Extent Of Proteolysis Research Articles

Bioactive peptides released by lactic acid bacteria fermented pistachio beverages

Fruit and vegetable are the raw materials for many popular fermented beverages with potential health benefits. In the present study, a novel pistachio beverage fermented with selected strains of lactic acid bacteria belonging to the species Leuconostoc pseudomesenteroides and Companilactobacillus paralimentarius was produced, and the extent of proteolysis was evaluated. Physico-chemical and microbiological analyses, together with peptidomics and proteomics, were carried out on the beverage after 24 h of fermentation, using both the non-inoculated and chemically acidified beverage as controls. Amino acid sequence of hundreds of peptides mainly released from 2S albumin, 11S and 7S globulin were characterized. The number and frequency of identified peptides were higher in beverage started with Leuconostoc pseudomesenteroides, followed by Companilactobacillus paralimentarius and acidic beverage. These results suggest that, in addition to endogenous proteases active at acidic pH, the proteolytic system of LAB directly participated to peptide degradation to some extent. According to the BIOPEP database, a group of 31 peptides were potentially bioactive, and primarily associated with antioxidative properties, ACE and DPP-IV inhibition. The beverage started with Leuconostoc pseudomesenteroides showed the highest amount and number of bioactive peptides. This study lays the foundations for the design of novel pistachio-fermented beverage with potential health properties.

The utility of a slice defect score method in understanding factors impacting the sliceability of commercial Cheddar cheese blocks

A slice defect score method was used to evaluate slicing ability of commercial Cheddar cheese blocks with varying extent of proteolysis. The extent of proteolysis was analysed using urea PAGE and pH 4.6 soluble nitrogen. Cheese blocks were sliced using a food slicer into 0.5, 1, 1.5 or 2-mm thicknesses and two shapes, i.e., square and triangular. Cheese slices were graded for slice quality using a five-point slice defect score. Slices with 2 mm thickness, square shape and less mature Cheddar had the lowest slice defects score due to rigidity, absence of cracks and pinholes due to fewer stress concentration points and higher amount of intact casein. The slice defect score method differentiated cheese blocks well for sliceability, depending upon the desired shape and thickness of the cheese slices. The finding of this work will help develop a more comprehensive quantitative tool for assessing the slicing ability of cheese blocks.

Investigating Structural Defects in Extra Hard Cheese Produced from Low-Temperature Centrifugation of Milk.

The present study investigated some physico-chemical and microbiological traits of 20-month ripened hard cheeses produced from low-temperature high-speed centrifuged raw milk that developed a structural defect consisting of eyes or slits in the paste. Cheeses obtained using the same process and that did not develop the defect were used as controls. The colour, texture, moisture, water activity, proton molecular mobility, microstructure, extent of proteolysis, and viable microorganisms have been evaluated in all the cheese samples, and the significant differences between the defective and non-defective cheeses have been critically discussed. At a microstructural level, the defects caused fat coalescence and an unevenly organised protein matrix with small cracks in the proximity of the openings. The different fat organisation was correlated to a different transverse relaxation time of 1H population relaxing at higher times. The textural and colour features were not different from those of the control cheeses and were comparable with those reported in the literature for other long-ripened hard cheeses. On the other hand, the defective cheeses showed a higher moisture level and lower lactobacilli and total mesophilic bacteria concentrations, but the microbial origin of the defect remains an open hypothesis that deserves further investigation.

Designing agricultural grasses to help mitigate proteolysis during ensiling to optimize protein feed provisions for livestock.

The efficient preservation of protein in silage for livestock feed is dependent on the rate and extent of proteolysis. Previous research on fresh forage indicated enhanced protein stability in certain Festulolium (ryegrass × fescue hybrids) cultivars compared to ryegrass. This is the first report of an experiment to test the hypothesis that a Lolium perenne × Festuca arundinacea var glaucescens cultivar had reduced proteolysis compared to perennial ryegrass (L. perenne) during the ensiling process. Forages were harvested in May (Cut 2) and August (Cut 4), wilted for 24 h and ensiled in laboratory-scale silos. Silage was destructively sampled at 0 h, 9 h, 24 h, 48 h, 72 h, 14 days and 90 days post-ensiling, and dry matter (DM), pH and chemical composition were determined. At Cut 2, there was no difference in crude protein between treatments but ryegrass had higher soluble nitrogen (SN) (P < 0.001) and grass × time interactions (p = 0.03) indicated higher rates of proteolysis. By Cut 4, Festulolium had (5.5% units) higher CP than ryegrass (p < 0.001) but SN did not differ. Ammonia-N did not differ between silages in either cut. DM differences (11.8% units) between treatments in Cut 4 (v.2.2% in Cut 2) may have masked effects on proteolysis, highlighting the importance of management on silage quality. This was despite higher WSC in ryegrass in both cuts (p < 0.001), with grass × time interactions (Cut 2; p = 0.03) showing slower WSC decline in ryegrass in Cut 4 (p < 0.001). Silage pH values did not differ between grasses in either cut, but grass × time interactions (p < 0.001) showed a slower decline in both ryegrass cuts, resulting in higher (p < 0.05) pH at 24 h and 72 h for Cuts 2 and 4, respectively. Overall, the hypothesis for an enhanced protein stability in Festulolium when ensiled as ruminant feed was evidenced by lower SN but not ammonia-N in an early-cut silage with a comparable DM to ryegrass.

How starter cultures affect the peptidomic profile and bioactive activities of the Asiago-PDO cheese throughout ripening

Our study investigated the chemical, microbiological, and bioactive peptide profiles of Asiago Protected Designation of Origin (PDO) cheese from two dairies (Dairy I and II) produced over two consecutive days (batches) and analysed during three months of ripening. The effect of different starter cultures was evaluated. The microbiome varied between the dairies and batches, with curds post-salting dominated by the starter culture-associated genera. During ripening, there was an increasing trend in the Lactobacillus genus, especially for Dairy I, which used an industrial starter. Bioactive peptide intensities differed throughout ripening due to the extent of proteolysis, and their intensity or concentration evolved, modifying, and differentiating profiles. The industrial starter used in Dairy I had the highest relative intensity (average value 76.50%) of bioactive peptides after three months of ripening. In contrast, the cheeses made with natural milk starter (Dairy II) had lower total relative intensity (average value 47.75%) but produced ACE-inhibitory peptides through sub-dominant strains and non-starter lactic acid bacteria. The importance of autochthonous strains of each micro-region even within a delimited PDO production area was highlighted.

In vitro digestion of two protein-rich dairy products in the ageing gastrointestinal tract.

It is still unclear if changes in protein digestibility and absorption kinetics in old age may affect the anabolic effect of high-protein foods. The objective of this study was to investigate the digestion of two high-protein (10% w/w) dairy products in vitro: a fermented dairy product formulated with a ratio of whey proteins to caseins of 80 to 20% (WBD) and a Skyr containing mainly caseins. The new static in vitro digestion model adapted to the general older adult population (≥65 years) proposed by the INFOGEST international consortium was implemented to investigate the digestion of these products and compared with the standard version of the protocol. Kinetics of proteolysis was compared between both models for each product, in the gastric and intestinal phases of digestion. Protein hydrolysis was studied by the OPA method, SDS-PAGE, and LC-MS/MS, and amino acids were quantified by HPLC. Protein hydrolysis by pepsin was slower with the older adult model than with the young adult model, and consequently, in spite of a longer gastric phase duration, the degree of proteolysis (DH) at the end of the gastric phase was lower. Two different scenarios were observed depending on the type of dairy product studied: -10 and -40% DH for Skyr and WBD, respectively. In the intestinal phase, lower concentrations of free leucine were observed in older adult conditions (approx. -10%), but no significant differences in proteolysis were observed overall between the models. Therefore, the digestion conditions used influenced significantly the rate and extent of proteolysis in the gastric phase but not in the intestinal phase.

Proteolysis of vaginally administered bovine lactoferrin: clearance, inter-subject variability, and implications for clinical dosing

This report describes proteolytic fragmentation and clearance of bovine lactoferrin (bLF) upon intravaginal administration in premenopausal women. Tablet formulations (MTbLF) containing 300 mg of bLF progressed through three phases: Pre-Dissolution, Dissolution, and Washout, over a 30-h time course. Tablets dissolved slowly, replenishing intact 80 kDa bLF in vaginal fluid (VF) as proteolysis occurred. bLF was initially cleaved approximately in half between its N- and C-lobes, then degraded into sub-fragments and small peptides. The extent of proteolysis was less than 10–20% across multiple subjects. Concentrations of both intact 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout not proteolysis was the main clearance mechanism. Concentrations of intact and/or nicked 80 kDa bLF peaked between 4 and 8 h after administration and remained above 5 mg/mL for approximately 24 h. Experiments with protease inhibitors in ex vivo VF digests suggested an aspartyl protease was at least partially responsible for bLF cleavage. However, digestion with commercial pepsin or in vivo in the human stomach, demonstrated distinctly different patterns of fragments compared to vaginal proteolysis. Furthermore, the 3.1 kDa antimicrobial peptide lactoferricin B was not detected in VF. This suggests pepsin-like aspartyl proteases are not responsible for vaginal proteolysis of bLF.

Impact of casein-to-whey protein ratio on gastric emptying, proteolysis, and peptidome profile of fermented milk during in vitro dynamic gastrointestinal digestion in preschool children

The effects of casein-to-whey ratios (i.e., 4:1, 2:1, 1:1, 1:1.5) in fermented milk on gastric emptying, proteolysis and intestinal peptidome were investigated using an in vitro dynamic stomach-intestine system mimicking preschool children digestion. The gastric emptying rate varied insignificantly among the milk samples. After 120 min digestion, the protein digestibility was found in the range of 73.3–93.5 %, with the highest extent of proteolysis observed at a casein-to-whey ratio of 2:1 due to the fewest gastric protein aggregates and relatively abundant caseins. Intestinal peptides derived from caseins or whey proteins showed a positive correlation with their parent protein content. The most abundant bioactive whey peptides were found at casein-to-whey ratio of 1:1.5 after intestinal digestion. These findings demonstrated the importance of protein compositions in fermented milk on the gastrointestinal proteolysis and peptide release in vitro. This will be meaningful for future development of milk products that are more suitable for children.

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.

Differences in bread protein digestibility traced to wheat cultivar traits

Wheat grain storage proteins, i.e. the gluten proteins, and alpha-amylase trypsin inhibitors are partially resistant to gastrointestinal digestion. They figure among the wheat components that are potential triggers of non-celiac wheat sensitivity. To explore the digestibility of bread wheat proteins in relation to heritable plant traits, old and modern wheat varieties grown in two environments were genotyped, phenotyped, and processed into breads, which were partially digested in vitro in a system resembling human digestion. The extent of proteolysis measured after 2 h of digestion was variable. Digestion of proteins present in bread made from old wheat cultivars registered before 1960 and modern cultivars registered after 1960 was similar. No major plant trait was correlated to partial protein digestibility of bread from cultivars with high grain protein content. The protein digestibility of bread from high yield cultivars was influenced by protein composition, as well as the genetic distance per chromosome from a reference variety. Improving high yield cultivars by breeding for highly digestible proteins might help to mitigate adverse reactions to a range of wheat proteins in susceptible individuals.

Influence of chymosin type and brine concentration on chemical composition, texture, microstructural and colour properties of Turkish white cheeses

The effects of chymosin type and brine concentration on chemical composition, texture, microstructure and colour of white cheese were studied during ripening. The cheeses were manufactured from cows’ milk using microbial rennet (Rhizomucor miehei), commercial calf rennet or recombinant chymosin (Kluyveromyces lactis), and ripened in whey brines containing 10% or 12% NaCl. Increasing the brine concentration increased the ratio of salt to moisture, hardness, gumminess, chewiness and whiteness values of cheeses and decreased the moisture, the ratio of moisture to protein and extent of proteolysis. The similar trends were observed in cheeses with microbial rennet compared with those with calf rennet or recombinant chymosin. pH values of all the cheeses unchanged during ripening but their brine pHs increased. A marked effect of rennet type on microstructure of the cheeses was visualised. In microbial rennet cheese brine salt content can be reducible to obtain satisfactory results in terms of quality parameters.

Characteristics and proteolysis of a Spanish blue cheese made with raw or pasteurised milk

Valdeón cheese is a Spanish Protected Geographical Indication of blue‐veined cheese produced on an industrial scale from raw or pasteurised cow and/or goat milk. The aim of this work was to evaluate the impact of pasteurisation on the microbiological, physicochemical and sensory characteristics of cheese. Cheeses made with raw milk showed higher counts of lactobacilli and enterococci, as well as lower values of pH and D‐lactic acid and salt/moisture ratio. Cheeses made with pasteurised milk showed greater extent of proteolysis, higher concentration of free amino acids and lower concentration of biogenic amines. Pasteurisation produced more elastic and harder cheeses, but with a lower sensory profile.

Proteolysis of Amyloid β by Lysosomal Enzymes as a Function of Fibril Morphology.

Aggregation of amyloid-β (Aβ) into extracellular plaques is a well-known hallmark of Alzheimer’s disease (AD). Similarly, autophagic vacuoles, autophagosomes, and other residual bodies within dystrophic neurites, though more difficult to detect, are characteristic features of AD. To explore the potential intersection between these observations, we conducted experiments to assess whether Aβ fibril formation disrupts proteolysis by lysosomal enzymes. Fibrils constituted by either Aβ 1–40 or Aβ 1–42 were grown under both neutral and acidic pH. The extent of proteolysis by individual cathepsins (L, D, B, and H) was monitored by both thioflavin T fluorescence and liquid chromatography combined with mass spectrometry. The results show that all Aβ fibril morphologies are resistant to cathepsin digestion, with significant amounts of the undigested material remaining for samples grown in either neutral or acidic pH. Further analysis revealed that the neutral-grown fibrils are proteolytically resistant throughout the sequence, while the acid-grown fibrils prevented digestion primarily in the C-terminal portion of the sequence. Fibrils grown from Aβ 1–42 are generally more resistant to degradation compared to Aβ 1–40. Overall, the results indicate that Aβ fibrils formed in the neutral pH environments found in intracellular or extracellular spaces may pose the greatest difficulty for complete digestion by the lysosome, particularly when the fibrils are comprised of Aβ 1–42.

Influence of Muscle Type on Physicochemical Parameters, Lipolysis, Proteolysis, and Volatile Compounds throughout the Processing of Smoked Dry-Cured Ham.

The influence of muscle type (biceps femoris, BF and semimembranosus, SM) on physicochemical parameters, volatile compounds, and the extent of proteolysis and lipolysis during the manufacturing of smoked dry-cured ham was investigated. A total of fifty smoked hams were sampled: raw ham, after salting, smoking, drying, and ripening. Almost all physicochemical parameters were affected by muscle type, manufacturing stage and their interactions. SM had lower water, ash, NaCl content, and water activity (aw), while fat and protein content were higher after ripening compared to BF. BF showed higher L*a*b* values compared to SM. The results of texture profile analysis showed that almost all analyzed parameters were influenced by muscle type and production stage. A total of 88 volatile compounds were identified, showing an increase in its number during processing: 31 volatile compounds were identified in raw ham and 72 after the ripening phase. Aldehydes and phenols were the predominant groups of compounds, followed by alcohols, ketones, aromatic hydrocarbons, aliphatic hydrocarbons, esters, and terpenes. Muscle type and production phase significantly affected lipid oxidation and the index of proteolysis: in SM, thiobarbituric acid reactive substances (TBARS) increased faster than in BF, while proteolysis had an opposite effect and was more pronounced in BF.

Age-related gastrointestinal alterations of legumes and cereal grains digestibility

Aging is accompanied by changes in gastrointestinal functions. The impact of the gastrointestinal (GI) conditions of the elderly on the extent of proteolysis and glycolysis as well as calcium bioaccessibility in some cooked legumes (chickpea, lentils, soya bean and white bean) and cereals/pseudocereals (oats, spelt and quinoa) were studied. Samples were digested in vitro using three GI models specifically focused on the elderly in which oral, gastric and intestinal conditions were altered (E1: altered oral conditions, E2: altered oral and gastric conditions and E3: altered oral, gastric and intestinal conditions). Samples were also subjected to a standardized GI digestion as a control (C). The extent of proteolysis was only significantly affected with suboptimal intestinal conditions (p < 0.05). Protein digestibility of cereal grains decreased to a greater extent than for legumes. The release of non-essential amino acids was more affected than that of essential ones, mainly in legumes such as soya bean, lentils and white bean. The extent of glycolysis was much higher in cereal grains than legumes regardless of GI digestion conditions. Glycolysis declined with altered intestinal conditions (E3) compared to the C, in all legumes and spelt. Calcium bioaccessibility was much higher in cereal/pseudocereals than in legumes. However, calcium bioaccessibility seems to be highly limited in elderly people suffering from oral, gastric or intestinal alterations (up to 53% reduction compared to C). Such data might be helpful to develop dietary strategies based on protein-rich vegetal foods, including alternative crops such as oats, quinoa and spelt, specifically used to mitigate sarcopenia and osteoporosis in elderly people.

Shelf-life Prediction Modeling and Physicochemical Changes of Canned African Giant Snail (Achachatina achatina) Based Products during Storage Using Sensory and Kinetic Data

Canned African giant snails (brine, egusi and sauce) of low acidity (pH 4.5) were produced using Thermal Death Time (TDT) techniques. Sixty cans of each product were stored for 6 months at normal temperature (30oC). The physicochemical and sensory properties of these products were evaluated monthly. And also, 28 cans of each product were equally evaluated at accelerated storage temperatures stress (50, 60, 70 &amp;amp; 80oC) for 6 weeks. Changes in pH and overall acceptability were described by first and second order kinetics respectively. The extent of proteolysis, peroxidase activities, and extent of fat hydrolysis i.e peroxide and thiobabituric acid values did not change significantly (P &amp;gt; 0.05) during storage. Regression analysis was used to fit models for Gibbs free energy of activation for physicochemical changes as a function of temperature and to predict models for overall acceptability as a function of pH. Shelf-life prediction models were fitted based on sensory and pH kinetic data. Shelf-lives of 27.6 months, 27-25 months and 18-16.8 months were estimated for snail in brine (SIB) snail in egusi (SES) and snail in sauce (SIS) respectively during storage at 30OC.

Evaluating the Authenticity of the Raw-Milk Cheese Fontina (PDO) with Respect to Similar Cheeses.

The implementation of quality assurance schemes for the assessment of PDO food authenticity is an issue involving manufacturers, traders, retailers and consumers. In this respect, reliable analytical methods are needed to integrate paper-trailing information. The feasibility of distinguishing the Italian Fontina PDO cheese from the generic Fontal cheese was preliminarily evaluated on a set of commercial samples by measuring selected parameters (pH, alkaline phosphatase activity, content of copper, volatiles, extent of proteolysis) related to the different manufacturing processes. The relative profile of free amino acids proved to be a promising tool. A new set of 41 samples of Fontina PDO cheese was collected at representative dairies within the recognized production area and analyzed for free amino acids. A chemometric model of Fontina PDO cheese was built based on the mean content and standard deviation of 15 free amino acids. On this basis, all of the PDO samples were correctly identified, whereas all of the Fontal cheeses were recognized as different cheeses.

Soy Protein Pressed Gels: Gelation Mechanism Affects the In Vitro Proteolysis and Bioaccessibility of Added Phenolic Acids.

In this study, a model system of firm tofu (pressed gel) was prepared to study how the coagulation mechanism—acidification with glucono δ-lactone (GDL) or coagulation with magnesium sulphate (MgSO4)—affected the physical properties of the gels along with their in vitro proteolysis (or extent of proteolysis). The two types of gels were also fortified with 3.5 mM protocatechuic (PCA) and coumaric acid (CMA) to test whether they can be used as bioactive delivery systems. Texture analysis showed that all MgSO4-induced gels (fortified and control) had a higher hydration capacity and a weaker texture than the GDL-induced gels (p < 0.05). MgSO4 gels had almost double proteolysis percentages throughout the in vitro digestion and showed a significantly higher amino acid bioaccessibility than the GDL gels (essential amino acid bioaccessibility of 56% versus 31%; p < 0.05). Lastly, both gel matrices showed a similar phenolic acid release profile, on a percentage basis (80% for PCA and 100% for CMA). However, GDL gels delivered significantly higher masses of bioactives under simulated intestinal conditions because they could retain more of the bioactives in the gel after pressing. It was concluded that the coagulation mechanism affects both the macro- and microstructure of the soy protein pressed gels and as a result their protein digestibility. Both pressed gel matrices are promising delivery systems for bioactive phenolic acids.

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.

Natural starter culture on artisanal cheese

The use of natural starter culture in the manufacture of artisanal Minas cheeses is required by the government legislation in Brazil. However, there are no scientific support on role of the natural starter culture in these cheeses. The aim of this work was to compare artisanal Minas cheeses produced with and without natural starter culture to verify the influence on the physico-chemical and microbiological characteristics during the ripening process. The use of natural starter culture did not have a significant effect on the physico-chemical (dry matter, humidity, fat, pH, chlorides, water activity, ash, total protein, depth and extent of proteolysis) and microbiological (Escherichia coli, coliforms and Staphylococcus aureus) properties of the cheeses. The significant changes that occurred in the cheeses were mainly because of the ripening time. According to obtained results, natural starter has shown to be innocuous to traditional Minas Serro cheese. The present study is pioneering in the scientific literature and opens new horizons for the understanding of the requirement to use natural starter culture.