Free Sulphydryl Research Articles

De-polymerisation and hydrolysis of seed storage protein is involved in the freezing tolerance of hydrated Brassica oleracea seeds

Hydrolysis of globulins might be involved in the freezing tolerance of hydrated lettuce seeds. The mechanism was further explored in hydrated brassica (Brassica oleracea) seeds, by application of 5,5'-dithiobis(2-nitro- benzoicacid) (DTNB), the Ellman's reagent, which can bind to the free sulphydryl side chain of cysteine. DTNB increased the viability of hydrated brassica seeds after slow cooling (-3°C hour-1 ) to -17°C. Two-dimensional electrophoresis and mass spectra analysis indicated the accumulation of small peptides derived from 12S globulins (cruciferin) in DTNB-treated seeds after slow cooling. The seed storage peptides with different molecular weight were cooled and the ice formation during cooling observed by cryomicroscopy, which proved that peptides of smaller molecular size produced smaller and rounder crystals. The smallest peptide in DTNB- treated seeds produced the smallest and roundest crystals during cooling. The levels of ASA, DHA and GSH were specifically up-regulated after DTNB treatment and following slow cooling, which indicates that DTNB enhanced the circulation of ASA and GSH to resist the oxidative stress. Consistent with this finding, RNA-seq and real time PCR indicated that glutathione reductase (GR) was up-regulated after DTNB treatment.

Pleurotus geesteranus mycelium proteins: physicochemical and functional properties

SummaryIn the present work, the physicochemical and functional properties, as well as the digestion behaviour of Pleurotus geesteranus mycelium protein isolates (PMPI), were investigated. SDS‐PAGE results found that the molecular weight of PMPI was mainly concentrated in >100 kDa and <25 kDa and a total of 365 proteins were detected with proteome analysis. The contents of secondary structures in PMPI were as follows: β‐sheet (27.15%) > α‐helix (23.23%) > random coil (21.92%) > β‐turn (17.21%) > β‐antiparallel (10.48%). The total sulphydryl, free sulphydryl and disulphide bond content of PMPI were 29.88, 9.77 and 10.05 μmol/g respectively. The denaturation temperature of PMPI was measured at 92.94 °C, with an enthalpy change of 39.76 J/g. However, PMPI showed poor solubility, leading to relatively low foaming properties and emulsion activity. The nitrogen release rate of PMPI was 21.78% after pepsin digestion and 74.60% at the end of trypsin digestion.

Effects of CaCl2 concentration on fibrils formation and characteristics of soybean protein isolate and β-conglycinin/glycinin

In this study, fibrillation of soybean protein isolate (SPI) and β-conglycinin (7S)/glycinin (11S) in acid-heating with CaCl2 concentration (0–200 mM) was explored. Fibril formation kinetics results showed the fastest fibril formation rate and the most fibrils with 80 mM CaCl2 addition. Fourier-transform infrared (FTIR) spectroscopy, size distribution and Transmission electron microscopy (TEM) results demonstrated SPI fibril structure became more complete and occurred entanglement with CaCl2 concentration increasing from 0 to 80 mM. When CaCl2 addition was above 80 mM, ζ-potential, surface hydrophobicity (H0) and free sulphydryl groups determination proved hydrophobic interaction among fibril built-units was hindered by charges shielded effect, and basic units of 11S were more exposed, aggregate morphology were changed to thicker fibrils or amorphous aggregates. The whole system with CaCl2 has preferable antioxidant property and no cytotoxicity. The study provided a basis for optimizing SPI fibrillation in food industry.

The effects of free radicals and metal ion on milk protein β‐lactoglobulin (BLG) glyco‐oxidation

SummaryIn this study, the effects of H2O2, 2,2′‐Azobis(2‐amidinopropane) dihydrochloride (AAPH) and metal ion Fe3+ on β‐lactoglobulin (BLG) glyco‐oxidation were investigated. The results demonstrated all the three inducers caused BLG morphology change and higher molecular weight BLG‐glucose (BLG‐Glu) adducts formation. H2O2 and AAPH significantly accelerated BLG oxidation by increasing carbonyl content and decreasing freesulphydryls (P < 0.05). However, Fe3+ showed little influence on carbonyl and free sulphydryls contents. All the three inducers promoted fluorescent‐advanced glycation end products (fl‐AGEs) and carboxymethyl‐lysine (CML) formation. Fe3+ and H2O2 (0.5% and 1%) could increase methylglyoxal (MGO) level and caused less time to reach its peak concentration than control group. Compared with the control group, new glycation sites were determined in H2O2, AAPH and Fe3+ treatments, indicating the inductive effect of those free radicals or metal ion on BLG glycation. The results will provide important information for the control of protein‐based products glyco‐oxidation during food processing and storage.

Physicochemical properties of fluid milk with different heat treatments and HS-GC-IMS identification of volatile organic compounds

To improve understanding of the effects of industrial heating processes on its physiochemical and flavour properties, fluid milk was evaluated for protein denaturation and flavour volatiles after high-temperature short-time (HTST), indirect ultra-high temperature (IND-UHT) and direct ultra-high temperature with direct steam infusion (INF-dUHT) treatments. Protein denaturation was evaluated by particle size, free sulphydryl content, fluorescence spectra, native whey protein retention rate and microstructure. Volatile organic compounds (VOCs) were compared by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). HTST treatment exerted the lowest negative effect on the aggregation and denaturation of native whey proteins including α-lactalbumin, β-lactoglobulin and lactoferrin, followed by INF-dUHT. IND-UHT caused the most severe denaturation of native whey proteins. HTST-treated milk was characterised by a high level of low molecular weight ketones and INF-dUHT milk possessed the greatest abundance of aldehydes, whereas IND-UHT milk exhibited the highest contents of high molecular weight methyl ketones, which contributed to an unpleasant aroma.

Multi-dimensional analysis of rennet-induced micellar casein gels after ultrasound

Multi-dimensional analyses (pH, particle size, zeta potential, surface hydrophobicity, protein structure, microstructure of gels, free sulphydryl content, and fluorescence intensity) were performed to determine the structural changes and gel properties of micellar casein concentrate (MCC) at different protein concentrations (8%, 12%, 16%) treated with ultrasound (40 kHz, 500 W, 0–60 min). The gel strength and water holding capacity were improved with the increase in ultrasound treatment time and protein concentration. A denser gel network with smaller pore size was formed. For gels made from MCC after ultrasound treatment for 60 min, the gel properties were especially improved. The particle size decreased, zeta potential and hydrophobicity increased after ultrasound treatment. Ultrasound treatment influenced the secondary structure of MCC while no change in the molecular weight of the protein was detected.

The gel properties and gastric digestion kinetics of a novel lactic acid bacteria fermented tofu: Focusing on the effects of transglutaminase

The purpose of this study was to evaluate the gel properties and gastric digestion of tofu made by combining transglutaminase (TGase) with lactic acid bacteria (LAB). TGase (0–7 U/g) significantly grew the tofu texture and water holding capacity, a significantly positive correlation between the hardness (R2 = 0.78, P < 0.05), chewiness (R2 = 0.72, P < 0.05), and WHC (R2 = 0.71, P < 0.05) with TGase amount was found. When the TGase was 4 U/g, the tofu had the highest hardness ((45.89 ± 0.88) g) and WHC ((76.56 ± 2.40)%). A negative correlations between surface hydrophobicity and free sulphydryl content values of tofu with TGase amount were detected (R2 = −0.90, −0.93, P < 0.01). With the extension of digestion time, the content of free amino acids (FAC) in tofu increased. It was found that the Logistic equation could be used to fit the relationship (an S-shaped curve) between FAC content (P) and the digestion time (t) of tofu. The equation was as follow: (P=KP0P0+(K−P0)e−rKt) (R2 = 0.964–0.998). Digestibility could be altered by controlling the amount of TGase and the structure of 2 U/g - tofu was in favor of the gastric digestion. The results of this study indicated that incorporation of TGase into tofu affects its digestibility and a theoretical foundation of tofu gastric digestion was established.

Stability of zein‐based films and their mechanism of change during storage at different temperatures and relative humidity

Mechanical properties are the important parameters of the film which reflected the resistance of the films during their shelf life. In order to evaluate the stability of zein films preserved at different temperature and relative humidity (RH), tensile strength (TS) and elongation at break (EB) of zein films plasticized with glycerol and PEG-400 were observed during storage. Results showed that the EB decreased and TS increased during the first 6 days of storage at different RH and temperatures. RH which brought a maximum change in TS and EB were 43% and 65%, respectively. Films preserved at RH of 80% for 18 days lost its extensibility. Statistical analysis results showed that the correlations between the free sulphydryl content and mechanical properties at RH 43%, and between the moisture content and mechanical properties at RH 80% were more significant. A model was proposed to explain the change in TS and EB during zein film storage. Practical applications Zein is commonly produced from the by-product of bio-ethanol and starch industries and has excellent film-forming properties. Pure zein film is brittle, which has limited their application. Many researches have focused on the improvement of the mechanical properties of zein films. As a packing material, zein film should have excellent stability during the shelf life of the packaged food. So a systematic study about the effect of temperature and relative humidity on the mechanical properties of zein film plasticized with glycerol and PEG-400 during storage is necessary. The results presented in this paper would highlight the suitable applying environment of zein films, either actual or potential.

Effect of dielectric barrier discharge (DBD) plasma on the structure and antioxidant activity of bovine serum albumin (BSA)

AbstractThis study was aimed to determine the influence of dielectric barrier discharge (DBD) plasma with a plasma intensity (20 and 40 kV) and treatment times (0, 2, 4, 6 and 8 min) on the physicochemical, structural and antioxidant properties of bovine serum albumin (BSA) solution. In results, the free sulphydryl content, pH and free amino group contents, while carbonyl groups and surface hydrophobicity were increased after the plasma treatment. After plasma treatment, there is also an increase in the DPPH and FRAP assay that was associated with the changes in the structure of the peptides chain. The complementary techniques were employed to identify the protein structural modifications by plasma treatment. These changes were more severe at the most extended treatment duration and plasma intensity. The high energetic and oxidising species carried by plasma may have significant associations on the structural and physical properties of BSA.

A unique mechanism for thiolation of serum albumins by disulphide molecules.

Protein S-thiolation is a reversible oxidative modification that serves as an oxidative regulatory mechanism for certain enzymes and binding proteins with reactive cysteine residues. It is generally believed that the thiolation occurs at free sulphydryl group of cysteine residues. Meanwhile, despite the fact that disulphide linkages, serving structural and energetic roles in proteins, are stable and inert to oxidative modification, a recent study shows that the thiolation could also occur at protein disulphide linkages when human serum albumin (HSA) was treated with disulphide molecules, such as cystine and homocystine. A chain reaction mechanism has been proposed for the thiolation at disulphide linkages, in which free cysteine (Cys34) is involved in the reaction with disulphide molecules to form free thiols (cysteine or homocysteine) that further react with protein disulphide linkages to form the thiolated cysteine residues in the protein. This review focuses on the recent finding of this unique chain reaction mechanism of protein thiolation.

Different routes of insulin administration do not influence serum free thiols in type 1 diabetes mellitus.

AimsIntraperitoneal (IP) insulin administration is a last‐resort treatment option for selected patients with type 1 diabetes mellitus (T1DM). As the IP route of insulin administration mimics the physiology more closely than the subcutaneous (SC) route, we hypothesized that IP insulin would result in less oxidative stress (expressed as systemic level of free sulphydryl (R‐SH) content) compared to SC insulin in subjects with T1DM.Materials and methodsProspective, observational case‐control study. Serum thiol measurements were performed at baseline and at 26 weeks in age‐ and gender‐matched patients with T1DM. Serum‐free thiols, compounds with a R‐SH group that are readily oxidized by reactive oxygen species, are considered to be a marker of systemic redox status.ResultsA total of 176 patients, 39 of which used IP and 141 SC insulin therapy were analysed. Mean baseline R‐SH concentration was 248 (31) μmol/L. In multivariable analysis, the route of insulin therapy had no impact on baseline R‐SH levels. The estimated geometric mean concentrations of R‐SH did not differ significantly between both groups: 264 (95% CI 257, 270) for the IP group and 258 (95% CI 254, 261) for the SC group with a difference of 6 (95% CI −2, 14) μmol/L.ConclusionsBased on R‐SH as a marker of systemic oxidative stress, these findings demonstrate that the route of insulin administration, IP or SC, does not influence systemic redox status in patients with T1DM.

Effect of baked wheat germ on gluten protein network in steamed bread dough

SummaryThe effect of baked wheat germ (BWG) on the gluten network structure in steam bread dough was investigated. The secondary structure, free sulphydryl (‐SH) content, disulphide (‐SS‐) bonds content and microstructure of gluten were analysed to evaluate gluten structural changes. The addition of different amounts of BWG (0, 2, 4, 6, 8, 10, and 12%) in dough resulted in decreased content of α‐helix and β‐sheet structures, but increased random coils, which indicated that a disordered structure was formed. The presence of BWG increased the ‐SH content but decreased the ‐SS‐ bonds content, which indicated fracture of disulfide bonds. Confocal laser scanning microscopy (CLSM) analysis indicated that steamed bread dough containing BWG had larger‐sized gas cell and granules of starch separated by the protein matrix, which weakened the gluten network structure. These changes may inevitably affect the viscoelastic properties of steamed bread dough.

Oxidant-Antioxidant balance in patients with coronary slow flow.

Objective:Recent studies have focused on the probable role of oxidative stress in cardiovascular diseases. We aimed to assess the oxidant/antioxidant biomarkers in coronary slow flow (CSF).Methods:The study included 51 subjects with CSF and age and sex matched 32 controls. Detailed anamnesis of the patients in the study was taken and routine physical examinations were performed. Routine biochemical blood tests were analyzed. Total oxidative status (TOS), oxidative stress index (OSI) and lipid hydroxyperoxide (LOOH) levels as oxidant biomarkers; paraoxonase (PON1), ceruloplasmin (CP), free sulphydryl (SH) groups, and total antioxidant capacity (TAS) levels as antioxidant biomarkers were studied.Results:Baseline demographic characteristics of the study population did not differ significantly between groups.TOS, OSI and LOOH concentrations were higher in study group than in control group. However, there was no significant difference detected in levels of TAS, PON1, SH and CP. Multivariate logistic regression analysis revealed that TOS, hsCRP and smoking were indepedent risk factors of CSF.Conclusions:Although there was not any significant difference in antioxidant biomarkers (TAS, PON1, SH and CP) in CSF patients, we detected increased TOS, OSI and LOOH levels which have oxidant properties. These data supported the possible involvement of oxidative stress in pathogenesis of CSF as previous studies reported.

Liposomal vesicles-protein interaction: Influences of iron liposomes on emulsifying properties of whey protein

In this study, whey protein (WP) was used as a model protein to interact with ferrous sulfate liposomes (FL). The interaction between FL and WP was researched using circular dichroism, intrinsic fluorescence, surface hydrophobicity, free sulphydryl groups, Fourier transform infrared spectroscopy, zeta potential, particle size, and interfacial tension. These observations indicated that FL interacted with WP via electrostatic force, hydrophobic force, and hydrogen bonds. WP secondary structure and tertiary structure could be changed by the interaction between FL and WP. The emulsifying properties of WP were improved by the interaction between FL and WP. Emulsifying activity index and emulsifying stability index of WP in the presence of FL increased from 39.69 to 65.63 m2/g, from 28.63 to 101.55 min, respectively. And the WP-stabilized emulsion droplet size decreased from 1.64 μm to 0.78 μm in the presence of FL. However, the emulsifying properties of WP were also influenced by liposomal composition, pH, and temperature. The changes of WP emulsifying properties could be attributed to the effects of liposomes on WP secondary structure and tertiary structure, zeta potentials, and particle size. This work provides new insights into the application of liposomes in food.

Pulsed light inactivation of polygalacturonase

Pulsed light is a non-thermal technology capable to inactivate enzymes. This study investigated the effect of pulsed light on the activity of polygalacturonase, and on the structure of the enzyme by means of fluorescence emission spectra, free sulphydryl detection and analysis of changes in parameter A and phase-diagram. The results showed that pulsed light is able to inactivate polygalacturonase in buffer, with >90% reduction of enzyme activity after applying 128 J/cm2 and a first-order kinetic constant of 0.0426 cm2/J under the experimental conditions. The free sulfhydryl detection revealed the rupture of sulfhydryl bridges. Fluorescence spectra analysis showed that the tertiary structure of polygalacturonase was changed. Phase diagram analysis shows the existence of only two populated states. It is suggested that the inactivation of polygalacturonase by pulsed light is an all-or-none process where disulfide bridges are broken and the enzyme is unfolded.

A novel process for peanut tofu gel: Its texture, microstructure and protein behavioral changes affected by processing conditions

This work reports, a novel method of peanut tofu preparation by combining transglutaminase (TG) and high-temperature pressure cooking (HTPC, 115 °C, 0.17 MPa) treatment, and its gel formation was studied. Texture and Scanning electron microscope (SEM) results showed TG addition (0.75 mg/g) significantly improved the tofu texture (p < 0.05), with a denser network composed of relatively even pores, whereas the excessive addition (1.25 mg/g) resulted in a softer tofu. At all concentrations (0.25–1.25 mg/g), TG treatment induced the crosslinking of protein molecules as proved by SDS-PAGE, particle size and apparent viscosity analyses. DSC measurement indicated that the subsequent HTPC treatment was able to denature peanut proteins completely. Moreover, by analyzing the changes in surface hydrophobicity (H0) and total free sulphydryl content (TFS) during processing, we found that hydrophobic interactions and disulfide bonds were the major forces involved in the formation of peanut tofu gel. This study suggests this novel method could be applied to make peanut tofu and provides a basis for understanding its gel formation process.

Biochemical Quality Indicators and Enzymatic Activity of Wheat Flour from the Aspect of Climatic Conditions

The contents of free sulphydryl groups (SH), disulphide bonds (SS), and free amino groups (NH2) were determined in order to estimate the extent of climatic condition influence on gluten quality. The analysis included four bread wheat varieties grown in two production years (2011 and 2012) with different climatic conditions in different locations. According to our previously reported results, the working hypothesis was that enzyme activity for breadmaking purpose was insufficient. The aim of this paper was to study the influence of naturally present enzymes on the bread quality by the addition of previously extracted and freeze-dried albumins to the base flour as an additive. The selection of samples was made on the basis of different combinations of proteolytic and α-amylolytic enzymes activity levels. For samples from 2012 production year, the content of SH groups was significantly higher. Regarding the SS content, the obtained results exhibited the opposite trend. Variations in NH2 content were dominantly caused by temperature treatment of tested samples. The addition of freeze-dried albumins to bread improved its specific volume in a lesser extent, while bread crumb texture was significantly improved.

Effects of amidated low methoxyl pectin on physiochemical and structural properties of polymerized whey proteins

ABSTRACTThis study aimed to investigate effects of amidated low methoxyl (ALM, 0.3%, w/v) pectin on physiochemical and structural properties of polymerized whey proteins (PWP, 10%, w/v) prepared by thermal aggregation at 85°C for 10–30 min. Particle size, zeta potential, isoelectric point (IEP), surface hydrophobicity, free sulphydryl group, rheological properties and the secondary structure of all samples were analysed. ALM pectin increased mean diameter and narrowed size distribution of PWP samples (p < 0.05) regardless of heating time. Addition of ALM pectin shifted the IEP of PWP‒pectin complex to lower pH, which decreased with heating time increased. ALM pectin decreased the surface hydrophobicity of PWP heated for 30 min pronouncedly. Whey protein (WP) complexed with ALM pectin showed decreased gelation time and rheological properties of improved elasticity. ALM pectin had no considerable effect on secondary structure of PWP. Data indicated that ALM pectin could alter physiochemical properties of heat-induced polymerized WPns.

High hydrostatic pressure (HHP) effects on antigenicity and structural properties of soybean β-conglycinin.

In this study, the effect of high hydrostatic pressure (HHP) on antigenicity, free sulfhydryl group (SH) content, hydrophobicity (Ho), fluorescence intensity and circular dichroism data of soybean β-conglycinin was studied. The antigenicity of soybean β-conglycinin was decreased significantly at pressures 200-400MPa. The antigenicity inhibition rate of β-conglycinin declined from 92.72 to 55.15%, after being treated at 400MPa for 15min. Results indicated that free sulphydryl (SH) groups and surface Ho of β-conglycinin were significantly increased at pressures 200-400MPa and 5-15min, whereas these properties decreased at the treatments above 400MPa and 15min. The maximum fluorescence intensity was noticed at 400MPa and 15min. The circular dichroism data analysis revealed that the amount of β-turns and unordered structure significantly increased, while the content of α-helix1 and β-strand1 noticeably decreased. These results provide evidence that HHP-induced the structural modification of β-conglycinin and could alter the antigenicity of β-conglycinin.

Whey protein film properties as affected by ultraviolet treatment under alkaline conditions

The effects of UV radiation treatment on film-forming solutions adjusted to two alkaline pH values (9 or 11) on the properties (water vapour permeability, solubility, mechanical properties, colour and microstructure) of whey protein concentrate films, and on some structural characteristics of proteins (free sulphydryl groups, degree of aggregation, denaturation and secondary structure) were evaluated. UV radiation increased protein aggregation at both pH values. Ultraviolet treatment of pH 9 solutions increased free sulphydryl groups and produced films with higher solubility, tensile strength, elastic modulus, and puncture properties and lower elongation at break; UV radiation at pH 11 decreased free sulphydryl groups and films showed higher solubility and elastic modulus and lower puncture deformation and elongation at break than untreated films. The treatment also affected film colour differently depending on the pH. In conclusion, ultraviolet treatment modifies the properties of the films in different ways depending on the pH of the solutions.