Concentrations Of Trypsin Research Articles

Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry.

The determination of protease activity is very important for disease diagnosis, drug development, and quality and safety assurance for dairy products. Therefore, the development of low-cost and sensitive methods for assessing protease activity is crucial. We report two approaches for monitoring protease activity: in a volume and at surface, via colorimetric and acoustic wave-based biosensors operated in the thickness-shear mode (TSM), respectively. The TSM sensor was based on a β-casein substrate immobilized on a piezoelectric quartz crystal transducer. After an enzymatic reaction with trypsin, it cleaved the surface-bound β-casein, which increased the resonant frequency of the crystal. The limit of detection (LOD) was 0.48 ± 0.08 nM. A label-free colorimetric assay for trypsin detection has also been performed using β-casein and 6-mercaptohexanol (MCH) functionalized gold nanoparticles (AuNPs/MCH-β-casein). Due to the trypsin cleavage of β-casein, the gold nanoparticles lost shelter, and MCH increased the attractive force between the modified AuNPs. Consequently, AuNPs aggregated, and the red shift of the absorption spectra was observed. Spectrophotometric assay enabled an LOD of 0.42 ± 0.03 nM. The Michaelis–Menten constant, KM, for reverse enzyme reaction has also been estimated by both methods. This value for the colorimetric assay (0.56 ± 0.10 nM) is lower in comparison with those for the TSM sensor (0.92 ± 0.44 nM). This is likely due to the better access of the trypsin to the β-casein substrate at the surface of AuNPs in comparison with those at the TSM transducer.

Trypsin Inhibitor Assay: Expressing, Calculating, and Standardizing Inhibitor Activity in Absolute Amounts of Trypsin Inhibited or Trypsin Inhibitors

AbstractFor expressing trypsin inhibitor activity (TIA), trypsin units inhibited (TUI), trypsin inhibited, and trypsin inhibitors have been used. Although the last two units are preferred, their calculations in current practices require refinement. With the proposed AOCS method Ba 12a‐2020, four experiments were conducted, using four trypsin preparations having specific activity of 11,625, 12,602, 13,728, and 14,926 Nα‐benzoyl‐L‐arginine ethyl ester (BAEE) units/mg protein, respectively. Experiment 1 determined the relationship between absorbance at 410 nm (A410) and trypsin concentration. Experiment 2 involved assaying raw and heated soybeans, expressing TIA as TUI/mg sample and μg trypsin inhibited/mg sample, and determining conversion factors between the two units. Experiment 3 resembled Experiment 2 except for using purified soybean Kunitz inhibitor (KTI) and Bowman‐Birk inhibitor (BBI). Conversion factors determined correlated highly with trypsin‐specific activity (R2 = 0.9789). After standardizing against a reference trypsin having 15,000 BAEE units/mg protein, a standardized conversion factor of 0.03 A410 (1.5 TUI) = 1 μg trypsin inhibited was determined. It remained consistent regardless of trypsin specific activity, with or without inhibitors, and type of inhibitor samples. By using purified inhibitors (Experiment 3), conversion values between TUI and μg trypsin inhibitor and between μg trypsin inhibited and μg trypsin inhibitor could also be calculated, enabling expression of TIA in amounts of pure KTI, BBI or their equivalents. Furthermore, when the AOCS method was modified with half substrate concentration, half trypsin concentration or half both (Experiment 4), TIA values in TUI could change with modifications but values in mg trypsin inhibited (standardized) or trypsin inhibitor remained consistent.

An anionic tetraphenyl ethylene based simple and rapid fluorescent probe for detection of trypsin and paraoxon methyl

In this study, we have developed an aggregation induced emission (AIE) based simple, sensitive, rapid and convenient biosensor for detection of trypsin and organophosphorous pesticides. The biosensor is composed of tetra-anionic sulphonyl derivative of tetraphenylethylene (Su-TPE) and cationic polyelectrolyte protamine (PrS), assembled by electrostatic interactions without any complex process of integration. The AIE property of Su-TPE/PrS system has been studied, in detail, using different spectroscopic techniques, such as, ground-state absorption spectroscopy, steady-state and time-resolved emission spectroscopy. The AIE phenomenon of Su-TPE molecules induced due to Su-TPE/PrS supramolecular complex formation has been supported by studying Su-TPE/PrS system under various environmental factors. After establishing the AIE phenomenon of Su-TPE/PrS complex, the potential of the sensing platform, for trypsin detection, has been explored based on the principle of trypsin dependent PrS hydrolysis. The detection system is highly sensitive in the linear trypsin concentration range of 0–16 nM with detection limit of 0.22 nM. Furthermore, the sensing platform also shows excellent selectivity towards trypsin. More importantly, based on trypsin inhibition, Su-TPE/PrS platform is also responsive towards organophosphorous pesticide detection. The commendable performance of the Su-TPE/PrS biosensor will enhance future development of rapid detection of trypsin along with fast high throughput screening of organophosphrous pesticides.

Fast in-line bottom-up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach.

For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.

Simple and Sensitive Method for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies by Combining Ultra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients, and BoxCar Mass Spectrometry Acquisition.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under nondenaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.5 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.

Morphological and biochemical effects of food deprivation during the early development of Pacific red snapper Lutjanus peru.

We report the effects of food deprivation on the early development of Pacific red snapper Lutjanus peru during the first days of development. The point of no return (PNR) was determined using the feeding incidence after a delay in first feeding. The gradual deterioration of the larvae during food deprivation was recorded using morphometric, histological, enzymatic and biochemical analysis. The time to reach the PNR was 120 h after hatching. Morphologically, the total length, muscle height, head length, tail length and pectoral angle showed the biggest reductions and their growth coefficients changed significantly during food deprivation. Histologically, enterocyte height also was reduced significantly. The protein concentration and activities of the digestive enzymes trypsin, cathepsin-like and lipase showed a significant decrease; meanwhile, amylase activity remained constant during food deprivation. The concentration of total essential free amino acids (EFAAs) decreased significantly while that of the nonessential free amino acids (NEFAAs) remain stable during food deprivation. The most abundant EFAAs were lysine, leucine, isoleucine and valine; the most abundant NEFAAs were alanine, glycine and glutamate, suggesting a more prominent role as energy substrates. At the time of the PNR the concentration of almost all the free amino acids showed a significant decrease. Early food deprivation has a significant impact on the morphology and biochemical characteristics of L. peru. These results suggest that initial feeding of L. peru should begin within 3 days of yolk sac depletion to avoid the PNR. Further studies are necessary to confirm and validate the characters identified in this study as biomarkers of starvation under culture conditions and evaluate their possible utility in ichthyoplankton surveys.

Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides

Although antimicrobial peptides (AMPs) have become powerful drug candidates in the post-antibiotic era, but their low protease stability hinders their clinical application. In the present study, the natural sunflower trypsin inhibitor 1 (SFTI-1) binding loop (CTKSIPPIC) was used to design and synthesize a specific anti-proteolytic sequence template ((RX)n W (RX)n CTKSIPPIC (n = 2, 3; X represents A, I, L, V, F, and W)). After several antibacterial, bactericidal, and toxicity tests, RV3 stood out from the variants and had the highest average selectivity index (SI all = 156.03). It is highly stable in serum, varying pH, temperature, and salt ions as well as under high trypsin, pepsin, or papain concentrations. In a mouse skin inflammation model, established by Pseudomonas aeruginosa infection, RV3 could effectively kill the pathogen, promote wound healing, inhibit inflammatory cell infiltration, and inhibit mRNA and protein expression of TNF-α, IL-6, and IL-1β inflammatory factors. The antibacterial mechanisms of RV3 include combining with lipopolysaccharides and increasing cell membrane permeability, leading to cell membrane rupture and death. These findings indicate that RV3 has great potential for the treatment of bacterial infections.

Peptide modified manganese-doped iron oxide nanoparticles as a sensitive fluorescence nanosensor for non-invasive detection of trypsin activity in vitro and in vivo.

Herein, a fluorescence turn-on nanosensor (MnIO@pep-FITC) has been proposed for detecting trypsin activity in vitro and in vivo through covalently immobilizing an FITC modified peptide substrate of trypsin (pep-FITC) on manganese-doped iron oxide nanoparticle (MnIO NP) surfaces via a polyethylene glycol (PEG) crosslinker. The conjugation of pep-FITC with MnIO NPs results in the quenching of FITC fluorescence. After trypsin cleavage, the FITC moiety is released from the MnIO NP surface, leading to a remarkable recovery of FITC fluorescence signal. Under the optimum experimental conditions, the recovery ratio of FITC fluorescence intensity is linearly dependent on the trypsin concentration in the range of 2 to 100 ng mL−1 in buffer and intracellular trypsin in the lysate of 5 × 102 to 1 × 104 HCT116 cells per mL, respectively. The detection limit of trypsin is 0.6 ng mL−1 in buffer or 359 cells per mL HCT116 cell lysate. The MnIO@pep-FITC is successfully employed to noninvasively monitor trypsin activity in the ultrasmall (ca. 4.9 mm3 in volume) BALB/c nude mouse-bearing HCT116 tumor by in vivo fluorescence imaging with external magnetic field assistance, demonstrating that it has excellent practicability.

The Efficiency of Decellularization of Bovine Pericardium by Different Concentrations of Sodium Dodecyl Sulfate

Background. In modern cardiovasclar surgery, it is a promising method to use xenotissues, which in their properties are close to human tissues, in order to restore the integrity of the heart chambers, its walls or valves. Decellularization of extracellular matrix (EMC) is applied in the process of creation of such bioprotheses. In EMC, the elastin and collagen components are preserved, and antigenic molecules are eliminated resulting in reduction the risk of rejection. The study is devoted to assessment of the histological, molecular-genetic and cytotoxic properties of decellularized bovine pericardium, processed with various concentrations of trypsin enzyme.Objective. The aim of the work is evaluation of efficiency of bovine pericardial decellularization, based on the use of trypsin enzyme with 1% Sodium Dodecyl Sulfate (SDS) and 0.1% SDS.Methods. Bovine pericardium was used as a biomaterial for decellularization. Decellularization protocol 1 envisages processing of samples with 0.25% Trypsin solution at 24 °C with constant shaking (200 rpm) along with processing with 1% ionic SDS detergent. The samples, prepared according to protocol 2, were processed with a low concentration of 0.1% SDS. Histological and morphological properties along with detection of nucleic acids concentration in the samples were studied. Matrix samples were cultured in a human fibroblasts cell culture suspension in order to determine cytotoxicity.Results. Histological examination has not revealed any presence of cells in tissues, decellularized in accordance with both protocols. More than 99% of the nucleic acids was removed from decellularized bovine matrix. During our study, we have not observed cytotoxic effect in vitro for protocol 2 matrix samples, decellularized with only 0.1% SDS. Focal destruction of fibroblasts was observed in conditions of long-term cultivation in protocol 1 samples (Trypsin + 1% SDS). Cells formed abnormal morphological aggregates. Samples of this group have also demonstated sructural changes in collagen and elastin fibers.Conclusions. Studies have shown that pericardial matrix tissue, decellularized with low-concentration of 0.1% SDS, has the same biological properties as the native pericardium. Decellularization of bovine pericardium, using trypsin enzyme with 1% SDS, has a cytotoxic effect on human cells.

Tryptophan Fluorescence and Time-Lag Hydrolysis of Peptide Bonds during Degradation of β-Lactoglobulin by Trypsin

The opening of protein globules and corresponding exposure of their internal peptide bonds, the so-called demasking effect, is required for successful hydrolysis of peptide bonds by proteases. Under the proteolytic action of trypsin on β-lactoglobulin (β-LG), the evolution of tryptophan fluorescence spectra showed that the demasking process consists of two stages with different demasking rate constants for each stage. It was found that the ratio of these constants depends on the concentration of trypsin and changes are approximately threefold when the concentration of trypsin changes in the range of 0.3–15 mg/L. Simulation of hydrolysis taking into account the demasking effect demonstrated how the apparent first-order rate constants obtained experimentally are related to the true hydrolysis rate constants and demasking parameters. The lag phase in the kinetic curves corresponding to the hydrolysis of various peptide bonds in β-LG was also analyzed. The increased lag times indicated sites that are hydrolyzed by a two-stage demasking mechanism.

Insulin Release from NPH Insulin-Loaded Pluronic® F127 Hydrogel in the Presence of Simulated Tissue Enzyme Activity

Background: Despite the widespread use of newer basal insulins, Natural Protamine Hagedorn (NPH) insulin still represents a well-established basal formulation with its long history of use, featuring the native form of human insulin. However, NPH insulin exhibits an undesirable peak within hours after a single subcutaneous (s.c.) injection, which may lead to hypoglycemia followed by insufficient basal insulin delivery. This may be attributed to the s.c. enzyme activities degrading the protamine in NPH microcrystals. Methods: A thermogelling block copolymer Pluronic® F127 (PF127) was utilized as a protective carrier for NPH microcrystals and as a modulator for insulin release from NPH. NPH insulin-loaded PF127 gel was prepared with varying concentrations of the polymer (15–25%) under mild conditions. The formulations were characterized for their gelling temperature, morphology, gel erosion, and in vitro insulin release, with trypsin concentrations up to 5 U/mL. Results: Scanning electron microscopy (SEM) showed that the integrity of NPH microcrystals was maintained after preparation. The burst release of insulin from NPH was significantly attenuated over the course of ~16h in the presence of PF127 with or without enzyme activity. Conclusion: NPH-PF127 successfully resisted the acceleration of NPH crystal dissolution and insulin release in vitro in the presence of protamine-degrading enzyme activity, warranting further testing.

The effect of proteolytic enzymes and pH on GII.4 norovirus, during both interactions and non-interaction with Histo-Blood Group Antigens

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes (1,500 units/mL pepsin, 100 mg/mL trypsin, and 100 mg/mL chymotrypsin). VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes.

Generic sensor platform based on electro-responsive molecularly imprinted polymer nanoparticles (e-NanoMIPs)

The present research describes the design of robust electrochemical sensors based on electro-responsive molecularly imprinted polymer nanoparticles (e-MIPs). The e-MIPs, tagged with a redox probe, combine both recognition and reporting functions. This system replaces enzyme-mediator pairs used in traditional biosensors. The analyte recognition process relies on the generic actuation phenomenon when the polymer conformation of e-MIPs is changing in response to the presence of the template analyte. The analyte concentration is measured using voltammetric methods. In an exemplification of this technology, electrochemical sensors were developed for the determination of concentrations of trypsin, glucose, paracetamol, C4-homoserine lactone, and THC. The present technology allows for the possibility of producing generic, inexpensive, and robust disposable sensors for clinical, environmental, and forensic applications.

Influence of ractopamine hydrochloride supplementation on pancreatic digestive enzyme activity in finishing steers

Sixty-nine finishing steers were randomly assigned to one of two treatment groups: supplementation to provide 0 or 267 mg·d−1 of ractopamine hydrochloride (RAC) for 42 d. Twelve steers were slaughtered for tissue and blood collection. Final body weight (BW) tended to be greater with RAC supplementation. Ractopamine hydrochloride supplementation decreased liver mass as a percentage of BW. Pancreatic protein concentration, digestive enzyme activities, serum glucose, and insulin concentrations were not influenced by RAC supplementation. Ractopamine hydrochloride supplementation decreased the pancreatic:α-amylase:trypsin and serum urea nitrogen concentrations. These data suggest that RAC may influence pancreatic exocrine function in cattle.

A Paper-Based Near-Infrared Optical Biosensor for Quantitative Detection of Protease Activity Using Peptide-Encapsulated SWCNTs

A protease is an enzyme that catalyzes proteolysis of proteins into smaller polypeptides or single amino acids. As crucial elements in many biological processes, proteases have been shown to be informative biomarkers for several pathological conditions in humans, animals, and plants. Therefore, fast, reliable, and cost-effective protease biosensors suitable for point-of-care (POC) sensing may aid in diagnostics, treatment, and drug discovery for various diseases. This work presents an affordable and simple paper-based dipstick biosensor that utilizes peptide-encapsulated single-wall carbon nanotubes (SWCNTs) for protease detection. Upon enzymatic digestion of the peptide, a significant drop in the photoluminescence (PL) of the SWCNTs was detected. As the emitted PL is in the near-infrared region, the developed biosensor has a good signal to noise ratio in biological fluids. One of the diseases associated with abnormal protease activity is pancreatitis. In acute pancreatitis, trypsin concentration could reach up to 84 µg/mL in the urine. For proof of concept, we demonstrate the feasibility of the proposed biosensor for the detection of the abnormal levels of trypsin activity in urine samples.

A Preliminary Study on the Ability of the Trypsin-Like Peptidase Activity Assay Kit to Detect Periodontitis.

This study aimed to explore whether the Trypsin-Like Peptidase Activity Assay Kit (TLP-AA-Kit), which measures the activity of N-benzoyl-dl-arginine peptidase (trypsin-like peptidase), can be used as a reliable tool for periodontitis detection in population-based surveillance. In total, 105 individuals underwent a full-mouth periodontal examination and provided tongue swabs as specimens for further analyses. The results of the TLP-AA-Kit were scored between 1 and 5; higher scores indicated higher trypsin concentrations. Receiver operating characteristic analyses were used to evaluate the predictive validity of the TLP-AA-Kit, where the periodontitis case definition provided by the Centers for Disease Control/American Academy of Periodontology served as the reference. Severe and moderate periodontitis were identified in 4.8% and 16.2% of the study population, respectively. The TLP-AA-Kit showed high diagnostic accuracy for severe periodontitis, with an area under the curve of 0.93 (95% confidence interval = 0.88–0.99). However, the diagnostic accuracy of the TLP-AA-Kit for moderate/severe periodontitis was not reliable. While further studies are necessary to validate our results, the results provided herein highlight the potential of the TLP-AA-Kit as a useful tool for the detection of periodontitis, particularly in severe cases, for population-based surveillance.

Assessment of grain and vegetable type soybean genotypes for nutritional composition

The present study was conducted to analyse the nutritional composition of fourteen soybean genotypes including seven each of grain and vegetable types. The study revealed that oil content was in the range of 17.51 to 20.59% at R8 stage, protein 18.11 % to 27.57% at R6 and 32.57 % to 39.09% at R8 stage, carbohydrate content at R6 stage 7.43 to 11.08 g/100g and 24.15 to 28.37 g/100g at R8 stage, sugar content 0.90 to 3.43 g/100g, iron content at R6 stage 3.24 to 7.12 mg/100g and 6.79 mg/100g to 9.79 mg/100g at R8 stage, zinc content at R6 stage 4.74 to 6.40 mg/100g and 3.93 mg/100g to 5.60 mg/100 g, The trypsin inhibitor content was ranged between 18.29 to 46.26 mg/g and 33.96 to 75.09 mg/g at R6 and R8 stage respectively. Vegetable types were found more nutritious than grain type soybean. Swarna vasundhara was found promising for protein content at R6 stage, the genotype AGS-450 exhibited high iron with low trypsin content whereas, AGS-459 exhibited high sugar and zinc content.

Production of Liquid Milk Protein Concentrate with Antioxidant Capacity, Angiotensin Converting Enzyme Inhibitory Activity, Antibacterial Activity, and Hypoallergenic Property by Membrane Filtration and Enzymatic Modification of Proteins

Liquid milk protein concentrate with different beneficial values was prepared by membrane filtration and enzymatic modification of proteins in a sequential way. In the first step, milk protein concentrate was produced from ultra-heat-treated skimmed milk by removing milk serum as permeate. A tubular ceramic-made membrane with filtration area 5 × 10−3 m2 and pore size 5 nm, placed in a cross-flow membrane house, was adopted. Superior operational strategy in filtration process was herein: trans-membrane pressure 3 bar, retention flow rate 100 L·h−1, and implementation of a static turbulence promoter within the tubular membrane. Milk with concentrated proteins from retentate side was treated with the different concentrations of trypsin, ranging from 0.008–0.064 g·L−1 in individual batch-mode operations at temperature 40 °C for 10 min. Subsequently, inactivation of trypsin in reaction was done at a temperature of 70 °C for 30 min of incubation. Antioxidant capacity in enzyme-treated liquid milk protein concentrate was measured with the Ferric reducing ability of plasma assay. The reduction of angiotensin converting enzyme activity by enzyme-treated liquid milk protein concentrate was measured with substrate (Abz-FRK(Dnp)-P) and recombinant angiotensin converting enzyme. The antibacterial activity of enzyme-treated liquid milk protein concentrate towards Bacillus cereus and Staphylococcus aureus was tested. Antioxidant capacity, anti-angiotensin converting enzyme activity, and antibacterial activity were increased with the increase of trypsin concentration in proteolytic reaction. Immune-reactive proteins in enzyme-treated liquid milk protein concentrate were identified with clinically proved milk positive pooled human serum and peroxidase-labelled anti-human Immunoglobulin E. The reduction of allergenicity in milk protein concentrate was enzyme dose-dependent.

Use of alternative plant and animal protein blends, in place of fishmeal, in diets for juvenile totoaba, Totoaba macdonaldi

The present study evaluated the replacement of fishmeal (FM) by blends of alternative protein sources in diets for juvenile (8.0 ± 0.5 g) Totoaba macdonaldi. Using a FM-based control diet containing 53% crude protein and 13% crude fat, a 3 × 3 factorial experiment was conducted replacing FM, at levels of 33, 66 and 100% by weight, with three blends of the test ingredients, as follows: 1) Arthrospira meal blend: 70% Arthrospira sp. meal, 15% blood meal, 15% soy protein concentrate (SPC); 2) blood meal blend: 70% blood meal, 15% Arthrospira sp. meal, 15% SPC; and 3) SPC blend: 70% SPC, 15% blood meal, 15% Arthrospira sp. meal. At the end of the 6-week feeding trial, significant effects of the alternative protein blends were detected, in which fish fed the Arthrospira meal and SPC blends had significantly (P < .05) better performance than fish fed the blood meal blend across all growth parameters (percent weight gain and thermal growth coefficient) and feed utilization indices (feed efficiency and protein efficiency ratio). Also, fish fed the former two blends deposited more crude protein and fat in whole body and muscle tissues, concurring with the growth response observed. However, the effects of protein blend on body condition indices (hepatosomatic index, condition factor, and muscle ratio) and on the concentrations of trypsin and chymotrypsin in anterior intestine and pyloric caeca, did not depict clear trends. A correspondence between levels of dietary amino acids (AA) in blends and accumulated AA in muscle, was not observed. However, significant effects of the level of FM replacement were also detected, in which the responses in growth, feed utilization, most body condition indices, and proximate composition of fish whole body, deteriorated as the level of FM replaced increased. All things considered, results indicate that the Arthrospira meal and SPC blends, but not the blood meal blend, can be used to replace at least 33% FM in the diet for totoaba, without adversely affecting growth and survival. The possible reasons for the decreased performance of fish fed the blood meal blend, or when fed any of the blends at levels higher than 33%, are discussed.

Combinatorial effects of antibiotics and enzymes against dual-species Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the wound-like medium.

Bacterial biofilms are one of the major issues in the treatment of chronic infections such as chronic wounds, where biofilms are typically polymicrobial. The synergy between species can occur during most polymicrobial infections, where antimicrobial resistance enhances as a result. Furthermore, self-produced extracellular polymeric substance (EPS) in biofilms results in a high tolerance to antibiotics that complicates wound healing. Since most antibiotics fail to remove biofilms in chronic infections, new therapeutic modalities may be required. Disruption of EPS is one of the effective approaches for biofilm eradication. Therefore, degradation of EPS using enzymes may result in improved chronic wounds healing. In the current study, we investigated the efficacy of trypsin, β-glucosidase, and DNase I enzymes on the degradation of dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in a wound-like medium. These species are the two most common bacteria associated with biofilm formation in chronic wounds. Moreover, the reduction of minimum biofilm eradication concentration (MBEC) of meropenem and amikacin was evaluated when combined with enzymes. The minimum effective concentrations of trypsin, β-glucosidase, and DNase I enzymes to degrade biofilms were 1 μg/ml, 8 U/ml, and 150 U/ml, respectively. Combination of 0.15 μg/ml trypsin and 50 U/ml DNase I had a significant effect on S. aureus-P. aeruginosa biofilms which resulted in the dispersal and dissolution of all biofilms. In the presence of the enzymatic mixture, MBECs of antibiotics showed a significant decrease (p < 0.05), at least 2.5 fold. We found that trypsin/DNase I mixture can be used as an anti-biofilm agent against dual-species biofilms of S. aureus-P. aeruginosa.