Low Molecular Weight Bands Research Articles

Antimicrobial Activity of Apis mellifera Bee Venom Collected in Northern Peru.

Due to the emergence of microorganisms resistant to antibiotics and the failure of antibiotic therapies, there is an urgent need to search for new therapeutic options, as well as new molecules with antimicrobial potential. The objective of the present study was to evaluate the in vitro antibacterial activity of Apis mellifera venom collected in the beekeeping areas of the city of Lambayeque in northern Peru against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Bee venom extraction was performed by electrical impulses and separated using the Amicon ultra centrifugal filter. Subsequently, the fractions were quantified by spectrometric 280 nm and evaluated under denaturant conditions in SDS-PAGE. The fractions were pitted against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. A purified fraction (PF) of the venom of A. mellifera and three low molecular weight bands of 7 KDa, 6 KDa, and 5 KDa were identified that showed activity against E. coli with a MIC of 6.88 µg/mL, while for P. aeruginosa and S. aureus, it did not present a MIC. No hemolytic activity at a concentration lower than 15.6 µg/mL and no antioxidant activity. The venom of A. mellifera contains a potential presence of peptides and a predilection of antibacterial activity against E. coli.

Partial enrichment of phospholipids by enzymatic hydrolysis and membrane filtration of whey protein phospholipid concentrate

Whey protein phospholipid concentrate (WPPC) contains high amounts of phospholipids (PL; 4.5 ± 1%) but there is interest in further enriching the PL content for nutritional and functional applications. Chemical methods were unsuccessful in separating PL from proteins due to the presence of protein-fat aggregates. Instead, we explored hydrolysis of the proteins to peptides with the objective of removing peptides, thereby concentrating the PL fraction. We used microfiltration (MF) with a pore size of 0.1 µm to help reduce protein/peptide retention. Hydrolyzing proteins should facilitate passage of low molecular weight peptides through the MF membrane, while concentrating fat and PL in the MF retentate. Bench-top experiments were performed to select the proteolytic enzyme that resulted in the most extensive hydrolysis of proteins in WPPC from among 5 different commercial proteases. Sodium dodecyl sulfate-PAGE analysis was performed to measure the extent of protein hydrolysis over a period of 4 h. Alcalase enzyme was found to exhibit the highest proteolytic activity at conditions of pH 8 and temperature 55°C. The intensity of major protein bands (milkfat globule membrane proteins, caseins, β-lactoglobulin) in WPPC decreased in sodium dodecyl sulfate-PAGE profiles as hydrolysis progressed, along with the appearance of low molecular weight bands. Pilot-scale MF production, coupled with diafiltration (DF), of the hydrolyzed sample aided in the removal of peptides that caused an ~18% reduction in protein content with the final retentate having a total PL content of 9.3% dry basis (db) with protein and fat contents at approximately 43.8 ± 0.4% (db) and 48.9 ± 1.2% (db), respectively. The MF permeate had minimal fat content, indicating that there was no transmission of lipids or PL through the membrane during the MF/DF process. Confocal laser scanning microscopy and particle size analysis of enzyme hydrolyzed solution revealed that protein aggregates were still present after 1 h of hydrolysis. Complete removal of proteins and peptides was not achieved by this process, suggesting that a combination of enzymes would be needed for further hydrolysis of protein aggregates in WPPC solution to further enrich the PL content.

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.

In vitro analysis of antioxidant peptides from an enzymatically hydrolysed Channa punctata protein isolate

Channa punctata, a freshwater fish consists of a good amount of protein (64.42 ± 0.51%) with essential amino acids. The degree of hydrolysis was maximum at 240 min with 82.72 ± 0.89% and 21.37 ± 1.21% of hydrolysis in the case of Alcalase 2.4 L (AlcHyd240) and pepsin (PepHyd240). Low molecular weight bands (<10 kDa) were more prominent in AlcHyd240. Hydrolysate shows improved functional properties in comparison with isolate. <3 kDa ultrafiltrate shows enhanced antioxidative efficacy than the hydrolysate and isolate. The fractions were analysed by MALDI-TOF MS/MS and two peptides PLRVGN and PLPNSK with the highest score of 70.4 and 56.5, respectively and hydrophobicity of 50% were selected for in vitro study. Molecular docking studies revealed that peptides interacted with the radicals by H-bond. The presence of proline, leucine, valine, glycine, arginine, serine, and lysine induces antioxidant activity. Thus, this study suggested the peptides identified from Channa punctata can be used as a nutraceutical with potent antioxidant activity. Novelty impact statement A potent antioxidant Channa punctata protein hydrolysates were generated by enzymatic hydrolysis and were purified by ultrafiltration. Amino acid sequences PLRVGN and PLPNSK were identified by using MALDI-TOF MS/MS. Docking study revealed H-bond interactions between the peptides and the free radical. According to the structure–activity relationship study, the presence of hydrophobic amino acid residues, glycine, and serine influences the antioxidant potential.

Porphyromonas gingivalis Conditioned Medium Induces Amyloidogenic Processing of the Amyloid-β Protein Precursor upon in vitro Infection of SH-SY5Y Cells

Background:Cleavage of the amyloid-β protein precursor (AβPP) mediated by host secretase enzymes, releases several fragments including amyloid-β (Aβ40 and Aβ42).Objective:To determine if Porphyromonas gingivalis conditioned medium cleaved AβPP to release Aβ40 and Aβ42.Methods:The SH-SY5Y cell line was challenged, in vitro, with P. gingivalis (Pg381) conditioned medium in the presence/absence of cytokines. The cells and their supernatants were assessed for AβPP cleavage fragments by immunoblotting and transmission electron microscopy.Results:Western blotting of the cell lysates with the anti-AβPP C-terminal antibody demonstrated variable molecular weight bands corresponding to full length and fragmented AβPP in lanes treated with the following factors: Tryptic soy broth (TSB), Pg381, IL-6, Pg381 + IL-1β, and Pg381 + TNF-α. The low molecular weight bands corresponding to the C99 dimerized fragment were observed in the Pg381 and interlukin-6 (IL-6) treated groups and were significantly more intense in the presence of Pg381 with either IL-6 or TNF-α. Bands corresponding to the dimerized C83 fragment were observed with cells treated with TNF-α alone and with Pg381 combined with IL-1β or IL-6 or TNF-α. The anti-Aβ antibody detected statistically significant Aβ40 and Aβ42, levels when these two Aβ species were pooled across test samples and compared to the untreated group. Electron microscopic examination of the supernatants demonstrated insoluble Aβ40 and Aβ42.Conclusion:These observations strongly imply that AβPP is an infection responsive protein cleaved via the amyloidogenic pathway on exposure to conditioned medium and in the presence of pro-inflammatory mediators.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

Characterisation of Salivary Gland Proteins and P-18 Gene of Camel Ticks from Bikaner

The main objective of this study was to investigate the various salivary gland proteins and gene analysis of camel ticks Hyalomma dromedarii found on one-humped camels (Camelus dromedarius) in Bikaner city, Rajasthan, India. For this purpose, Salivary glands were collected from 5 adult ticks (engorged females). Various protein analysis from salivary gland using SDS-PAGE for determination of protein bands and their molecular weights for each fraction was done. The molecular weight of resolved major bands ranged from 14.4 to 96.0 KDa. Out of them large majority of protein molecular weight was around 18 KDa. This low molecular weight band of around 18 KDa was detected in the all 5 fractions of salivary gland of Hy. dromedarii ticks. The protein gene P–18 of Hy. dromedarii of present study had 90.4% sequence identity with that of Hy. asiaticum from China.

Three phase purification of milk clotting protease from Wrightia tinctoria fruit and studies on its casein subunit specificity

Crude aqueous extract of Wrightia tinctoria fruit was subjected to three phase partitioning (TPP) with three different ammonium sulphate concentrations (40, 60, 80%) and their effect on concentrating milk clotting proteases were examined with 1:1 ratio of crude extract to t-Butanol. Proteases that concentrated in the interphase (IP) fraction of 60 and 80% salt concentration exhibited high milk clotting activity. TPP contributed to reduction in Caseinolytic Activity (CA) and enhancement of Milk Clotting Index (MCI) in these IP fractions compared to the crude enzyme. Casein (whole and kappa) hydrolytic pattern was analyzed by tricine SDS PAGE. Electrophoretic pattern of hydrolysate after 1 hr incubation revealed hydrolysis of κ-CN by CE, IP 60 and IP 80 fractions. Appearance of two low molecular weight bands approximately around 15 kDa indicated the specific affinity and controlled hydrolytic behavior by the proteases. Study provides evidence towards the utility of TPP in concentrating these milk clotting proteases and in improving their MCI. Keywords : Milk clotting • Wrightia tinctoria • Proteases• cheese

The effects of camel chymosin and Withania coagulans extract on camel and bovine milk cheeses

Withania coagulans (W. coagulans) extract and camel chymosin have aspartic protease capable of coagulating milk for cheese production. This study investigated the quality of camel and bovine milk cheeses coagulated using Withania extracts, came chymosin, and their mixture in two experiments. In Experiment (1), a factorial design with four factors (W. coagulans, camel chymosin, incubation time, and incubation temperature) was performed. The effect of these factors on cheese’s yield and hardness were assessed. An enzyme concentration corresponding to a 36 µg/L of milk of W. coagulans, 50 IMCU/L of camel chymosin, holding time of 4 h, and incubation temperature of 60 °C provided the optimal textural hardness for both camel and bovine milk cheeses. Seven treatments were analyzed in experiment (2) were analyzed for physicochemical properties, yield, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGEitation). The results showed that pure Withania extract exhibited the lower coagulating effect resulting in cheeses with low yield, hardness, fat, protein, and total solids. The SDS-PAGE electropherograms of camel cheese showed several low molecular weight bands as compared to bovine cheese. This phenomenon is due to excessive proteolysis in camel cheese, which we believed is caused by the presence of endogenous enzymes.

Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator.

Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown. Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl-enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed. These data suggest that under physiological conditions a fraction of the tPA-PAI-1 population exists as nonacylated-enzyme inhibitor complex.

Chromium (VI) tolerance and bioaccumulation by Candida tropicalis isolated from textile wastewater

In the present study a yeast strain isolated from industrial wastewater, identified as Candida tropicalis, showed chromium (Cr) tolerance level up to 5 mM. Yeast grown in minimal salt medium containing Cr (VI) ions for 48 h and crude enzyme extracts were tested for chromate reductase activity. Optimum temperature and pH of chromate reductase were 30 °C and pH of 7. The enzyme activity was greatly enhanced in the presence of divalent metal cations. Total protein profile revealed some protein bands were present in hexavalent chromium [Cr (VI)] treated samples but were absent in non-treated samples, especially low molecular-weight protein bands in the mass range of < 25 kDa with greater intensity in Cr (VI) treated samples. Yeast cells were able to uptake Cr (VI) between 21 and 80 mg g− 1 within 2–12-d of time, indicating yeast strain promising potential for Cr (VI) removal from the wastewater. The present study results suggest that C. tropicalis is a suitable candidate for bioremediating chromium ions from the contaminated-environment.

Assessment of high yielding local Pakistani bread wheat genotypes for improved chapati making quality

The experiment was conducted to classify the maximum glutenin protein possessed Pakistani bread wheat genotype for superlative chapati making quality by ten yield-related parameters. The studied germplasm was acquired from NARC, Pakistan, and planted in randomized-complete-block-design with four replicates at the screen house of the Genetics Department. Data were assessed via Duncan’s test, correlation analysis, SDS-PAGE, and cluster analysis. Duncan’s test conceded that Pirsabak-85 had the highest plant height, flag leaf area, biomass, grain yield plantˉ1, harvest index, and protein content. While, the correlation studies showed that plant height, tillers plantˉ1 (r = 0.649), fertile tillers plantˉ1 (r = 0.713),biomass (r = 0.861), spike length (LS), thousand-grain weight and harvest index had a positive higher significant association with grain yield plantˉ1. The SDS-PAGE analysis resolved 30 diverse high and low molecular weight bands, ranging from 200 kDa to 28 kDa glutenin subunits. Among genotypes, Pirsabak-85 showed maximum protein content and 10 Glu-1 scores. The dendrogram analysis revealed that Pirsabak-85 associated with cluster-II, which was a major and most diverged cluster. The Pirsabak-85 can be utilized to enhance bread wheat production and better chapati making quality

Gelatinases Cleave Dentin Sialoprotein Intracellularly.

Dentin sialoprotein (DSP), the NH2-terminal fragment of dentin sialophosphoprotein (DSPP), is essential for dentin formation and further processed into small fragments inside the odontoblasts. Gelatinases, including matrix metalloproteinases 9 (MMP9) and MMP2, were able to cleave DSP(P) in tooth structures. We hypothesized that gelatinases may also cleave DSP intracellularly in the odontoblasts. In this study, the co-expression and physical interaction between DSP and gelatinases were proved by double immunofluorescence and in situ proximity ligation assay (PLA). Intracellular enzymatic activity of gelatinases was verified by gelatin zymography and in situ zymography. To confirm whether DSP was cleaved by active gelatinases intracellularly, lysates of wild-type (WT) odontoblastic cells treated with a MMP2 inhibitor or a MMP9 inhibitor or a MMP general inhibitor and of Mmp9–/– odontoblastic cells were analyzed by western blotting. Compared with the WT odontoblastic cells without inhibitor treatment, all these groups exhibited significantly higher ratios of high molecular weight to low molecular weight band density. FURIN was verified to be co-localized and physically interacted with MMP9 by double immunofluorescence and in situ PLA. The ratio of proMMP9 to activated MMP9 inside the odontoblastic cells were increased when function of endogenous FURIN was inhibited. And overexpressed proMMP9 was intracellularly cleaved by FURIN in the HEK293E cells, which was completely blocked by the mutation of proMMP9 with R96TPR99 substituted by A96AAA99. Taken together, these results indicate that DSP is intracellularly processed by gelatinases, and FURIN is involved in the intracellular activation of proMMP9 through cleavage of its R96TPR99 motif.

Phytochemical Compounds and Antioxidant Activity Modified by Germination and Hydrolysis in Mexican Amaranth.

Amaranth (Amaranthus spp.) grains have become essential for human health and nutrition; due to the presence of bioactive compounds that have shown some biological activities. This study aimed to evaluate the effect of germination, enzymatic hydrolysis, and its combination on the phytochemical compounds and antioxidant activity in Mexican amaranth. Germinated amaranth flours (GAF) exhibited increases in the concentrations of soluble protein (SP), total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC) and antioxidant activity (AOX) by 35.7, 17.2, 163.0, 1472.2, and 54.3%, respectively, compared with ungerminated amaranth flours (UAF). In SDS-PAGE, both hydrolysates of UAF and GAF exhibited low molecular weight bands (< 10kDa). The hydrolysates of UAFH and GAFH had the highest degree of hydrolysis at 205min of sequential hydrolysis (pepsin with pancreatin) time with 73.4 and 60.3%, respectively. Both hydrolysates obtained from GAF and UAF released significantly SP, TPC, TFC after sequential enzymatic hydrolysis (up 205min), which led to a remarkable improvement of AOX when compared to nonhydrolyzed amaranth samples. The UAFH and GAFH had the best AOX at 270min of enzymatic hydrolysis with 983.1 and 1304.9μmol TE/mg SP, respectively. Hence, the combination of germination and enzymatic hydrolysis could be used to produce functional ingredients for food product development.

High seroprevalence of Toxocara antibodies in pregnant women attending an antenatal clinic at a university hospital in Ile-Ife, Nigeria

Human toxocariasis is a neglected zoonotic disease and its impact on human health is underestimated. Studies investigating the occurrence of toxocariasis in pregnancy are very scarce in Nigeria. This study investigated the seroprevalence of Toxocara spp. antibodies as well as the possible risk factors in pregnant women who routinely attended an antenatal clinic in a university hospital in Ile-Ife, Osun state. Blood samples were collected from 413 participating pregnant women between October 2017 and February 2018. Epidemiological data were obtained through a structured questionnaire. The sera were screened for anti-Toxocara IgG antibodies by western blot technique based on Toxocara canis larval excretory-secretory antigens, targeting low molecular weight bands of 24-35 kDa, specifically for T. canis infection. The overall seroprevalence was 92.49% (382/413). There was no significant difference in the seroprevalence rate of Toxocara spp. infection between pregnant women aged ≤30 y and those aged >30 y (91.63 vs 93.33%; OR 1.28, 95% CI 0.61 to 2.67, p=0.511). The seroprevalence rate increased with the number of miscarriages but there was no statistical significance (p>0.05). Moreover, no significant associations were found between Toxocara spp. seropositivity and other studied risk factors (p>0.05). The high seroprevalence of Toxocara spp. in pregnant Nigerian women needs comprehensive health education regarding personal hygiene and how to avoid exposure to this parasite infection.

Physicochemical state and in vitro digestibility of heat treated water-soluble protein from Pacific oyster (Crassostrea gigas)

Oyster water-soluble protein (OWSP) was extracted from the Pacific oyster (Crassostrea gigas) and the impact of heat treatment on the physicochemical properties, structural changes and in vitro digestibility (proteolysis using pepsin, trypsin and α-chymotrypsin) of OWSP at different temperatures (35, 45, 55, 65, 75, 85, 95, 100 °C) were studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the intensity of low molecular weight bands decreased while the intensity of high molecular weight bands increased with increased temperature, indicating the bonding of proteins. The differential scanning calorimetry curve showed that the denaturation temperature and enthalpy of OWSP were 39.3 °C and 4.42 J/g, respectively. Heating resulted in significantly increased surface hydrophobicity and electronegativity, and decreased protein solubility of OWSP (p < 0.05). However, the dynamic light scattering, confocal microscopy and Nile red fluorescence showed that protein aggregates repelled each other and dispersed more evenly in solution due to the increase of surface hydrophobicity and electronegativity at 65–95 °C. The secondary structure determined using circular dichroism spectrometer showed that the α-helix content decreased and β-sheet content increased with heating. In vitro digestion showed that the proteolysis of OWSP increased with the denaturation of proteins at lower heating temperatures (45, 55 °C), while protein aggregation and the formation of a more compact structure inhibited the digestion of OWSP at higher heating temperatures (65–100 °C).

Analysis of Chromosomal DNA Fragmentation in Apoptosis by Pulsed-Field Gel Electrophoresis.

Double-strand DNA break (DSB) formation is a key feature of apoptosis called chromosomal DNA fragmentation. However, some apoptosis inducers introduce DNA damage-induced DSBs prior to induction of apoptotic chromosomal DNA fragmentation. To analyze these distinct breaks, we have developed a method using pulsed-field gel electrophoresis (PFGE) with a rotating gel electrophoresis system (RGE) that enables us to distinguish between apoptotic DSBs and DNA damaging agent-induced DSBs based on their mobility in the electrophoresis gel. Apoptotic DSBs appear as smeared low-molecular weight bands (less than 500kb), while damage-induced DSBs result in a compact single band (more than 500kb). Furthermore, using a caspase inhibitor, Z-VAD-FMK, we can confirm whether broken DNA fragments are produced as part of an apoptotic response. Overall, we succeeded in characterizing two individual apoptosis inducers and showed the different effects of those compounds on the induction of DNA breaks.

Physicochemical, molecular and thermal properties of high-intensity ultrasound (HIUS) treated protein isolates from album (Chenopodium album) seed

Protein isolates from album seed were prepared and subjected to HIUS treatment using a probe method. The effect of ultrasonication on physicochemical, molecular and thermal characteristics were investigated and compared with those of the control. The color characteristics (L*, a*, b* values), whiteness index, solubility, foaming capacity, and foam stability and molecular weight loss of album protein isolates (APIs) have been increased significantly (p ≤ 0.05) than the native API as a result of HIUS treatment. However, HIUS treatment reduced the denaturation temperatures (Td), enthalpies of denaturation (ΔH), thermal stability, particle size, and whiteness index. With increase in ultrasonication treatment from 5 to 25 min, the Td decreased from 84.56 to 75.90 °C and the ΔH from 44.87 to 38.75 J/g. This decrease might be related to some structural and conformational changes that had occurred in APIs due to break down of molecular bonds by sonication. The highest reduction in particle size from 245.63 μm to 134.28 μm was observed in album protein isolates treated for 25 min. Most importantly, probe sonication had imparted the structural and conformational changes in API which were confimed form the results of SDS-PAGE, surface hydrophobicity, and weight loss analyses. SDS-PAGE and weight loss showed splitting of high molecular bands into lower molecular weight bands whereas, surface hydrophobicity of HIUS treated APIs was found significantly (p ≤ 0.05) higher than that of native APIs. The changes in SDS-PAGE patten, surface hydrophobicity, and weight loss subsequently enhanced solubility along with improvement in thermal and other functional properties of album protein isolates.

Effect of ultrasound pretreatment on the antioxidant capacity and antihypertensive activity of bioactive peptides obtained from the protein hydrolysates of Erythrina edulis

The aim of this research was to evaluate the effect of ultrasound pretreatment on enzymatic hydrolysis with Flavourzyme® and Alcalase® enzymes and the ACE-inhibitory activity and the antioxidant capacity of protein hydrolysates of Erythrina edulis. A protein concentration of 78.8% was obtained after sonicating the protein solutions (10%, w/v) for 10 min at 80 kHz and 100% amplitude. The ultrasonic pretreatment (UP) increased the degree of hydrolysis (47.7%) up to 70 min, the IC50 values in both samples [with (UP) and without pretreatment (WP)] were 100 μg/mL, and the UP samples presented the highest percentage of inhibition at 57.3%. The UP hydrolysates showed the highest (p &lt; 0.05) antioxidant (ABTS*) and radical (DPPH*) - scavenging activities, with IC50 values ranging from 64.52 to 77.62 μg/mL and from 151.13 to 173.22 μg/mL, respectively. In SDS-PAGE, the hydrolysates UP exhibited low molecular weight bands (8 - 20 kDa). The results of both, antioxidants and antihypertensive activities obtained in vitro, showed a higher percentage of activity for the peptides obtained after pretreatment with ultrasound than for those obtained without the use of ultrasound prior to enzymatic hydrolysis.

The enhancement of gastrointestinal digestibility of β‐LG by dynamic high‐pressure microfluidization to reduce its antigenicity

SummaryDynamic high‐pressure microfluidization (DHPM) can increase the antigenicity of β‐lactoglobulin (β‐LG) in previous researches. We observed in this study that the antigenicity of DHPM‐modified β‐LG at pressures of 0.1, 80 and 160 MPa after in vitro digestion declined from 13.41 to 12.27 and 7.19 μg mL−1, gradually. The enhancement of gastrointestinal digestibility of β‐LG was related to the aggregation state and conformational changes induced by DHPM and was reflected by the electrophoretic bands of low molecular weight (5 and 10 kDa) shown in electrophoresis, the reduction of particle size, the generation of smaller peptide fragments (m/z 837.444, 955.492 and 1215.616) detected by mass spectrum and the increase in surface hydrophobicity. These changes contributed to the decrease in antigenicity. Simultaneously, a schematic diagram was proposed to demonstrate the conceivable mechanism of antigenicity changes of modified β‐LG after in vitro digestion.