Proteases as Tools for Modulating the Antioxidant Activity and Functionality of the Spent Brewer's Yeast Proteins.

The functionality of the peptides obtained through enzymatic hydrolysis of spent brewer's yeast was investigated. Hydrolysis was carried out for 4-67 h with bromelain, neutrase and trypsin. The resulting hydrolysates were characterized in terms of physical-chemical, antioxidant and techno-functional properties. The solid residues and soluble protein contents increased with the hydrolysis time, the highest values being measured in samples hydrolyzed with neutrase. Regardless of the hydrolysis time, the maximum degree of hydrolysis was measured in the sample hydrolyzed with neutrase, while the lowest was in the sample hydrolyzed with trypsin. The protein hydrolysate obtained with neutrase exhibited the highest DPPH radical scavenging activity (116.9 ± 2.9 µM TE/g dw), followed by the sample hydrolyzed with trypsin (102.8 ± 2.7 µM TE/g dw). Upon ultrafiltration, the fraction of low molecular weight peptides (<3 kDa) released by bromelain presented the highest antioxidant activity (50.06 ± 0.39 µM TE/g dw). The enzymes influenced the foaming properties and the emulsions-forming ability of the hydrolysates. The trypsin ensured the obtaining of proteins hydrolysate with the highest foam overrun and stability. The emulsions based on hydrolysates obtained with neutrase exhibited the highest viscosity at a shear rate over 10 s-1. These results indicate that the investigated proteases are suitable for modulating the overall functionality of the yeast proteins.

Identification and preparation of selenium-containing peptides from selenium-enriched Pleurotus eryngii and their protective effect on lead-induced oxidative damage in NCTC1469 hepatocytes.

BACKGROUND: Lead (Pb) is a highly toxic and persistent substance that easily accumulates in living organisms, eliciting cellular toxicity and oxidative stress. Some selenium-containing proteins and peptides prepared from plant extracts are beneficial for protecting the body's health and resisting external disturbances. In the present study, selenium-containing peptide species were prepared from selenium-enriched Pleurotus eryngii protein hydrolysates and to evaluate the benefits of selenium-containing peptides on Pb-induced oxidative stress in NCTC1469 hepatocytes. RESULTS: Trypsin was selected as primary enzyme to hydrolyze the selenium-enriched protein (SPH). The optimal hydrolysis conditions were: hydrolysis time, 1.5 h; initial pH 8.0. The SPH was digested by trypsin and then purified by ultrafiltration, gel filtration chromatography and reversed-phase HPLC to obtain the selenium-containing peptides SPH-I-2. Furthermore, SPH-I-2 was analyzed and a number of total 12 selenium-containing peptides were identified by liquid chromatography-tandem mass spectroscopy. The NCTC1469 cell culture study showed that selenium-containing peptides were capable of reducing reactive oxygen species levels and regulating the Keap1/Nrf2 pathway by upregulating Nrf2, HO-1, GCLC, GCLM and NQO1 genes and downregulating Keap1 genes. Moreover, selenium-containing peptides were also able to suppress Pb-induced elevated levels of nitric oxide (NO), lactate dehydrogenase (LDH), malondialdehyde (MDA), increase antioxidant enzyme activity and alleviate cell apoptosis. CONCLUSION: The present study indicated that the selenium-containing peptides could protect cells from Pb2+ -induced oxidative stress. © 2023 Society of Chemical Industry.

In vivo developmental studies of Helicoverpa armigera and in silico molecular interactions with trypsin reveal the bio-insecticidal potential of trypsin inhibitor (SSTI) isolated from Solanum surattense.

We report the impact of gut protease inhibition on the development of Helicoverpa armigera by trypsin inhibitor and the use of molecular modeling to understand the mechanism of trypsin inhibition. Larvae of H. armigera fed on an artificial diet containing 150 and 300 µg/ml SSTI showed a negative impact on the insects' development in terms of mean larval weight, larval fatality, survival rate, and nutritional indices. Prominent physical abnormalities like curled wings, malformed appendages, and small body size were observed during the development. Gene expression studies revealed down regulation in trypsin (HaTry 1, 2, 3, 4, 6, 8) and chymotrypsin (HaChy 1, 2, 3, 4) genes of the larval gut upon treatment of SSTI. Homology modeling has been used to build the three-dimensional structure of SSTI, which showed ß-sheets having a stable canonical inhibitory loop (CIL) with conserved lysine residue. Molecular docking studies showed the strong binding of SSTI at the active site of trypsin. Molecular dynamic (MD) simulation revealed the stable interactions of the rigid CIL of SSTI at the active site of trypsin, leading to its destabilization. Conserved lysine63 of the P1 site in SSTI forms a strong hydrogen bonding network with residues Asp189 and Ser190 of trypsin.

Effects of autochthonous strains mixture on gut microbiota and metabolic profile in cobia (Rachycentron canadum).

The fish immune system is a topic or subject that offers a unique understanding of defensive system evolution in vertebrate heredity. While gut microbiota plays several roles in fish: well-being, promoting health and growth, resistance to bacterial invasion, regulation of energy absorption, and lipid metabolism. However, studies on fish gut microbiota face practical challenges due to the large number of fish varieties, fluctuating environmental conditions, and differences in feeding habits. This study was carried out to evaluate the impacts of supplemented three autochthonous strains, Bacillus sp. RCS1, Pantoea agglomerans RCS2, and Bacillus cereus RCS3 mixture diet on cobia fish (Rachycentron canadum). Also, chromatography, mass spectrometry and high throughput sequencing were combined to explore composition and metabolite profile of gut microbiota in juvenile cobia fed with supplemented diet. In the trial group, juvenile cobia received diets supplemented with 1 × 1012 CFU mL-1 autochthonous strains for ten weeks and a control diet without supplementation. Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without additives (control). Haematological indices, such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and mean corpuscular haemoglobin, were higher in the supplemented group. Similarly, digestive enzymes (trypsin, lipase, amylase, pepsin and cellulose, activities) activities were higher in supplemented diet with an indigenous isolates mixture. Serum biochemical parameters albumin, globulin, and total protein were significantly higher, while triglyceride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and cholesterol showed no significant difference. On the other hand, glucose was significantly (P < 0.05) higher in the group without supplementation. On gene expression in the midgut, Immunoglobulin, Colony-stimulating factor receptor 1, major histocompatibility complex 1 were up-regulated by native isolates while T cell receptor beta, and Major histocompatibility complex 2 showed no significant difference. Gut bacterial composition was altered in fish receiving supplemented diet with autochthonous strains. Metabolomics also revealed that some metabolic pathways were considerably enriched in fish fed with supplemented diet; pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that differentially expressed metabolites were involved in galactose metabolism, tryptophan metabolism, carbohydrate digestion and absorption, purine metabolism, and ABC transporters. Functional analysis of bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites, nucleotide and its metabolites, amino acid and its metabolites, heterocyclic compounds, and tryptamines, cholines, pigments. The current investigation results showed that autochthonous strains mixture has significantly enhanced the growth, survival, and innate and adaptive immunities of juvenile cobia.

[Trypsin-inhibitor activity in colostrum - an overview]. / Trypsin-Inhibitor-Aktivität im Kolostrum ­ eine Übersicht.

The crucial role of colostrum for the neonatal immune system is well recognized. Following ingestion, proteins and especially immunoglobulins must pass the gastrointestinal tract and its proteolytic enzymes intact in order to be absorbed into the neonatal blood circulation. For this reason colostrum exhibits trypsin-inhibitor activity. This activity is not exerted by a single molecule but represents a general characteristic of the first colostrum. In artiodactyl species, high-level trypsin inhibition has been demonstrated along with a rapid decrease during the first days of lactation. In equine colostrum, trypsin-inhibitor activity has also been detected. Its importance is however controversially discussed in the literature due to the fact that the anti-trypsin activity is less pronounced in comparison to artiodactyl species and exhibits reduced stability in acidic environment. In the colostrum of carnivores, anti-trypsin activity has also been proven, this however is less prominent than in ungulates. The presented overview of the literature aims at summarizing the current understanding of trypsin inhibition in the colostrum of different species.

Energy values evaluation and improvement of soybean meal in broiler chickens through supplemental mutienzyme.

This study measured the metabolizable energy of soybean meal (SBM) and evaluated effects of soybean meal specific enzymes supplementation in corn-soybean diets on growth performance, intestinal digestion properties and energy values of 28-day-old broilers. A total of 336 one-day-old male AA broiler chickens were distributed to 7 groups in a completely random design. The birds were given 7 diets containing 6 diets with different combined soybean meals and a fasting treatment, 8 replicates per treatment and 6 birds per replicate (Trial 1). A total of 672 one-day-old male AA broiler chickens were randomly allocated to 7 dietary treatments including a control diet and 6 diets supplemented with 300 mg/kg α-galactosidase, 200 mg/kg ß-mannanase, and 300 mg/kg protease individually or in combination (Trial 2). Apparent metabolizable energy (AME) of broilers was measured from d 25 to 27 in both trial 1 and trial 2. The results showed that AME values of combined soybean meals averaged 2,894 kcal/kg. Dietary ß-mannanase and protease supplementation increased body weight gain (P < 0.05) during d 0 to 14, whereas did not affect the growth performance (P > 0.05) during d 14 to 28. Addition of ß-mannanase in combination with other enzymes significantly increased lipase and trypsin content (P < 0.05) in ileum. In addition, dietary ß-mannanase and protease supplementation individually or in combination enhanced trypsin enzyme content in jejunum (P < 0.05). The ß-mannanase enzyme enhanced villus height and villus height to crypt depth ratio (P < 0.05) of ileum compared with control diet. Moreover, supplementation of enzyme except for protease enhanced raffinose and stachyose degradation ratio (P < 0.05). Dietary ß-mannanase supplementation individually or in combination enhanced AME and AMEn values (P < 0.05). This study demonstrated that dietary enzyme supplementation especially ß-mannanase improved intestinal digestion properties and contributed to high energy values.

Modulatory effects of laurel-leaf cistus (Cistus laurifolius) ethanolic extract on innate immune responses and disease resistance in common carp (Cyprinus carpio).

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

Structural insights and molecular dynamics into the inhibitory mechanism of a Kunitz-type trypsin inhibitor from Tamarindus indica L.

Trypsin inhibitors from tamarind seed have been studied in vitro and in preclinical studies for the treatment of obesity, its complications and associated comorbidities. It is still necessary to fully understand the structure and behaviour of these molecules. We purifed this inhibitor, sequenced de novo by MALDI-TOF/TOF, performed its homology modelling, and assessed the interaction with the trypsin enzyme through molecular dynamics (MD) simulation under physiological conditions. We identified additional 75 amino acid residues, reaching approximately 72% of total coverage. The four best conformations of the best homology modelling were submitted to the MD. The conformation n°287 was selected considering the RMSD analysis and interaction energy (-301.0128 kcal.mol-1). Residues Ile (54), Pro (57), Arg (59), Arg (63), and Glu (78) of pTTI presented the highest interactions with trypsin, and arginine residues were mainly involved in its binding mechanism. The results favour bioprospecting of this protein for pharmaceutical health applications.

Optimization of trypsin extraction technology of Allium cepa L. polysaccharide by response surface methodology and the antitumor effects through immunomodulation.

The trypsin-assisted extraction of polysaccharides from Allium cepa L. was optimized using the response surface methodology (RSM). The optimum extraction conditions were extraction temperature, extraction time, extraction pH, and enzyme amount of 37.16°C, 180 min, 8.57, and 5.16%, respectively. Under the optimized conditions, the yield of A. cepa L. polysaccharides (ACP) reached 9.69%, which was comparable with the predicted yield (9.73%). Mid- and high-dose ACP significantly inhibited the tumor growth (43.93%) and the tumor inhibition percentage (38.05%), which were more than 30%. The ACP could extend the survival time of H22 ascites tumor-bearing mice. Furthermore, the ACP could reduce the thymus and the spleen atrophy and significantly promoted the Con A-induced proliferation of splenocytes and elevated the serum IFN-γ and IL-2 levels. Therefore, the ACP could inhibit the tumor growth in tumor-bearing mice and regulated the immune function of mice. Practical ApplicationsThe trypsin-assisted extraction has high efficiency, is carried out through the polysaccharide extraction and the deproteinization at the same time, and is more convenient and fast than traditional methods. No detailed study on the optimization of the trypsin extraction of onion polysaccharides is available. Thus, this experiment aims to use the BBD (4 factors and 3 levels) to optimize the roles of extraction temperature, extraction time, extraction pH, and amount of enzyme on the yield of polysaccharides obtained from the fruit of A. cepa L. In addition, when looking for high-quality biological functional principles for the pharmaceutical industry, the antitumor activity of ACP was evaluated. A. cepa L. is one of the most widely cultivated and consumed crops worldwide. Polysaccharides are the main active ingredient, and studies have shown that a high intake of Allium vegetables is associated with reduced risk of cancers.

Effect of Anadenanthera colubrina protease inhibitors as an antiinflamatory mediator.

This work aimed to obtain and characterize protease inhibitors from A. colubrina leaves and evaluate their potential as inflammatory mediator and cell viability. The protein extract was analyzed and characterized by SDS-PAGE, RP-HPLC-PDA, MALDI-TOF/MS and Zeta potential. Bioassays were conducted in order to evaluate the cell viability in RAW 264.7, in vitro (NO and TNF-α production inhibition) and in vivo anti-inflammatory potential, inhibition rate of trypsin and hemagglutination activity from protein extract. The results revealed the presence of bands at 14, 21 and 30 kDa in SDS-PAGE, the RP-HPLC-PDA analysis showed peaks at 12, 13, 28 and 40 minutes and MALDI-TOF/MS showed peaks with 3.4, 4.7, 5.6, 9.4 and 11.2 kDa. The protein extracts presented enzymatic activity inhibition of trypsin (IC50 59.2 µgmL-1), did not show any cytotoxicity to RAW264.7 cells, hemagglutination 8HU and insignificant reduction in NO and TNF-α production and reduced anti-inflammatory potential in vivo compared to dexamethasone.

Expression analysis of protease-activated receptor-2 in cats.

Chronic kidney disease (CKD) is a common disease in geriatric cats. Despite its high prevalence, the pathogenesis of feline CKD is poorly understood. Recently, there has been increasing evidence for the role of protease-activated receptor-2 (PAR-2) in the progression of CKD in humans and rodents. However, the role of PAR-2 in feline CKD has not been evaluated. In this study, we determined nucleotide sequence of feline PAR-2 from the kidney, evaluated PAR-2 mRNA and protein expression in normal feline tissues, and analyzed functional expression in the feline kidney epithelial cell line Crandell-Rees Feline Kidney (CRFK). The open reading frame of feline PAR-2 comprised 1,194 bp and encoded 397 amino acids, showing 90%, 90%, and 85% identities to human, dog, and mouse PAR-2, respectively. In healthy cats, expression levels of the PAR-2 mRNA and protein were relatively higher in the gastrointestinal tract and kidney, and was lowest in the heart. The feline PAR-2 protein expression was confirmed, and stimulation of trypsin and PAR-2 agonists induced a prompt increase in the intracellular calcium ion concentration in CRFK cells. The present study will provide fundamental information for investigation of the involvement of PAR-2 in the pathogenesis of CKD in cats.

Effects of dietary asafoetida (Ferula sinkiangensis K. M. Shen) levels on feeding attraction activity, growth performance, healthiness, and digestive enzyme activity in juvenile Lateolabrax japonicus.

The study was to investigate effects of asafoetida (Ferula sinkiangensis K. M. Shen) powder on feeding attraction activity (FAA), growth performance, healthiness, and digestive enzyme activity of juvenile Lateolabrax japonicus. Six concentration levels (0, 5, 10, 15, 20, and 25 g/kg diets) were formulated for luring and feeding experiment. Results showed that asafoetida could stimulate the appetite of L. japonicus at the dietary levels from 10 to 25 g/kg; reduce the feed conversion ratio (FCR) and feed intake (FI) at 10-20 g/kg; increase the weight gain (WG) and specific growth rate (SGR) at 5-10 g/kg; increase the hepatosomatic index (HSI), body crude lipid content, serum total protein (TP) content, and lysozyme activity at 10-15 g/kg; decrease the moisture at 10-15 g/kg; and increase the serum total superoxide dismutase (T-SOD) activity at 5-15 g/kg, when compared with the control group (p < 0.05). Digestive enzyme activities including amylase (AMS) and trypsin in the intestine were significantly affected by the asafoetida powder (p < 0.05). Regression analyses between the FAA, FCR, WG, SGR, HSI, LZM, T-SOD, AMS, trypsin, and the dietary asafoetida powder levels showed that the optimal additional amount was 16.95, 13.65, 8.36, 8.15, 15.45, 9.94, 8.75, 11.77, and 7.07 g/kg, respectively, indicating that the optimal amount of asafoetida powder was located in 7.07-16.95 g/kg diet. However, combined with the significant difference analyses obtained from the current study, we would suggest the additive suitable dosage was 10 g/kg.

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis-based immobilized enzyme microreactor with chromogenic substrate.

In this study, a capillary electrophoresis-based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S-2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis-Menten constant (Km ) was (0.47 ± 0.08) mM, and the half-maximal inhibitory concentration (IC50 ) and inhibition constant (Ki ) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate-binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.

A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

Structural and functional relationship of Cassia obtusifolia trypsin inhibitor to understand its digestive resistance against Pieris rapae.

Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic ß-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.

Exploration of anti-insect potential of trypsin inhibitor purified from seeds of Sapindus mukorossi against Bactrocera cucurbitae.

Peptidase inhibitors (PIs) are defense proteins of plants which are active against gut peptidases of different insects. Sapindus mukorossi was identified as a source of bioactive PIs which could confer resistance against Bactrocera cucurbitae, a most devastating pest of several economically important crops. In the present study, a trypsin inhibitor was purified from mature dry seeds of S. mukorossi and characterized for its biochemical properties as well as its potential for bio control of B. cucurbitae. The purified fractions from RP- HPLC through SDS-PAGE gave an apparent molecular weight of ~29 kDa. S. mukorossi trypsin inhibitor (SMTI) was found to be a non-competitive inhibitor which was active over a broad range of temperature (10-100 °C) and pH (6-11). SMTI when incorporated in artificial diet inhibited the growth and development of B. cucurbitae larvae. Gene expression analysis of trypsin and chymotrypsin genes via qRT-PCR indicated that their mRNA expression was down-regulated while that of other genes namely, Catalase, Elastase, Superoxide Dismutase, Glutathione -S-transferase and Alkaline Phosphatase was up regulated. SMTI also showed deleterious effects against different bacterial strains. The results of this study indicated that S. mukorossi trypsin inhibitor has potential to be used as a bio control agent that can reduce the harm caused by melon fruit fly and other devastating pests.

Tamarind Trypsin Inhibitor in Chitosan-Whey Protein Nanoparticles Reduces Fasting Blood Glucose Levels without Compromising Insulinemia: A Preclinical Study.

In vivo studies show the benefits of the trypsin inhibitor isolated from tamarind (Tamarindusindica L.) (TTI) seeds in satiety and obesity. In the present study, TTI nanoencapsulation (ECW) was performed to potentialize the effect of TTI and allow a controlled release in the stomach. The impact on glycemia, insulin, and lipid profile was evaluated in Wistar rats overfed with a high glycemic index diet (HGLI). Characterization of the nanoparticles and in vitro stability in simulated gastrointestinal conditions, monitored by antitrypsin activity and HPLC, was performed. ECW and empty nanoparticles (CW) were administered by gavage, using 12.5 and 10.0 mg/kg, respectively. Both nanoformulations presented a spherical shape and smooth surface, with an average diameter of 117.4 nm (24.1) for ECW and 123.9 nm (11.3) for CW. ECW maintained the antitrypsin activity (95.5%) in the gastric phase, while TTI was completely hydrolyzed. In Wistar rats, the nanoformulations significantly reduced glycemia and HOMA IR, and ECW increased HDL-c compared to CW (p < 0.05).Pancreas histopathology of animals treated with ECW suggested an onset of tissue repair. Thenanoencapsulation provided TTI protection, gradual release in the desired condition, and improvement of biochemical parameters related to carbohydrate metabolism disorders,without compromising insulinemia.

Enzymatic Isolation, Amplification and Characterization of Dental Pulp Stem Cells.

The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.

Controlled enzymatic hydrolysis of pollen protein as promising tool for production of potential bioactive peptides.

In the present study, response surface method was used to optimize hydrolysis condition to generate potential bioactive peptides from pollen protein using pepsin (pepsin hydrolysated pollen-PHP) and trypsin (trypsin hydrolysated pollen-THP). Then PHP and THP prepared under optimized conditions were analyzed by size-exclusion chromatography. The fractions possessing the maximum ACE-inhibitory, DPPH radical scavenging, and ferric-reducing power were further purified by RP-HPLC. A heterogeneous composition of hydrophobic and hydrophilic peptides in both fractions was obtained. Finally, peptide sequences in active fractions of PHP and THP were identified by mass spectrometry in tandem. All the identified peptides had herbal protein origins. These were 6-21 amino acids in length, and Glycine and Alanine were two main hydrophobic amino acids present in their sequences. The results proved that using controlled enzymatic hydrolysis of pollen protein is possible to generate bioactive peptides with high ACE-inhibitory and antioxidant activity in final product. PRACTICAL APPLICATIONS: Pollen is well-known as an interesting protein source. Compared to other types of hydrolysis, enzymatic hydrolysis of vegetable proteins has few or no undesirable side reactions or products. In this study, controlled enzymatic hydrolysis of pollen protein was applied as a suitable method to produce bioactive peptide. The results proved that using controlled enzymatic hydrolysis of pollen protein is possible to generate bioactive peptides with high ACE-inhibitory and antioxidant activity in final product. This product can be used as functional and health promoting ingredient in different food formulations.