Rate Of Proteolysis Research Articles

Mechanistic study on in vitro disintegration and proteolysis of whey protein isolate gels: Effect of the strength of sodium ions

In this study, the influence of the strength of sodium ions on the mechanical properties, microstructure and proteolysis of whey protein isolate (WPI) gels during in vitro gastrointestinal digestion was investigated. The results showed that heat treatment of 15% (w/v) WPI solutions in the presence of 0–0.1 M NaCl resulted in fine-stranded gels with a smaller coarseness length scale, while the presence of higher salt concentrations (0.15–0.5 M) induced coarse-stranded gels with larger particle sizes of the protein aggregates. The elastic shear modulus was found to increase with increasing NaCl concentration, followed by a reduction under higher ionic strength. During in vitro gastrointestinal digestion, the kinetics of disintegration of all the gels were dominated by a surface erosion mechanism. The half disintegration time ( t 1/2 ) significantly increased from 136.2 min to 416.0 min with the increase of NaCl concentration from 0 to 0.5 M, respectively. The coarse-stranded gels were more resistant to digestion with less mass loss and a lower rate and extent of proteolysis throughout the in vitro digestion than the fine-stranded gels. This could be attributed to the limited swelling and more severe protein aggregation found in the coarser gels with more compact structures, which would retard the accessibility of acids and enzymes during digestion. The present work has provided mechanistic insights into the microstructural changes induced by sodium salts on disintegration and proteolysis of WPI gels during in vitro digestion. This is meaningful for future designing protein-based structured food products with designated digestion rates for targeted dietary demands. • Formation of coarse-stranded WPI gels with protein aggregation at higher NaCl concentration. • Elastic shear modulus of WPI gels showed an optimum at 0.15 M NaCl. • All gels were disintegrated by surface erosion mechanism during in vitro digestion. • Half disintegration time increased with increasing NaCl concentration in WPI gels. • Coarser gels showed less mass loss and a lower extent and rate of proteolysis.

Low-moisture part-skim mozzarella cheese made from blends of camel and bovine milk: Gross composition, proteolysis, functionality, microstructure, and rheological properties.

Camel (CM) milk is used in variety of ways; however, it has inferior gelling properties compared with bovine milk (BM). In this study, we aimed to investigate the physicochemical, functional, microstructural, and rheological properties of low-moisture part-skim (LMPS) mozzarella cheese, made from BM, or BM mixed with 15% CM (CM15%) or 30% CM (CM30%), at various time points (up to 60 d) of storage at 4°C after manufacture. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% had high moisture and total Ca contents, but lower soluble Ca content. Compared with BM cheese, CM15% and CM30% LMPS mozzarella cheese exhibited higher proteolysis rates during storage. Adding CM affected the color properties of LMPS mozzarella cheese manufactured from mixed milk. Scanning electron microscopy images showed that the microstructure of CM15% and CM30% cheeses had smooth surfaces, whereas the BM cheese microstructures were rough with granulated surfaces. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% showed significantly lower hardness and chewiness, but higher stringiness than BM cheese. Compared with BM cheese, CM15% and CM30% cheeses showed lower tan δ levels during temperature surges, suggesting that the addition of CM increased the meltability of LMPS mozzarella cheese during temperature increases. Camel milk addition affected the physicochemical, microstructural, and rheological properties of LMPS mozzarella cheese.

Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases.

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide-peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for an MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side-chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography-mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well tolerated in the P3 and P3' substrate sites and that the identity of the peptoid in these sites displays a moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with side-chain identity than side-chain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate the practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

Carcass Characteristics and Quality Attributes of Beef from Cattle Supplemented Zinc and Ractopamine Hydrochloride

The objective of this experiment was to identify the impact of supranutritional zinc (SUPZN) and ractopamine hydrochloride (RAC) supplementation of beef steers on postmortem protein degradation and tenderness development of extended (>14 d) aged steaks. It was hypothesized that RAC and SUPZN supplementation would influence meat quality development during extended aging of the longissimus thoracis muscle. Crossbred steers (n=27) were fed in a 2 × 2 factorial: control (CON; analyzed 36 mg Zn/kg dry matter) or supranutritional Zn supplementation (SUPZN; CON diet + 60 mg Zn/kg dry matter [from ZnSO4] + 60 mg Zn/kg dry matter [from Zn–amino acid complex]) dietary treatments for the entire 91-d trial. Before harvest (30 d), steers were blocked by body weight within Zn treatments to RAC treatments of 0 or 300 mg per steer per day. Steers were harvested at a commercial processing facility. Carcass characteristics were collected 2 d postmortem. Warner-Bratzler shear force of the Longissimus thoracis was measured at 7, 14, 28, and 42 d postmortem. Calpain-1 autolysis (2 d postmortem) and desmin degradation (2, 7, 14, 28, and 42 d postmortem) were analyzed. RH resulted in greater (P<0.02) Warner-Bratzler shear force values at 7, 14, and 28 d postmortem, lesser (P<0.01) calpain-1 autolysis (76-kDa band) at 2 d postmortem, and lesser (P<0.02) desmin degradation at 2, 7, 14, and 28 d postmortem. Supplementation of Zn resulted in greater (P<0.01) calpain-1 autolysis (78- & 76-kDa band) at 2 d postmortem and a trend for greater (P=0.08) desmin degradation at 2 d postmortem. Tougher steaks from RH supplemented steers were explained by slowed postmortem proteolysis, and Zn supplementation showed evidence to enhance proteolysis early postmortem (2 d). These results demonstrate that nutritional supplementation can impact the rate of tenderness development and postmortem proteolysis in muscle.

Cooking affects pork proteins in vitro rate of digestion due to different structural and chemical modifications

The effect of thermal processing on the in vitro digestibility of pork proteins was studied. Raw samples were considered the control group, while the thermal treatments included 58, 80, 98 and 160 °C for 72 min, 118 °C for 8 min and 58 °C for 17 h, resembling a range of different cooking procedures. Samples were subsequently subjected to pepsin digestion at pH 3.00 in the gastric phase followed by trypsin and α-chymotrypsin at pH 8.00 in the intestinal phase. Pork cooked at 58 °C for 72 min had a significantly higher pepsin digestibility rate than meat cooked at 80 °C or 160 °C. The trend was similar in the intestinal phase, with samples cooked at 58 °C for 72 min having enhanced digestion rate over other treatments after 120 min of digestion. A PLS model pointed out to an inverse relationship between in vitro proteolysis rate and variables like Maillard reaction compounds or protein structural changes.

Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction.

Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases protease activity in neuronal cell bodies prior to DCD. Treatment of rat cortical neurons with excitotoxic (100 µM) glutamate increased the low baseline rate of intracellular cA-TAT proteolysis by approximately three-fold prior to DCD and by approximately seven-fold upon calcium deregulation. The glutamate-induced rate enhancement prior to DCD was suppressed by glutamate receptor antagonists, but not by calpain or proteasome inhibitors, whereas DCD-stimulated proteolysis was partly attenuated by the proteasome inhibitor MG132. Further suggesting that cA-TAT cleavage is calpain-independent, cA-TAT fluorescence was observed in immortalized Capn4 knockout fibroblasts lacking the regulatory calpain subunit. About half of the neurons lost calcium homeostasis within two hours of a transient, 20 min glutamate receptor stimulation. These neurons had a significantly (49%) higher mean baseline cA-TAT proteolysis rate than those maintaining calcium homeostasis, suggesting that the unknown protease(s) cleaving cA-TAT may influence DCD susceptibility. Overall, the results indicate that excitotoxic glutamate triggers the activation of calpain-independent neuronal protease activity prior to the simultaneous loss of calcium homeostasis and mitochondrial bioenergetic function.

Influence of Aging Temperature and Duration on Spoilage Organism Growth, Proteolytic Activity, and Related Chemical Changes in Vacuum-Packaged Beef Longissimus

The objective was to evaluate the influence of vacuum-packaged aging temperature, duration, and their inter-action on spoilage organism growth, proteolytic activity, and resulting beef tenderness. Paired strip loins were collected from 60 USDA Low Choice beef carcasses (n=60), then assigned a storage temperature (−2°C, 0°C, or 4°C). Loins were portioned into half loins and assigned to an aging duration (14, 28, 42, or 56 d) and vacuum packaged. Loins were aged in commercial upright refrigerators. Half-loin packages, at their respective aging duration, were aseptically opened and cut surface swabbed for microbial analysis before fabrication into 2.54 cm strip steaks (n=5). Steaks assigned to slice shear force (SSF) were cooked to 71°C. A raw steak was used for microbial, proteolytic, and volatile analyses. Two-way interactions were observed for all spoilage organisms (P < 0.001). Aging for 42 and 56 d at−2°C produced lower microbial counts compared to individual aging durations at 4°C (P < 0.05). Loins aged for 14 d at 4°C had increased desmin and troponin-T degradation compared to aging for 14 at−2°C and 0°C (P < 0.05). Loins aged at 4°C produced more tender steaks compared to−2°C and 0°C (P = 0.001). Steaks aged for 42 and 56 d possessed the lowest SSF values (P < 0.05). Aging for 56 d at 4°C produced the greatest amount of total free amino acids (P < 0.001). Two-way interactions were observed for 7 compounds (alcohols, aldehydes, carboxylic acids, ketones, and sulfur-containing compounds; P < 0.05). Aging for 56 d at 4°C had the greatest ethanol concentration (P < 0.05). These data indicate aging at 4°C increases the rate of proteolysis and subsequent tenderness development and flavor precursor accumulation. However,extended aging at 4°C resulted in increased microbial counts. Many traits peaked at 42 d of aging.

Influence of different types of fermentation-produced chymosin on quality of soft cheeses

The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content, proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe. The main proteolytic agent in soft cheeses is the milk-clotting enzyme (MCE). Increasing the shelf life of cheeses can be achieved by using MCE types having low proteolytic activity (PA). We have studied the effect of MCE based on different types of fermentation-produced chymosin: Chy-max® Extra (bovine chymosin), Chy-max® M (camel chymosin), Chy-max® Supreme (“modified” chymosin) on the dynamics of proteolysis in soft cheeses and related changes in the structure of cheeses during their storage. All 3 types of studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose of MCE also increases the rate of proteolysis in cheeses. To increase the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is promising to use MCE with low PA based on camel chymosin (Chy-max® M) and “modified” chymosin (Chy-max® Supreme).

Actinidin reduces gluten-derived immunogenic peptides reaching the small intestine in an in vitro semi-dynamic gastrointestinal tract digestion model

Actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), has been identified as a potential enzyme to hydrolyse gluten within the lumen of the gastrointestinal tract (GIT). The present study aimed to further evaluate the effect of purified actinidin sourced from green kiwifruit on the digestion of gluten and the release of immunogenic peptides during GIT digestion using an in vitro semi-dynamic GIT digestion model. Purified gluten was digested for 180 min with or without actinidin and subsequently analysed for free amino groups (o-phthaldialdehyde) to determine the degree of hydrolysis (DH), gluten R5 epitopes (ELISA), and peptide profiles (mass spectrometry). Strong interactions were observed between treatment (GIT digestion with or without actinidin) and digestion time for the DH of gluten (P < 0.01), amount of free amino groups released into the small intestine (P < 0.01), and amount of gluten epitopes present in the small intestine (P < 0.001). The rate of increase of DH of gluten and the amount of R5 epitopes present in the small intestine during the first 30 min of GIT digestion with actinidin was 0.3%/min and 4.8 ng/g of gluten respectively, whereas it was 0.01%/min and 60.9 ng/g of gluten respectively without actinidin. These results were corroborated by untargeted peptidomics, with a 1.5-fold lower number of known immunogenic epitopes reaching the small intestine at 30 min of GIT digestion when actinidin was present compared to the control. Present results demonstrate that actinidin enhanced the rate of proteolysis of gluten and reduced the number of immunogenic gluten epitopes reaching the small intestine during simulated semi-dynamic GIT digestion.

A metal ion–dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.

Discovery of the Xenon-Protein Interactome Using Large-Scale Measurements of Protein Folding and Stability.

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low-throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein target discovery. Here, we report the application of stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5000 methionine-containing peptides and over 5000 semi-tryptic peptides, mapping to ∼1500 and ∼950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified are tied to previously established modes of action related to xenon's anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein-gas interactions in complex biological mixtures, such as cell lysates.

APPLICATION OF BOX-WILSON CENTRAL COMPOSITE EXPERIMENTAL DESIGN TO OPTIMIZE THE PROTEOLYTIC ACTIVITY IN MILK BY LACTOCOCCUS LACTIS LCL STRAIN

In this work, the individual and interactive effects of temperature and pH on the proteolytic activity of Lactococcus lactis LCL strain in milk were studied by quadratic Response Surface Methodology. The effects of the selected variables have been analysed using a Central Composite Design and the maximum response (proteolysis rate) under the optimum values of variables and of a satisfactory fit model was realized. The mathematical relationship of the proteolytic activity on the two significant independent variables can be approximated by a nonlinear polynomial model. Predicted values were found to be in a good agreement with experimental values (R2 = 0.937) demonstrating the good significance of the model. The result of optimization predicted by the model has shown that the maximal result for the proteolysis rate revolved around of 43.7 ± 1.4 % at the optimal condition (30°C and pH 7).

Impact of different starter cultures and Lactobacillus helveticus on volatile components, chemical and sensory properties of pasta filata cheese

L.helveticus is known to follow mainly similar metabolic pathways to contribute to cheese flavor with S. thermophilus and L. delbrueckii subsp. bulgaricus. In this study, the flavor contributions of commercial S. thermophilus starter cultures of different brands and L. helveticus adjunct culture to pasta filata type fresh Kashar cheese were investigated. L. helveticus affected the buttery aroma components of fresh Kashar cheese and resulted in low diacetyl content. In addition, acetaldehyde and other aroma components of L. helveticus added cheese was found to be higher than control and modified control cheeses. On the other hand, the modified control sample containing S. thermophilus from Danisco instead of Chr-Hansen in the control was closer to the control sample in terms of volatile profile. As the shelf-life progressed, the contribution of alcohols and hydrocarbons to volatile components decreased, while the contribution of ketones, which was the dominant group, increased in all products. When the proteolysis and lipolysis levels were examined, the control sample differed from the other two cheese samples with its high proteolysis and lipolysis rate. In summary, the rates of increase in water-soluble nitrogen and free fatty acid contents in 8 weeks of storage (from 18 to 72 days) were determined as 61% and 47%, respectively, in the control Kashar cheese, while it was 39% and 27% in the L. helveticus added sample, and 37% and 28% in the modified control sample. Finally, the sensory scores revealed that cheese flavor and texture preferences could be increased with the addition of L. helveticus.

Optimization of Enzymatic Preparation Process of Hypoglycemic Peptide from Moringa oleifera Seeds by Response Surface Methodology and Its Hypoglycemic Activities Evaluation in Vitro

In order to improve the resource utilization of Moringa oleifera seeds powder, enzymatic method was used to prepare hypoglycemic peptide from Moringa oleifera seeds. Taking the the degree of proteolysis and α-glucosidase inhibition rate as the response value, single-factor experimental screening and response surface method was used to optimize the optimum enzymatic hydrolysis time, liquid-to-material ratio, hydrolysis pH, enzyme addition, and enzymatic hydrolysis temperature. The hypoglycemic peptide with a molecular weight of <3 kDa were prepared by coarse separation by ultrafiltration, and their inhibitory effects on human liver cancer cells (HepG2 cells) were analyzed by MTT method. The results showed that the optimal conditions of enzymatic hydrolysis were as follows: Time 4.6 h, ratio of liquid to material 40.5:1, pH8.3, enzyme dosage 5.5%, temperature 55 ℃, and the inhibition rate of α-glucosidase was 23.62%±0.14%. The α-glucosidase inhibition rate of the ultrafiltration fraction with a molecular weight of less than 3 kDa had an IC50 value of 5.56 mg/mL. When the concentration of ultrafiltration fraction was 300 μg/mL, the proliferation of HepG2 cells was significantly inhibited after 48 h(P<0.05). The research can lay the foundation for the further separation and purification of hypoglycemic peptide from Moringa oleifera seeds.

Computational modeling of unphosphorylated CtrA:Cori binding in the Caulobacter cell cycle

SummaryIn the alphaproteobacterium, Caulobacter crescentus, phosphorylated CtrA (CtrA∼P), a master regulatory protein, binds directly to the chromosome origin (Cori) to inhibit DNA replication. Using a mathematical model of CtrA binding at Cori site [d], we provide computational evidence that CtrAU can displace CtrA∼P from Cori at the G1-S transition. Investigation of this interaction within a detailed model of the C. crescentus cell cycle suggests that CckA phosphatase may clear Cori of CtrA∼P by altering the [CtrAU]/[CtrA∼P] ratio rather than by completely depleting CtrA∼P. Model analysis reveals that the mechanism allows for a speedier transition into S phase, stabilizes the timing of chromosome replication under fluctuating rates of CtrA proteolysis, and may contribute to the viability of numerous mutant strains. Overall, these results suggest that CtrAU enhances the robustness of chromosome replication. More generally, our proposed regulation of CtrA:Cori dynamics may represent a novel motif for molecular signaling in cell physiology.

Effect of Probiotic Strains on Sensory Attributes of Buffalo Milk Cheddar Cheese

Probiotics are living micro-organisms which impart certain health benefits like anti-oxidant, anti-hypertensive, immune-modulatory, opioid, anti-inflammatory, anti-microbial and consumer related inherit nutrition with the utilization in recommended quantity. The study aimed to determine the effect of Lactobacillus acidophilus, Bifidobacterium bifidum and Lactobacillus helveticas and their adjuncts on sensory attributes of cheddar cheese. The cheddar cheese having commercially available mesophilic starter cultures and with probiotics strains were prepared and assessed for acetic acid production, proteolysis and descriptive sensory analysis. The probiotics adjuncts Lb. acidophilus + Bifidobacterium bifidum (La + Bb) and Lb. acidophilus + Bifidobacterium bifidum + Lb. helveticus (La + Bb + Lh) showed remarkable results in production of acetic acid 489.33 (ppm) and as compared to control respectively. Among probiotic strains, the probiotic strain Lactobacillus helveticas showed the highest water-soluble nitrogen as 28.50 (%) and free amino acid 5220 µg/g. non-significant effect was observed for overall acceptability for control and probiotic adjuncts. Higher rates of organic acids and proteolysis was observed in probiotic adjuncts as compared to control samples. The current study reveals that the addition of probiotics adjuncts to cheddar cheese does not affects its overall acceptability.

Acquired von Willebrand syndrome in patients with monoclonal gammopathy of undetermined significance investigated using a mechanistic approach.

Acquired von Willebrand syndrome (AVWS) has been reported to occur in association with monoclonal gammopathy, usually of undetermined significance (MGUS). It may present as a type 1 or type 2 von Willebrand factor (VWF) defect depending on the patient's representation of large VWF multimers. The mathematical model by Galvanin et al., already employed for studying inherited von Willebrand disease (VWD), was used to explore the pathogenic mechanisms behind MGUS-associated AVWS. The patients studied showed significantly reduced VWF levels and function; an increased VWF propeptide to VWF antigen ratio; and all VWF multimers present but in reduced quantities, with the low-molecular-weight VWF forms being significantly more represented than those of higher molecular weight. Our mathematical model revealed a significantly increased VWF elimination rate constant, with values similar to those of type Vicenza VWD. An even more increased VWF proteolysis rate constant was observed, with values one order of magnitude higher than in type 2A VWD but, in contrast, no loss of large multimers. The model predicted the same elimination rate for high- and low-molecular-weight VWF multimers, but proteolysis of the high-molecular-weight forms also contributes to the pool of low-molecular-weight oligomers, which explains why they were relatively over-represented. In MGUS-associated AVWS the increase of both clearance and proteolysis contributes to the circulating levels and multimer pattern of VWF, with a phenotype that appears to be a combination of type Vicenza and type 2A VWD. Hence, the mechanisms behind the onset of AVWS seem to differ from those of inherited VWD.

English

Restaurants and pizza makers in Pakistan demand a cheese that has ability to melt, stretch with a characteristics flavor and less free oil formation while applied on pizza dough. The desired characteristics can be obtained with proper amalgamation of fresh and ripened cheeses. Therefore, the present research was planned to prepare Pizza cheese blends (PCB) from fresh Mozzarella and ripened (2 and 4 months) Cheddar cheese. Seven Pizza cheese blends were prepared with fresh Mozzarella and ripened (2 and 4 months) Cheddar cheese. The quality of Pizza cheese blends were evaluated by measuring chemical composition, proteolysis, intact casein and organic acids contents. The rate of proteolysis (pH 4.6-soluble and TCA-soluble nitrogen) was rapid in PCB made with higher level of four months ripened Cheddar cheese. Electrophoresis (Urea PAGE) and High Performance Liquid Chromatography (HPLC) analysis indicated reduced intact casein in PCB that has higher level of aged (4 months) Cheddar cheese. Mean abundances indicated significant change in organic acid contents of PCB. In conclusion, significant variation was observed for proteolysis, intact casein and organic acids production with the difference in percentages and ages of cheeses. The prevalence of a comparatively large amount of variability in technological properties of Pizza cheese was confirmed. This blending of cheeses provides new insight to cheese industries which directs new strategies to improve the characteristics of Pizza cheese.

Quantitative NMR Study of Insulin-Degrading Enzyme Using Amyloid-β and HIV-1 p6 Elucidates Its Chaperone Activity.

Insulin-degrading enzyme (IDE) hydrolyzes monomeric polypeptides, including amyloid-β (Aβ) and HIV-1 p6. It also acts as a nonproteolytic chaperone to prevent Aβ polymerization. Here we compare interactions of Aβ and non-amyloidogenic p6 with IDE. Although both exhibited similar proteolysis rates, the binding kinetics to an inactive IDE characterized using relaxation-based NMR were remarkably different. IDE and Aβ formed a sparsely populated complex with a lifetime of milliseconds in which a short hydrophobic cleavage segment of Aβ was anchored to IDE. Strikingly, a second and more stable complex was significantly populated with a subsecond lifetime owing to multiple intermolecular contacts between Aβ and IDE. By selectively sequestering Aβ in this nonproductive complex, IDE likely increases the critical concentration required for fibrillization. In contrast, IDE and p6 formed a transient, submillisecond complex involving a single anchoring p6 motif. Modulation of intermolecular interactions, thus, allows IDE to differentiate between non-amyloidogenic and amyloidogenic substrates.

Development and characterization of antimicrobial and antioxidant whey protein-based films functionalized with Pecan (Carya illinoinensis) nut shell extract

Aim of this study was the development of an active packaging based on whey proteins (WP) functionalized with Pecan nut shell extract (PNSE). To this purpose, aqueous solutions of WP, PNSE and glycerol were mixed and characterized, whereas corresponding films were prepared by casting. Zeta-potential measurement revealed that the film forming solutions were stable, and the particle size was reduced further to the incorporation of PNSE, as a result of tannins-WP interactions. Films were handleable and homogeneous, and PNSE was able to improve their mechanical and barrier properties. PNSE-containing films inhibited the growth of the foodborne bacteria Enterococcus faecalis and Salmonella enterica subsp. enterica ser. Typhimurium. Ferric reducing/antioxidant power assay clearly highlighted the ability of PNSE to impart antioxidant properties to the films. Finally, simulated digestion experiments showed a significant lowering of the proteolysis rate in the presence of PNSE, although 40 % of the protein was still digested after 60 min incubation. Overall, these results put the basis for a possible use of PNSE functionalized WP-based films as new environmentally friendly candidates for increasing the shelf-life of foods.