Protease Activity Research Articles

Transcriptomic and Metabolomic Analyses of Soybean Protein Isolate on Monascus Pigments and Monacolin K Production.

Monascus pigments (MPs) and monacolin K (MK) are important secondary metabolites produced by Monascus spp. This study aimed to investigate the effect of soybean protein isolate (SPI) on the biosynthesis of MPs and MK based on the analysis of physiological indicators, transcriptomes, and metabolomes. The results indicated that the growth, yellow MPs, and MK production of Monascus pilosus MS-1 were significantly enhanced by SPI, which were 8.20, 8.01, and 1.91 times higher than that of the control, respectively. The utilization of a nitrogen source, protease activity, the production and utilization of soluble protein, polypeptides, and free amino acids were also promoted by SPI. The transcriptomic analysis revealed that the genes mokA, mokB, mokC, mokD, mokE, mokI, and mokH which are involved in MK biosynthesis were significantly up-regulated by SPI. Moreover, the glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid degradation, tricarboxylic acid (TCA) cycle, and amino acid metabolism were effectively up-regulated by SPI. The metabolomic analysis indicated that metabolisms of amino acid, lipid, pyruvate, TCA cycle, glycolysis/gluconeogenesis, starch and sucrose, and pentose phosphate pathway were significantly disturbed by SPI. Thus, MPs and MK production promoted by SPI were mainly attributed to the increased biomass, up-regulated gene expression level, and more precursors and energies.

Exploring the potential of water channels for developing genetically encoded reporters and biosensors for diffusion-weighted MRI

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on synthetic contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for synthetic contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

Reciprocal inhibition of autophagy and Botrytis cinerea-induced programmed cell death in ‘Shine Muscat’ grapes

Botrytis cinerea causes gray mold, decreasing the quality of table grapes. The berry response to B. cinerea infection was explored in present study, focusing on the relationship between presence of autophagy and programmed cell death (PCD). Results demonstrated B. cinerea infection decreased cell viability, triggering cell death, possibly resulting in PCD occurrence. It was further verified by increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive nuclei, heightened caspase 3-like and caspase 9-like protease activity, and elevated expression of metacaspase genes. Additionally, autophagy was indicated by the increased VvATG expression and autophagosome formation. Notably, the autophagy activator rapamycin reduced TUNEL-positive nuclei, whereas the autophagy inhibitor 3-methyladenine increased caspase 9-like protease activity. The PCD activator C2-ceramide inhibited autophagy, whereas the PCD inhibitor Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) enhanced autophagy gene expression. Autophagy and B. cinerea-induced PCD in berry cells are reciprocally negatively regulated; and the rapamycin and Ac-DEVD-CHO could potentially maintain table grape edible quality.

Targeted-metabolomic and untargeted-proteomic approaches reveal the effects of muscle fibre type and postmortem ageing on taste-active compounds in beef

The taste of beef is caused by taste-active compounds detected in the mouth during mastication. We hypothesised that the concentration of taste-active compounds in beef is influenced by muscle-fibre-type and postmortem ageing. To test this, and unravel the underlying mechanisms, we investigated the taste-active compounds, and proteomic profiles, in beef masseter [oxidative muscle, all type I fibres) and cutaneous trunci (glycolytic muscle, mostly type II fibres) before and after 14-days postmortem ageing. Our results showed that nucleotides were initially higher and degraded slower in cutaneous trunci (P < 0.05 for both), which could be explained by the profile of nucleotide metabolism enzymes. In contrast, free amino acids were initially higher and increased more in masseter compared to cutaneous trunci (P < 0.05 for all), which might be explained by the profile and activity of proteases in these two muscles. Our results indicate the taste of beef is affected by the muscle-fibre-type and postmortem ageing.

Variability of soil enzyme activities and nutrients with forest gap renewal interacting with soil depths in degraded karst forests

Forest gaps serve as crucial starting points for forest self-renewal. Within these gaps, soil enzymes play an essential role in driving nutrient transformation and species renewal. However, how forest gap renewal regulates soil fertility through soil enzyme across various soil depths remains unclear. This investigation delved into the dynamics of soil enzymes and nutrients within karst forest gaps at four developmental age stages: Infancy (<10 years), Youth (10–20 years), Middlescence (20–30 years), and Elder (>30 years) across different soil depths (0–10 cm, 10–20 cm, and 20–30 cm). The study determined the contents of CNP and the activities of soil enzymes such as polyphenol oxidase, catalase, protease, and alkaline phosphatase. Two-way ANOVA was employed to assess the variations in gap ages and soil depths, along with their interaction. Redundancy analysis (RDA) was used to reveal the correlations between soil enzyme activities and CNP fertility indicators. The results indicated that gap age significantly influenced the activities of alkaline phosphatase, polyphenol oxidase, and protease, as well as the contents of total and available nitrogen, phosphorus, and organic carbon, alongside the ratios of N/P, C/N, and C/P. Catalase activity, however, did not vary significantly with gap age, although soil depth influenced these parameters, excluding total phosphorus and the C/N ratio. Notably, in the 0–10 cm soil layer, alkaline phosphatase activity, total nitrogen, available nitrogen, and organic carbon contents significantly increased with gap age increase, followed by a significant decrease after the middlescence stage. In the 10–20 cm and 20–30 cm soil layers, Alkaline phosphatase and polyphenol oxidase activities, along with N/P and C/N ratios initially decreased and then increased with gap age increase. During the Infancy, Youth, and Middlescence stages, Polyphenol oxidase and protease activities, CNP contents, as well as N/P and C/P ratios generally decreased with soil depth increase or after an increase in the 10–20 cm soil layer. RDA highlighted the variational positive and negative correlations between soil enzyme activities and CNP fertility indicators across soil depths during forest gap renewal, with explanatory rates ranging from 88.05 % to 94.67 % across soil layers. Overall, this study suggests that forest gap renewal significantly influences soil enzymes, CNP content and stoichiometry, modifying soil CNP content through stoichiometric regulation across different soil depths, and ultimately sustaining soil fertility via soil enzymes.

Exploring the suitability of Hermetia illucens meal in Clarias magur catfish: effect on growth, physiology and flesh quality

Abstract The effect of substituting Hermetia illucens (HI) meal for fish meal (FM) on the growth performance, survival rate, feed utilization, digestive enzyme activity, immunological function, and flesh quality of Clarias magur (average weight = 3.3 ± 0.05 g) is investigated in this work. Four iso-nitrogenous diets with 35% crude protein were formulated, each with varying levels of HI substitution: HI25%, HI50%, HI75%, and HI100%, then compared to a control diet (HI0%). Following a 90-day experiment, it was observed that the growth performance and feed efficiency were significantly improved at the 50% level (). However, the control group exhibited greater levels of digestive enzymes, specifically protease and lipase activity. The fish’s health status remained unaffected by the substitution of fishmeal, while the 50% HI group showed significant improvements. The high levels of the antioxidant enzymes SOD and CAT, as well as the metabolic product MDA, suggested enhanced antioxidant activity in the groups that received HI inclusion. However, as the substitution level reached 100%, the quality of the flesh decreased, as indicated by increased lipid and adhesiveness, as well as decreased L* and WI (). The findings of the study suggest that HI larvae can replace FM in diet of C. magur. Overall, at 50% level of HI larvae meal inclusion in diet, better growth and immune function was evident, but it does lead to a decrease in flesh quality. This study enhances the existing knowledge on substituting fish meal with insect meal in catfish that are of regional importance. Additionally, it emphasizes the necessity for additional investigation to examine the underlying mechanisms employing biotechnological approaches.

Enhancement of high-density lipoprotein-associated protease inhibitor activity prevents atherosclerosis progression

Background and aimsInflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. MethodsWe designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. ResultsHTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe−/− mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr−/− mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. ConclusionsThese data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.

COVID-19 and Prostatitis: A Review of Current Evidence.

Coronavirus disease 2019 (COVID-19), a highly contagious viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a global health threat. The virus enters host cells by binding with angiotensin-converting enzyme 2 (ACE2), which is then facilitated by the protease activity of transmembrane serine protease 2 (TMPRSS2). It triggers a cytokine storm that eventually leads to cell apoptosis, tissue damage, and organ failure. Therefore, any organs in the human body that have both receptors are highly susceptible to COVID-19 infection, potentially resulting in multiple-organ failure. The prostate has been reported to express high levels of ACE2 and TMPRSS2. While there are limited studies regarding the association between COVID-19 and prostatitis, the possibility that SARS-CoV-2 could cause prostatitis cannot be denied. Thus, through this review, a better insight into the associations of SAR-CoV-2 can be provided.

Effects of different packaging method on oxidation stability and tenderness of chicken breast injected with different types of kiwifruit during storage

SummaryThis study aimed to investigate the antioxidant and protease activities of red kiwifruit (RK), green kiwifruit (GRK), and gold kiwifruit (GOK) and determine the effects of various packaging methods on the quality of chicken breast injected with various kiwifruit types during storage. The physicochemical and textural properties of chicken breasts containing various kiwifruit types were evaluated. Chicken breasts were injected with brine solution containing 5% kiwifruit powder, and their product quality was measured every 3 days until day 9. GOK and RK addition to chicken breast delayed oxidation during storage. Adding GOK and GRK to chicken breast was found to increase tenderness. Modified‐atmosphere packaging (MAP) delayed microbial growth more effectively than over‐wrap packaging (OWP). These findings suggest that the combination of GOK and MAP potentially extends shelf‐life without deteriorating quality.

Isolation, Identification, and Characteristics of Local Yeast Isolates with Proteolytic Capability Obtained from Traditional Processed Indonesian Duck Eggs

Various microorganisms can grow in egg-based food products, and yeast is among the microorganisms that thrive in egg products. Yeasts naturally produce large enzymes by breaking down organic materials during growth. Yeast has a great biotechnological potential owing to its extracellular enzymatic activity. Therefore, it is important to study yeast species, especially those from food sources and the environment. Protease production and activity are affected by various factors such as temperature, time, and pH. In this study, 47 yeast isolates were identified and purified from traditionally processed Indonesian duck eggs via repeated sub-culturing and quadrant streaking. Screening of the yeast isolates on skim milk agar medium showed that 23 isolates exerted proteolytic activity, whereas the remaining 25 did not. Yeast isolates with proteolytic capabilities belonged to the Saccharomyces, Pichia, and Candida genera. The highest protease activities were observed in isolates TA-17 and TPi-08, with activity values of 0.618 and 0.098 U/mL, respectively.

Characterization of protease-producing bacteria from garden soil and antagonistic activity against pathogenic bacteria

Bacteria play an essential role in various industrial processes, including food, medicine, and detergents, due to their ability to produce protease, a hydrolytic enzyme. This study aimed to identify and characterize protease-producing bacterial strains isolated from garden soil, evaluate their antibiotic susceptibility, and optimize conditions for enhanced protease production. Using skimmed milk agar medium and the gelatin hydrolysis method, 7 bacterial strains (S1, S2, S3, S4, S5, S6, and S7, respectively) were identified for protease production in garden soil. The bacteria were identified based on morphological studies, biochemical characterization, and the BIOLOG™ system, and the isolates were determined to be Bacillus cereus (S1), B. thuringiensis (S2), B. subtilis (S3), B. amyloliquefaciens (S4), Pseudomonas aeruginosa (S5), Macrococcus brunensis (S6), and B. schlegelii (S7). The antibiotic susceptibility test, performed using the Kirby-Bauer agar disc diffusion method, revealed that all isolates were resistant to penicillin, nitrofurantoin, and metronidazole. Additionally, B. subtilis and P. aeruginosa demonstrated antagonistic activity against pathogens. Optimal conditions for protease production were found to be a temperature range of 30°C to 40°C and a pH range of 6.5–7.5. Under these conditions, B. cereus (2.19 U/ml) and B. thuringiensis (2.12 U/ml) exhibited the highest protease activity. Further characterization of protease activity in B. cereus, P. aeruginosa, and M. brunensis under different physiological parameters revealed that maximum activity occurred within a pH range of 5.0–6.0 and temperatures between 30°C and 40°C. Protease activity increased in the presence of calcium (CaCl2) but decreased with the addition of urea. These findings underscore the significant industrial applications of proteases produced by these bacterial strains.

Candida spp. isolated from recreational coastal waters of Rio de Janeiro – Brazil: Focus on antifungal resistance and virulence attributes

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1–5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87–3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

A Bilingual Nanobiosensor for Cross‐Category Integrated Decoding of the Beta‐Site Amyloid Precursor Protein‐Cleaving Enzyme 1–Aβ Oligomer Signaling Pathway

Herein, a sequentially responsive peptide DNA bilingual nanobiosensor is developed, which allows integrated quantification of amyloid signaling pathway. In this system, upstream beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) protease and downstream Aβ oligomer (AβO) are designed as two inputs for the AND DNA logic gate. In the existence of both inputs, peptide substrate with aptamer can be sequentially cleaved, reporting electrochemical and fluorescence dual‐mode outputs. In comparison with conventional single protease activity assay based on peptide nanotechnology, this strategy permits accurate diagnosis of Alzheimer's disease (AD) from normal subjects. More importantly, it can achieve distinguished diagnosis between AD and type 2 diabetes mellitus patients. This bilingual nanobiosensor is successfully applied to detect BACE1 (1–100 U mL−1) and AβO (5–1000 pg mL−1) with limit of detections as low as 0.10 U mL−1 and 0.76 pg mL−1, respectively. Furthermore, this strategy inspires advanced nanobiosensors to target the activation of other signaling pathways, which are potential tools for future biology and medicine investigation.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Effects of N levels on land productivity and N2O emissions in maize-soybean relay intercropping.

Relay intercropping of maize and soybean can improve land productivity. However, the mechanism behind N2O emissions in this practice remains unclear. A two-factor randomized block field trial was conducted to reveal the mechanism of N2O emissions in a full additive maize-soybean relay intercropping. Factor A was three cropping systems - that is, monoculture maize (Zea mays L.), monoculture soybean (Glycine max L. Merr.) and maize-soybean relay intercropping. Factor B was different N supply, containing no N, reduced N and conventional N. Differences in N2O emissions, soil properties, rhizosphere bacterial communities and yield advantage were evaluated. The land equivalent ratio was 1.55-2.44, and the cumulative N2O emission ( ) was notably lower by 60.2% in intercropping than in monoculture, respectively. Reduced N declined without penalty on the yield advantages. The relay intercropping shifted soil properties - for example, soil organic matter, total N, and protease activity - and improved the soil microorganism community - for example, Proteobacteria and Acidobacteria. Intercropping reduced by directly suppressing nirS- and amoA-regulated N2O generation during soil N cycling, or nirS- and amoA-mediated soil properties shifted to reduce indirectly. Reduced N directly reduced by decreasing soil N content and reducing soil microorganism activities to alleviate N2O produced in soil N cycling. Conducting a full additive maize-soybean relay intercropping with reduced nitrogen supply provides a way to alleviate N2O emissions without the penalty on the yield advantage by changing rhizosphere bacterial communities and soil N cycling. © 2024 Society of Chemical Industry.

Peptide production through enhanced solid-state fermentation of soybean meal with Bacillus subtilis SBM_1: Fermentation process and product characteristics

This study aimed to assess the impact of additional inoculation of Bacillus subtilis SBM_1 on solid-state fermentation (SSF) of soybean meal (SBM). The peptide yield of fermented SBM increased to 220.93 mg/g under the optimal SSF conditions, marking a 47.74 % rise compared to natural SSF. Throughout enhanced SSF, the physicochemical properties of metabolites were analyzed. The results showed that the pH and total bacterial counts increased in the initial 8 h, along with elevated levels of peptide, soluble protein, and free amino acid (FAA). Protease activities is maintained at a relatively high level (103.37–241.93 U/g), leading to the substantial degradation of trypsin inhibitor (TI) and macromolecular protein. Following enhanced SSF, the TI activity of the fermented SBM decreased by 76.11 %, and small peptides with molecular weight (MW) < 3000 Da dominated the fermented products (71.64 %). Additionally, the enhanced SSF process demonstrated an improvement in nutrient content in the fermented products. Structural analysis of the fermented SBM showed that macromolecular soybean globulin and allergenic proteins were degraded, with the unfolded and loosened protein spatial structures and the loosely packed and dispersed surface microstructure. Biosafety analysis revealed no detectable microbial contaminants, including mold, salmonella, and coliforms, in compliance with national standards.

Integrated biofloc technology in red tilapia aquaculture: Salinity-dependent effects on water quality, parental stock physiology, reproduction, and immune responses

The study examines the impact of integrated biofloc technology (BFT), different salinity levels, and their combined effects over 90 days on various physiological parameters. The investigation includes growth performance and feed utilization, water quality, the chemical composition of biofloc and fish, digestive enzymes, reproductive performance, stress and biochemical indicators, and antioxidant-immune responses in red tilapia (Oreochromis spp.) broodstock. The fish were initially weighed (males: n = 270; 104 ± 0.96 g; females: n = 270; 93.2 ± 0.66 g) and subsequently divided into 12 treatment groups (6 for males and 6 for females) spread across 36 separate tanks (3 tanks per treatment; 45 fish per treatment; 15 fish/tank). The treatments involved three salinity levels (18, 28, and 36 ppt) in both clear water (CW) and BFT systems. The outcomes demonstrated that fish in the 36 ppt salinity with BFT treatment demonstrated significant improvements (P < 0.05) in growth parameters (final body weight, weight gain, and specific growth rate, feed intake, and feed conversion ratio). The condition factor in BFT groups increased in all salinity situations. The survival rates of broodstock were consistently high in all experimental conditions The study found that BFT and salinity significantly impacted (P < 0.05) whole body contents (moisture, protein, lipid, and ash) in both males and females. Water quality parameters showed variations between BFT and CW, with notable impacts (P < 0.05) on dissolved oxygen and pH. The BFT and salinity influenced digestive enzyme activities (protease, amylase, and lipase) and reproductive performance (males) and the 36 ppt salinity with BFT recorded the highest values. The hemato-biochemical and antioxidant-immune responses were also impacted by BFT and salinity exposure. The study highlights the potential benefits of incorporating BFT into red tilapia aquaculture systems, particularly in optimizing growth, health, and reproductive performance under various salinity conditions, which can enhance sustainable intensification, disease control, and environmental stewardship.

Platelet-Rich Plasma: Characteristics and Current Review of its Use in Surgery.

Platelet-rich plasma (PRP) is presently employed across various medical disciplines, including surgical specialties. It is primarily used in the healing of chronic wounds, burn medicine, tissue regeneration support, and scar correction as well as in other surgical and orthopedic indications. Wounds, in general, possess a pro-inflammatory biochemical environment characterized by high protease activity that diminishes the effective concentration of growth factors. In contrast, PRP serves as a source of growth factors and exhibits mitogenic, angiogenic, and chemotactic properties. PRP is a biological product defined as a portion of the autologous blood&apos;s plasma fraction with a higher than physiological platelet concentration. PRP is obtained through the centrifugation of blood samples collected from patients. The insights into the biology, mechanism of action, and classification of PRP presented in this review can assist medical professionals in orienting themselves and comprehending the possibilities of the clinical application of this therapy, which remains a subject of investigation and validation. This review summarizes up-to-date information regarding the use of PRP, primarily in burn and reconstructive surgery, plastic surgery, traumatology, and general surgery.

The effect of various dietary MUFA content and n-6/n-3 ratios on rainbow trout (Oncorhynchus mykiss) fed on a whole plant protein based diet: growth performance, digestive enzymes activity, intestinal morphometry and fatty acids profile of muscle and liver.

The effect of diets with various dietary MUFA content and n-6/n-3 ratios, diets 1 (55.33, 1.00), 2 (25.30, 1.00), 3 (55.13, 2.05), 4 (24.92, 2.03), 5 (54.94, 8.06) and 6 (24.91, 8.06) and a control diet with fish oil (FO), diet 7 (33.60, 5.97), was studied on growth and digestive physiology of rainbow trout, Oncorhynchus mykiss. Juveniles, 14.65g ± 0.17, were fed the experimental diets for eight weeks. Those fish fed diet 1 (55.33, 1.00) had the lowest growth and nutritional indices, while fish fed diet 6 (24.91, 8.06) possessed the highest indices. However, fish fed D7 (33.60, 5.97) was intermediate in this regard. Proximate body composition results revealed no significant differences among various experimental groups (P > 0.05). Fish fed on diet 1 possessed the highest alkaline protease activity, while fish raised on diet 2 showed the lowest enzyme activity (P < 0.05). Fish fed on diets 1, 3 and 4 had the highest lipase activity (P > 0.05). Amylase activity and intestinal parameters did not differ among groups (P > 0.05). MUFA contents of liver and muscle reflected the MUFA contents of the diets; however, liver PUFA contents was not affected by dietary PUFA contents (P < 0.05). However, 18:3n-3 contents of liver decreased as dietary n-6/n-3 ratio increased (P < 0.05). Furthermore, the liver 20:4n-6 contents considerably increased as dietary n-6/n-3 increased to 8.06 (P < 0.05). Muscle 22:6n-3 content of groups 1 to 6 was lower than group 7 (P < 0.05). In conclusion, rainbow trout was considerably capable of de novo synthesis of 22:6n-3. Meanwhile, increasing dietary n-6/n-3 ratio to 8.06 decreased the muscle 22:6n-3 content which requires further considerations regarding fish immune competence and nutritional quality for human consumption.

Qualitative and quantitative protease activity tests based on protein degradation in three-dimensional structures

The pattern of the activity of proteases is related to distinct physiological states of living organisms. Often activity changes of a certain protease can be assigned to a specific disease. Hence, they are useful biomarkers and a simple and fast determination method of their activity could be a valuable tool for the efficient monitoring of numerous diseases. Here, two different methods for the qualitative and quantitative determination of protease activity are demonstrated using the model system of proteinase K. The first test system is based on a protein-modified and colored 3D silica structure that changes color when exposed to the enzyme. This method has also been used for the detection of matrix metallo-protease 2 (MMP2) with gelatine as protease substrate on the plates. The second detection system uses the decrease in the voltammetric signal of a cytochrome c/DNA multilayer electrode after incubation with a protease to quantitatively determine its proteolytic activity. While activities down to 0.15 U/ml can be detected with the first method, the second one provides detection limits of about 0.03U/ml (for proteinase K.) The functionality of both systems can be demonstrated and ways for further enhancement of sensitivity have been elucidated.