) and LAn12a was Lactiplantibacillus pentosus. The tolerance of all strains to gastric acid and bile salts was more than 90%; they showed a high auto-aggregation capacity and a variable antagonistic property against pathogenic strains. Strain LAn12a presented the best biotechnological characteristics (proteolytic, acidifying and lactose hydrolysis), while the LAn91 and LAn110 strains were not able to acidify milk. All the strains exhibited a survival count in fermented milk exceeding 7 log CFU/ml during storage at 4°C on day 21, which surpasses the requirement of 6 log CFU/ml to provide health benefits. These bacteria can also be employed for the creation of functional foods and to validate their suitability as probiotic starter cultures.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Evidence of a divided nutritive function in rainbow Trout (Oncorhynchus mykiss) midgut and hindgut microbiomes by whole shotgun metagenomic approach

The nutritive role and ecology of gut-dwelling microbes in rainbow trout remain enigmatic. To improve our understanding of the rainbow trout gastrointestinal tract (GIT) microbiome, we performed whole shotgun metagenomic analyses on the assembled contigs from luminal contents from both mid- and hind-GIT regions for taxonomic and functional classifications of fish-fed animal and plant protein dietary sources. Our study revealed that trout respond well to the two diets containing animal and plant protein sources when supplemented with essential amino acids to meet the requirements of the fish. Microbes present were predominantly bacteria (89.9%) and mainly of the phyla Tenericutes, Firmicutes, Fusobacteria, and Proteobacteria. Eukaryotic (8.8%) microbes were mainly from phyla Ascomycota and Basidiomycota, while Archaea (<1%) were also present and predominantly from the phylum Euryarchaeota. Comparisons of genus-level classifications and functional profiles revealed compositional differences in these GIT locations that appear modulated by differences in the dietary treatments. The functional analysis provided evidence of amino acid biosynthesis/catabolism and methane production in the mid-GIT, while in the hind-GIT, proteolytic hydrolysis and butyrate metabolism were expressed in the trout fed with plant protein diet. The animal protein-based diet provided metabolites for microbial protein fermentation in the hind-GIT. Our report highlights and identifies the potential nutritive contributions of GIT microbes to trout and a potentially crucial functional division along the GIT. Finally, the plant-based diet enhanced amino acid catabolism in the midgut section, while the hindgut section supports evidence of methanogen fermentation.

Extraction, Composition, Functionality, and Utilization of Brewer’s Spent Grain Protein in Food Formulations

In recent years, brewer's spent grain (BSG) has gained attention as a plant-based protein source because it occurs in large quantities as a by-product of beer brewing. BSG can contribute to future food requirements and support the development of a circular economy. In light of the dynamic developments in this area, this review aims to understand the proteins present in BSG, and the effect of extraction techniques and conditions on the composition, physicochemical, and techno-functional properties of the obtained protein extracts. The water-insoluble hordeins and glutelins form the major protein fractions in BSG. Depending on the beer brewing process, the extraction technique, and conditions, the BSG protein isolates predominantly contain B, C, and ϒ hordeins, and exhibit a broad molecular weight distribution ranging between <5 kDa and >250 kDa. While the BSG isolates obtained through chemical extraction methods seem promising to obtain gelled food products, physical and enzymatic modifications of BSG proteins through ultrasound and proteolytic hydrolysis offer an effective way to produce soluble and functional protein isolates with good emulsifying and foaming capabilities. Specifically tailored protein extracts to suit different applications can thus be obtained from BSG, highlighting that it is a highly valuable protein source.

Potential Application of High Hydrostatic Pressure on the Production of Hydrolyzed Proteins with Antioxidant and Antihypertensive Properties and Low Allergenicity: A Review.

The high hydrostatic pressure (HHP) process has been studied for several applications in food technology and has been commercially implemented in several countries, mainly for non-thermal pasteurization and shelf-life extension of food products. HHP processing has been demonstrated to accelerate proteolytic hydrolysis at a specific combination of pressure and pressure-holding time for a given protein source and enzyme. The enzymatic hydrolysis of proteins is a well-known alternative to producing biologically active peptides, with antioxidant and antihypertensive capacity, from different food protein sources. However, some of these protein sources contain allergenic epitopes which are often not degraded by traditional hydrolysis. Moreover, the peptide profile and related biological activity of a hydrolysate depend on the protein source, the enzymes used, the parameters of the proteolysis process (pH, temperature, time of hydrolysis), and the use of other technologies such as HHP. The present review aims to provide an update on the use of HHP for improving enzymatic hydrolysis, with a particular focus on studies which evaluated hydrolysate antihypertensive and antioxidant capacity, as well as residual allergenicity. Overall, HHP has been shown to improve the biological properties of hydrolysates. While protein allergenicity can be reduced with traditional hydrolysis, HHP can further reduce the allergenicity. Compared with traditional hydrolysis methods, HHP-assisted protein hydrolysis offers a greater opportunity to add value to protein-rich products through conversion into high-end hydrolysate products with enhanced nutritional and functional properties.

Extrahepatic in vitro metabolism of peptides; comparison of human kidney and intestinal S9 fraction, human plasma and proximal tubule cells, using cyclosporine A, leuprorelin, and cetrorelix as model compounds

Peptide therapeutics showcase number of advantages compared to the traditional small molecule drugs, e,g. they usually have higher affinity to target and lower toxicity profiles. Endogenous peptides are mostly cleared from the body through renal clearance or proteolytic hydrolysis. As a part of drug discovery, metabolite identification is an important part in their development to identify metabolic hot spots and to further improve their stability. As the catabolism of the peptides and peptide-like drugs is often considered to be extrahepatic, the use of in vitro systems derived from these organs might be beneficial. In this study, multiple extrahepatic metabolic systems were evaluated for the applicability for peptide metabolism studies. Three peptide drugs (leuprorelin, cetrorelix, cyclosporin) were incubated in kidney and intestinal S9 fraction ( ± NADPH), fresh plasma (anticoagulants EDTA and heparin separately), and plated proximal tubule cells. Additionally, leuprorelin was also incubated with human kidney microsomes and cytosol to further investigate the NADPH-dependent metabolism detected in kidney S9 fraction. Both substrate disappearance and metabolite formation were monitored, using UPLC/HR-MS analysis of the collected samples.Overall, the largest number of metabolites was formed in the incubation with kidney S9 fraction, followed by intestinal S9, while incubations with proximal tubule cells produced lower number of metabolites All investigated peptides were stable in plasma and only a few metabolites were detected, likely because the studied peptide drugs have been optimized to be stable in plasma. Leuprorelin showed NADPH-dependent metabolite formation in kidney S9 fraction, while the metabolism of cetrorelix was more NADPH independent. As expected, formation of cytochrome P450 (CYP) catalyzed metabolism of cyclosporine was not observed with the employed extrahepatic systems. The NADPH-dependent metabolism of leuprorelin was detected also in the incubation with kidney cytosol, but not with kidney microsomes, and was thus not caused by CYPs or FMOs, but with cytosolic NADPH-dependent drug metabolizing enzymes. These enzymes could, in principle, activate the amide bond via reductive or oxidative metabolism outside the amide bond. The identity of the involved drug metabolizing enzymes in this process is still unknown.

Opposites Attract: Electrostatically Driven Loading of Antimicrobial Peptides into Phytoglycogen Nanocarriers.

Antimicrobial peptides, such as GL13K, have a high binding selectivity toward bacterial membranes, while not affecting healthy mammalian cells at therapeutic concentrations. However, delivery of these peptides is challenging since they are susceptible to proteolytic hydrolysis and exhibit poor cellular uptake. A protective nanocarrier is thus proposed to overcome these obstacles. We investigate the potential to employ biodegradable phytoglycogen nanoparticles as carriers for GL13K using a simple loading protocol based on electrostatic association rather than chemical conjugation, eliminating the need for control of chemical cleavage for release of the peptide in situ. Both the native (quasi-neutral) and carboxymethylated (anionic) phytoglycogen were evaluated for their colloidal stability, loading capacity, and release characteristics. We show that the anionic nanophytoglycogen carries a greater cationic GL13K load and exhibits slower release kinetics than native nanophytoglycogen. Isotope exchange measurements demonstrate that the antimicrobial peptide is entrapped in the pores of the dendritic-like macromolecule, which should provide the necessary protection for delivery. Importantly, the nanoformulations are active against a Pseudomonas aeruginosa clinical isolate at concentrations comparable to those of the free peptide and representative, small molecule antibiotics. The colloidal nanocarrier preserves peptide stability and antimicrobial activity, even after long periods of storage (at least 8 months).

Effect of processing treatments on the allergenicity of nuts and legumes: A meta-analysis.

The effective food processing to reduce nuts and legumes allergenicity could not be easily and directly concluded from reading a few published reports. Therefore, we conducted a meta-analysis to investigate this issue. A literature search was conducted in eight electronic databases from January 2000 to June 11, 2021. The primary outcome of interest was the allergenicity of processed nuts or legumes determined by enzyme-linked immunosorbent assay from in vitro studies. Data with the standardized mean difference (SMD) of 95% confidence interval (CI) were pooled using a random-effect model by RevMan 5.4 software. Heterogeneity was assessed using Cochran's Q (PQ ) and I2 tests. The search strategy identified 18,793 articles. However, only 61 studies met the inclusion criteria and were included in this meta-analysis. There were 21 and 15 types of respective single and combined food processing treatments analyzed for their effects on reducing allergenicity. In single processing treatment, the extrusion and fermentation had the largest reduction in allergenicity, considering their SMD value, that is, -20.19 (95% CI: -22.22 to -18.17; the certainty of evidence: moderate) and -20.8 (95% CI: -24.10 to -17.50; the certainty of evidence: moderate), respectively. Whereas in the combination, the treatment of fermentation followed by proteolytic hydrolysis showed the most significant reduction (SMD: -53.34; 95% CI: -70.18 to -36.5) and the evidence quality of this treatment was considered moderate. In conclusion, these three food processing methods showed a desirable impact in reducing nuts or legumes allergenicity. PRACTICAL APPLICATION: Nuts and legumes play an essential role as protein sources in food consumption worldwide, but they usually contain allergens. Our study has investigated the food processing methods that effectively reduce their allergenicity by meta-analysis. The result gives valuable information for further laboratory investigation on allergens and can be used by food industries in providing foods from nuts and legumes with lower allergenicity.

Corn distillers solubles by two-step proteolytic hydrolysis as a new source of plant-based protein hydrolysates with ACE and DPP4 inhibition activities

Proteins of low-value and underexplored corn distillers solubles (CDS) have not been considerably valorized. Hence, the influence of one-step enzymatic hydrolysis of proteins with alcalase (A), trypsin (T) or flavourzyme (F) and two steps with AT, TA, AF, FA, TF, or FT was assessed to release peptides with angiotensin-I converting enzyme inhibition (ACEi) and dipeptidyl peptidase4 inhibition (DPP4i). The AF hydrolysate was the best sample in terms of yield, protein content, degree of hydrolysis, ACEi (97.68 ± 1.09 %), and DPP4i (51.51 ± 0.28 %). Mass spectrometry of the most active AF hydrolysate (<3 kDa) identified new major peptides like APLA, PLFP, LFLP, LPPYL, PLYPLP, NDWHTGPL, LPPYLPS, GSPFLGQ, SWQQPIVGG. Bioinformatic analysis showed these can inhibit both ACE and DPP4. This is because peptides contain functional groups and adopt conformations significantly binding with other functional groups at enzyme active sites (p < 0.05). This establishes dual bioactivity of peptides, which may have applications in food, feed, and pharmaceutical industries.

Isolation and characterization of amyloid-like protein aggregates from soya beans and the effect of low pH and heat treatment on their stability.

Purified soya bean proteins (glycinin and conglycinin) are known to form amyloid-like aggregates in vitro at a higher temperature. Soya beans (chunks) are textured proteinaceous vegetables made from defatted soya flour by heating it above 100°C and extruding under high pressure. Therefore, it was assumed that subjecting the soya bean proteins to high temperatures raises the possibility of forming amyloids or amyloid-like protein aggregates. Hence, the present study aimed to examine the presence of amyloid-like protein aggregates in soya beans. The isolated protein aggregates from hydrated soya beans displayed typical characteristics of amyloids, such as the red shift in the absorption maximum (λmax ) of Congo red (CR), high Thioflavin T (ThT), and 8-Anilinonapthalene-1-sulfonate (ANS) binding, and fibrilar morphology. Furthermore, these aggregates were found to be stable against proteolytic hydrolysis, confirming the specific property of amyloids. The presence of amyloid-like structures in soya beans raises concerns about their implications for human nutrition and health. PRACTICAL APPLICATIONS: Protein aggregation has usually been considered detrimental. The traditional food-processing conditions, such as thermal processing, are associated with protein denaturation and aggregation. The formation of ordered protein aggregates with extensive β-sheet are progressively evident in various protein-rich foods known as amyloid, which expands food safety concerns. Instead, it is also associated with poor nutritional characteristics. The present study concerns the presence of amyloid-like protein aggregates in widely consumed native soya beans, which are manufactured by extensive heat treatment of defatted soy flour. Although there is no indication of their toxicity, these aggregates are found to be proteolytically resistant. The seminal findings in this manuscript suggest that it is time to adapt innovative food processing and supplementation of bioactive molecules that can prevent the formation of such protein aggregates and help maximize the utilization of protein-based nutritional values.

Next-generation nutraceuticals: bioactive peptides from plant proteases.

Bioactive peptides are short and specific fragments of proteins with a wide range of biological activities that provide health benefits to the host. These natural peptides are safe and nontoxic and do not show any side effects. Nowadays, the production and characterization of bioactive peptides have been a key area of research as they show great potential as nutraceuticals and functional foods. Thus, bioactive peptides are considered next-generation therapeutic agents that can replace pharmaceutical products with profound adverse effects in the near future. So far, proteolytic hydrolysis has been used as the method of choice for the large-scale production of bioactive peptides. Studies have reported that peptides with specific characteristics can be generated using a particular type of protease. Microbial proteases are the predominantly used ones because of the ease in their production and purification. However, recently, plant proteases have gained a renewed interest as they offer diversity and better specificity compared with other proteases. This review highlights the potential of plant proteases for the production of bioactive peptides and also describes the benefits of bioactive peptides as nutraceuticals.

Modulating Angiogenesis by Proteomimetics of Vascular Endothelial Growth Factor.

Angiogenesis, formation of new blood vessels from the existing vascular network, is a hallmark of cancer cells that leads to tumor vascular proliferation and metastasis. This process is mediated through the binding interaction of VEGF-A with VEGF receptors. However, the balance between pro-angiogenic and anti-angiogenic effect after ligand binding yet remains elusive and is therefore challenging to manipulate. To interrogate this interaction, herein we designed a few sulfono-γ-AA peptide based helical peptidomimetics that could effectively mimic a key binding interface on VEGF (helix-α1) for VEGFR recognition. Intriguingly, although both sulfono-γ-AA peptide sequences V2 and V3 bound to VEGF receptors tightly, in vitro angiogenesis assays demonstrated that V3 potently inhibited angiogenesis, whereas V2 activated angiogenesis effectively instead. Our findings suggested that this distinct modulation of angiogenesis might be due to the result of selective binding of V2 to VEGFR-1 and V3 to VEGFR-2, respectively. These molecules thus provide us a key to switch the angiogenic signaling, a biological process that balances the effects of pro-angiogenic and anti-angiogenic factors, where imbalances lead to several diseases including cancer. In addition, both V2 and V3 exhibited remarkable stability toward proteolytic hydrolysis, suggesting that V2 and V3 are promising therapeutic agents for the intervention of disease conditions arising due to angiogenic imbalances and could also be used as novel molecular switching probes to interrogate the mechanism of VEGFR signaling. The findings also further demonstrated the potential of sulfono-γ-AA peptides to mimic the α-helical domain for protein recognition and modulation of protein-protein interactions.

High-Phytate Diets Increase Amyloid β Deposition and Apoptotic Neuronal Cell Death in a Rat Model.

Amyloid-β (Aβ) accumulation in the hippocampus is an essential event in the pathogenesis of Alzheimer’s disease. Insoluble Aβ is formed through the sequential proteolytic hydrolysis of the Aβ precursor protein, which is cleaved by proteolytic secretases. However, the pathophysiological mechanisms of Aβ accumulation remain elusive. Here, we report that rats fed high-phytate diets showed Aβ accumulation and increased apoptotic neuronal cell death in the hippocampus through the activation of the amyloidogenic pathway in the hippocampus. Immunoblotting and immunohistochemical analyses confirmed that the overexpression of BACE1 β-secretase, a critical enzyme for Aβ generation, exacerbated the hippocampal Aβ accumulation in rats fed high-phytate diets. Moreover, we identified that parathyroid hormone, a physiological hormone responding to the phytate-mediated dysregulation of calcium and phosphate homeostasis, plays an essential role in the transcriptional activation of the Aβ precursor protein and BACE1 through the vitamin D receptor and retinoid X receptor axis. Thus, our findings suggest that phytate-mediated dysregulation of calcium and phosphate is a substantial risk factor for elevated Aβ accumulation and apoptotic neuronal cell death in rats.

Akt activation‐dependent protective effect of wild ginseng adventitious root protein against UVA‐induced NIH‐3T3 cell damage

Prolonged skin exposure to ultraviolet radiation can lead to development of several acute and chronic diseases, with UVA exposure considered a primary cause of dermal photodamage. We prepared a wild ginseng adventitious root extract (ARE) that could alleviate UVA irradiation-induced NIH-3T3 cell viability decline. After employing a series of purification methods to isolate main active components of ARE, adventitious root protein mixture (ARP) was identified then tested for protective effects against UVA irradiation-induced NIH-3T3 cell damage. The results showed that ARP treatment significantly reduced UVA-induced cell viability decline and confirmed that the active constituent of ARP was the protein, since proteolytic hydrolysis and heat treatment each eliminated ARP protective activity. Moreover, ARP treatment markedly inhibited UVA-induced apoptosis, cell cycle arrest and DNA fragmentation, while also significantly reversing UVA effects (elevated Bax levels, reduced Bcl-2 expression) by reducing Bax levels and increasing Bcl-2 expression. Mechanistically, ARP promoted Akt phosphorylation regardless of UVA exposure, thus confirming ARP resistance to inactivation by UVA light. Notably, in the presence of Akt inhibitor SC0227, ARP could no longer counteract UVA-induced cell viability decline and DNA fragmentation. Additionally, our results demonstrated that ARP treatment protected UVA-irradiated NIH-3T3 cells by preventing UVA-induced reduction of collagen-I expression. Taken together, these results suggest that ARP treatment of NIH-3T3 cells effectively mitigated UVA-induced cell viability decline by activating intracellular Akt to reduce UVA-induced DNA damage, leading to reduced rates of apoptosis and cell cycle arrest after UVA exposure and restoring collagen expression to normal levels.

Triticum monococcum amylase trypsin inhibitors possess a reduced potential to elicit innate immune response in celiac patients compared to Triticum aestivum

ScopeSeveral studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. Methods and ResultsATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. ConclusionTM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

Alfalfa Ribulose-1,5-Bisphosphate cCarboxylase/oxygenase: Extraction, Biochemical Characterization, and Proteolytic Hydrolysis Optimization Process for Plausible Biotechnological Tools

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO2 into the biosphere. In this work, we report the extraction, the purification of the Alfalfa Rubisco. Indeed, the purification steps of the last one lead to the purified protein composed of two subunits (13 kDa and 55 kDa) as indicated in SDS-PAGE. The Rubisco proteolytic process via proteinase-K was optimized. The Box-Behnken Response Surface Methodology was applied to optimize three factors for increasing peptide production. So, the proteinase-K concentration (6, 13 and 20 mg/mL), the reaction incubation time (6 h, 8 h and 10 h) and the reaction temperature (30°C, 43°C and 56°C) were selected for the experimental design. A best condition of 20 mg/mL of proteinase-K, 10 h of time incubation, and 30°C of temperature treatment resulted in a 79.83%. This yield was 2.66 times higher than the one achieved before optimization. The resulting hydrolysate was injected in gel filtration column coupled to HPLC giving rise to four fractions (F1, F2, F3 and F4) eluted respectively after 12.54 min, 12.47 min, 12.44 min and 12.60 min. Also, the ROS scavenging system was monitored like the CAT, SOD and also the DPPH capacity. Eventually, those findings will give birth to the opportunity to look for plausible biotechnological investigations.

Magnetic nanoparticle decorated anodic alumina nanotubes for fluorescent detection of cathepsin B

In this work, we present the process to provide anodic alumina nanotubes with magnetic responsivity based on magnetic nanoparticles. We demonstrate the possibility to cause the motion of these composite nanotubes under magnetic field, providing them with guided mobility. The obtained magnetic anodic alumina nanotubes are completely characterized and their potential to undergo selective and effective functionalization, and stimuli-responsive load release is demonstrated. For this purpose, protease-triggered release of fluorescent molecules loaded inside the magnetic anodic alumina nanotubes (MAANTs) by selective functionalization is performed. The inner walls of the MAANTs were selectively covered with protein padding of albumin-fluorescein isothiocyanate conjugate (FITC-BSA) through means of silanization. Protein functionalization was designed to undergo proteolytic hydrolysis in presence of cathepsin B- protease highly expressed during growth and initial stages of tumor metastasis – in order to cleave peptide bond of albumin and release fluorescent fragments of the protein. Proteolytic reaction with the enzyme is performed under acidic conditions. Presented arrangement is an exemplary combination of functionalities – which are vast – and of value for applications like drug delivery and biosensing applications.

Photothermal Effect-based Cytotoxic Ability of Melanin from Mytilus edulis Shells to Heal Wounds Infected with Drug-resistant Bacteria in vivo

ObjectiveOwing to antibiotic abuse and the subsequent development of antibiotic resistance, bacterial infection has become one of the most persistent unresolved problems. New antibacterial agents, especially those that are environmental-friendly, are urgently needed. MethodsMelanin extracted by filtration centrifugation and acid and proteolytic hydrolysis was characterized using UV, FTIR, TEM, and XPS. Photothermal conversion was calculated, and the bacteriostatic effects, in vitro and in vivo, were assessed by plate counting and ratios (%) of wound areas. ResultsNatural melanin hydrolyzed by trypsin had good photothermal conversion effects, which resulted in superior bacteriostatic activities. The extracted melanin along with laser NIR irradiation at 808 nm promoted the healing of wounds infected by drug-resistant bacteria in vivo and was biocompatible according to toxicity tests in vivo and in vitro. ConclusionThe present findings indicated a safe and efficient method of developing natural antibacterial agents.

Effects of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on proteolytic changes and bioactivities (antioxidant and antihypertensive activities) in fermented pork sausage

This research explored the influences of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on protein degradation and bioactivities (antioxidant and antihypertensive activities) of peptides in sausages fermented for 21 days. Hydrolysis of sarcoplasmic and myofibrillar proteins, especially intense in inoculated group, was evidenced by polyacrylamide gel electrophoresis. A significant increment (P < 0.05) of hydrophobic amino acid concentration and ACE inhibitory activity was also observed in inoculated group. Based on DPPH, hydroxyl radical, superoxide anion scavenging activities and Ferric-reducing antioxidant power values, the inoculated group exhibited higher antioxidant ability, but possessed lower ABTS+· scavenging activity than control group (P < 0.05). LC-MS/MS totally identified 22 peptides, which were from troponin T, myosin, and particularly actin. These peptides shared joint characteristics with other bioactive peptides. We concluded mixed starters promoted proteolytic hydrolysis and peptide formation with high ACE inhibitory and antioxidant activities.