Sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

Extraction and Characterization of Type I Collagen from Parrotfish (Scarus sordidus Forsskål, 1775) Scale solubilized with the Aid of Acetic Acid and Pepsin.

Waste from marine fish processing is an important source of valuable products. Fish collagen is considered a alternative biomaterial due to its excellent properties, and it is widely used for industrial purposes. Thus, this present study aimed to characterize acid and pepsin-soluble collagens from the waste of parrotfish (Scarus sordidus Forsskål, 1775) scales. The yields (p > 0.05) of acid-soluble collagen (ASC-PFS) and pepsin-soluble collagen (PSC-PFS) were 1.17 g/100 g and 1.00 g/100 g, respectively. Both collagen samples were categorized as type I owing to the presence of two alpha chain subunits (α1 and α1) after being confirmed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under the fourier transform infrared (FTIR) test, the triple helical structure of type I collagens from the ASC-PFS and PSC-PFS was maintained. Moreover, the study of UV visible spectra and X-ray diffraction (XRD) showed the similarity of collagens derived from different fish species, and the thermostability (T max) evaluation of all extracted collagens was in the range of 36.22-37.78°C, and their values were comparable to previous research on the fish scale collagens. The effect of various pH and sodium chloride (NaCl) treatments on solubility exhibited that the ASC-PFS and PSC-PFS were highly soluble in an acidic condition (pH < 5.0) and low concentration of sodium chloride (<30 g/L). Taken together, collagens extracted from parrotfish scale waste can be an alternative source for industries.

Effect of Microwave Vacuum Freeze-Drying Power on Emulsifying and Structure Properties of Egg White Protein.

The study investigated the effects of different microwave vacuum freeze-drying powers (100-500 W) on the emulsifying properties and structural characteristics of egg white protein, which is of great significance in enhancing the added value of EWP and promoting its application. Emulsification analysis revealed that the emulsification performance was significantly influenced by microwave power and reached its maximum at 300 W. Fourier-transform infrared spectroscopy (FT-IR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses showed that microwave vacuum freeze-drying treatment altered the secondary structure of EWP without changing its peptide structure. Fluorescence measurements indicated that the maximum fluorescence emission intensity decreased, and the maximum emission wavelength shifted towards blue as the power increased. Particle size, zeta potential, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) analyses showed that the average particle size of EWP reached the minimum value of 1203.66 nm, the absolute value of zeta potential reached the maximum value of 41.35 mV, and the thermal stability was strongest, with a more uniform and loose structure observed at 300 W. Texture profile analysis (TPA) showed that appropriate power treatment significantly enhanced the chewiness and viscoelasticity of egg white protein. Therefore, appropriate power treatment could effectively improve the emulsifying properties and stability.

New method to detect a diagnostic marker anti-NH2–terminal of α–enolase autoantibody for Hashimoto’s encephalopathy (P5-5.018)

<h3>Objective:</h3> To develop a new rapid and automatic method to detect a diagnostic marker, anti-NH₂–terminal of α–enolase (NAE) autoantibody (Ab) for Hashimoto’s encephalopathy (HE). <h3>Background:</h3> HE is a steroid-responsible autoimmune disorder associated with chronic thyroiditis. Patients with HE present with various neuropsychiatric symptoms. We discovered a serum autoantibody against the NAE (anti-NAE Ab) to be a useful diagnostic marker for HE and analyzed more than 4,700 samples by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot system. However, this conventional detection method is time-consuming (2 days for 10 samples). Thus, we newly developed a rapid and automatic method to detect the NAE Ab (less than 4 h for 24 samples) by a capillary electrophoresis/blot system using the Wes™ system (Protein Simple, CA). <h3>Design/Methods:</h3> We prepared a recombinant NAE protein after the electroporation of the NAE cDNA into human cultured cells HEK293, purified through a Ni column, and used this protein for the detection of anti-NAE Ab. Subsequently, we investigated the specificity/sensitivity and reproducibility by the Wes method as compared to that of the conventional method in patient sera from 11 positives or six negatives for anti-NAE Ab. <h3>Results:</h3> This study demonstrated the positive (81.8%; 9/11), negative (100%; 6/6) and judgment match rates (88.2%; 15/17) of the two methods, leading to good specificity and sensitivity. These data also showed good reproducibility. Thus, the Wes method is useful and reliable in the detection of the NAE Ab. <h3>Conclusions:</h3> The Wes method to detect a diagnostic marker, anti-NAE Ab, for HE is rapid and automatically performed with good specificity, sensitivity, and reproducibility compared to the conventional method. <b>Disclosure:</b> The institution of Makoto Yoneda has received research support from Cosmic Corporation. Ms. Nakayama has nothing to disclose. AKIKO MATSUNAGA, MD has nothing to disclose. Dr. Togashi has nothing to disclose.

Soluble laticifer proteins from Calotropis procera as an effective candidates for antimicrobial therapeutics

Calotropis procera is a latex-producing plant with plenty of pharmacologically active compounds. The principal motivation behind this study was to separate and characterize laticifer proteins to check their antimicrobial potential. Laticifer proteins were separated by gel filtration chromatography (GFC) and investigated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE assay detected proteins of molecular weights of 10 to 30 kDa but most of them were in the range of 25 to 30 kDa. The soluble laticifer proteins (SLPs) were tested against Gram-positive bacteria i.e., Streptococcus pyogenes and Staphylococcus aureus whereas Escherichia coli and Pseudomonas aeruginosa were tested as Gram-negative bacteria, we determined a profound anti-bacterial activity of these proteins. In addition, SLPs were also investigated against Candida albicans via the agar disc diffusion method which also showed significant anti-fungal activity. SLP exhibited antibacterial activity against P. aeruginosa, E. coli, and S. aureus with a minimum inhibitory concentration (MIC) of 2.5 mg/mL for each, while MIC was found at 0.625 mg/mL for S. pyogenes and 1.25 mg/mL for C. albicans. Moreover, enzymatic activity evaluation of SLP showed the proteolytic nature of these proteins, and this proteolytic activity was greatly enhanced after reduction which might be due to the presence of cysteine residues in the protein structure. The activity of the SLPs obtained from the latex of C. procera can be associated with the involvement of enzymes either proteases or, protease inhibitors and/or peptides.

SDS Electrophoresis on Gradient Polyacrylamide Gels as a Semiquantitative Tool for the Evaluation of Proteinuria.

Proteinuria is an important sign of kidney diseases. Different protein patterns in urine associated with glomerular, tubular and overload proteinuria may be differentiated using the immunochemical detection of indicator proteins or via urinary proteins electrophoresis. Our aim was to characterize sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using commercially available 4-20% gradient gels as a method to detect and differentiate proteinuria. Our laboratory-based study used excess urine samples collected for routine diagnostic purposes from adult patients of a tertiary-care hospital, including patients with albumin/creatinine < 30 mg/g and patients with dipstick proteinuria. The limit of albumin detection was estimated to be 3 mg/L. In 93 samples with albumin/creatinine < 30 mg/g, an albumin fraction was detected in 87% of samples with a minimum albumin concentration of 2.11 mg/L. The separation of 300 urine samples of patients with proteinuria revealed distinct protein patterns differentiated using the molecular weights of the detected proteins: glomerular (albumin and higher molecular weights) and two types of tubular proteinuria ("upper" ≥20 kDa and "lower" with lower molecular weights). These patterns were associated with different values of the glomerular filtration rate (median 66, 71 and 31 mL/min/1.72 m2, respectively, p = 0.004) and different proportions of multiple myeloma and nephrological diagnoses. As confirmed using tandem mass spectrometry and western blot, the SDS-PAGE protein fractions contained indicator proteins including immunoglobulin G, transferrin (glomerular proteinuria), α1-microglobulin, retinol-binding protein, neutrophil gelatinase-associated lipocalin, cystatin C, and β2-microglobulin (tubular), immunoglobulin light chain, myoglobin, and lysozyme (overflow). SDS-PAGE separation of urine proteins on commercially available 4-20% gradient gels is a reliable technique to diagnose proteinuria and differentiate between its main clinically relevant types.

Effect of Ultrasound Pre-Treatment on Extraction and Characterization of Collagen from Bactrian Camel Skin.

The objective of this study was to evaluate the effect of ultrasound pre-treatment on the characterization from Bactrian camel skin. It was possible to produce and characterize collagen extracted from Bactrian camel skin. The results showed that the yield of collagen was higher in ultrasound pre-treatment (UPSC) (41.99%) than the pepsin-soluble collagen extraction (PSC) (26.08%). All extracts were identified as type I collagens using sodium dodecyl sulfate polyacrylamide gel electrophoresis and retained their helical structure, as confirmed through Fourier transform infrared spectroscopy. The scanning electron microscopy analysis of UPSC revealed that some physical changes were caused by sonication. UPSC had smaller particle size than PSC. The viscosity of UPSC always plays a leading role in the range of 0-10 Hz. However, the contribution of elasticity to the solution system of PSC increased in the range of 1-10 Hz. Moreover, ultrasound-treated collagen had superior solubility property at pH 1-4 and at <3% (w/v) NaCl than non-ultrasound treated collagen. Therefore, the utilization of ultrasound for the extraction of pepsin soluble collagen is a good alternative technology to expand the application at industrial level.

Blue agave inulin-soluble dietary fiber: effect on technological quality properties of pangasius mince emulsion-type sausage.

The aim of the work was to investigate the influence of supplementing pangasius mince-based emulsion sausages with blue agave-derived inulin at 1% (T1), 2% (T2), 3% (T3), 4% (T4), and 5% (T5) on its technological quality attributes and acceptability. The cooking yield of T-2, T-3, and T-4 sausages (96-97%) exhibited no significant difference (P > 0.05), which was higher than the other lots. The T-2 batter exhibited a significant difference with all other treatments, showing the lowest total expressible fluid (12.20%) value, indicating the highest emulsion stability of the batter. There was a significant effect on the diameter reduction of the cooked sausages as the level of inulin increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the proteolysis of raw mince without inulin and new bands in cooked sausage samples were observed. Increasing inulin content increased the hardness of the sausages from 2510.81 ± 114.31 g to 3415.54 ± 75.88. The differential scanning calorimetry melting temperatures of peak 2 of the T-1, T-2, T-3, and T-4 increased as the inulin content increased from 1 to 4%. The scanning electron microscope images exhibited a smooth appearance on the surface as the inulin level increased. The sausages incorporated with the 2% and 3% blue agave plant-derived inulin (T-2 and T-3) showed better sensory overall acceptability scores than the control. The results suggested that the blue agave plant-derived inulin could be efficiently utilized at the 2% and 3% levels to enhance the quality of emulsion-type pangasius sausage. © 2023 Society of Chemical Industry.

A novel method for the component identification of human blood products: Mass spectrometric analysis of human fibrinogen digested after SDS-PAGE in-gel digestion

Human fibrinogen, as a blood product of special origin, is relatively simple to prepare and purify. Therefore, completely isolating and removing the relevant impurity proteins is difficult. Further, which impurity protein components are present is not clear. In this study, human fibrinogen products from seven enterprises were collected from the market, and the presence of impurity proteins was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Subsequently, the major 12 impurity proteins were identified and screened by in-gel enzymolysis mass spectrometry, and 7 major impurity proteins with different peptide coverage were identified by enzyme-linked immunosorbent assay, in agreement with the mass spectrometry results. The seven major impurity proteins included fibronectin, plasminogen, F-XIII, F-VIII, complement factor H, cystatin-A, and α-2-macroglobulin. The final test results were in the range of undetectable to 50.94 µg/mL, with correspondingly low levels of impurity proteins between different companies and a manageable risk. Moreover, we found that these impurity proteins existed in the form of polymers, which might also be an important cause of adverse reactions. This study established a protein identification technique applicable to fibrinogen products, which provided new ideas for studying the protein composition of blood products. In addition, it provided a new means of testing for companies to monitor the flow of proteomic fractions and improve the purification yield and product quality. It laid the foundation for reducing the risk of clinical adverse reactions.

Biochemical, Histological Changes, Protein Electrophoretic Pattern, and Field Application of CuPb-Ferrite/TiO2 Nanocomposites for Controlling Terrestrial Gastropod Eobania vermiculata (Müller).

Eobania vermiculata is a hazardous snail that can damage ornamental plants and cause significant harm to plant sections in Egyptian areas. Herein, the molluscicidal activity of CuPb-Ferrite/TiO2 and TiO2 nanoparticles (NPs) against E. vermiculata was evaluated using the poisonous bait method. LC50 values were determined using the leaf dipping and contact methods, with values of 631.23 and 1703.49 ppm for CuPb-Ferrite/TiO2 and 193.67 and 574.97 ppm for TiO2. Exposure to both NPs resulted in a significant increase in the biochemical parameters of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as well as a decrease in total protein (TP) percentage of E. vermiculata. Histological examinations revealed that many digestive cells had ruptured, and their contents had been lost, while the foot's epithelial layer became ruptured. The average reduction was 66.36% for CuPb-Ferrite/TiO2 NPs compared to the recommended molluscicide, Neomyl, with a 70.23% reduction in the field application. Electrophoretic separation of total protein using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with LC50 concentrations of TiO2 and CuPb-Ferrite/TiO2 demonstrated the potency of these synthetic compounds as molluscicidal agents. Therefore, we recommend the use of CuPb-Ferrite/TiO2 NPs as a novel land snail molluscicide because it is safe to use, and the baits are arranged to not affect irrigation water, with a high molluscicidal effect.

Effects of anthocyanins and microbial transglutaminase on the physicochemical properties of silver carp surimi gel.

The objective of this study was to investigate effects of anthocyanins (AC) and microbial transglutaminase (MTGase) on the physicochemical properties of surimi gels from silver carp. The addition of AC and MTGase significantly increased gel strength and water holding capacity (WHC) of surimi gels, but the effect of MTGase was much stronger (p < .05). There were the highest gel strength, storage modulus (G') and WHC with 0.1g/100 g AC and 0.4g/100 g MTGase, while they were higher than that with AC or MTGase alone. AC promoted the cross-linking mainly by covalent and non-covalent bonds in surimi gels, while MTGase did mainly through covalent bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the results of gel strength, WHC, chemical interactions and G' of surimi gel or paste with AC and MTGase. In general, AC and MTGase could synergistically improve the physicochemical properties of surimi gels and potentially enhance the quality of surimi-based product from silver carp.

PAMUK ÇEKİRDEĞİ PROTEİN KONSANTRESİNİN KARAKTERİSTİK ÖZELLİKLERİNİN BELİRLENMESİ

The current study focused on characteristic attributes of protein concentrate obtained from oil-free cottonseed. For this, the physicochemical properties namely moisture content, water activity, color, flowability, wettability, and protein solubility of cottonseed protein concentrate (CSPC) were investigated. Water holding capacity (WHC), oil binding capacity (OBC), foaming capacity, foam stability (10 and 30 min), emulsion activity index (EAI) and emulsion stability index (ESI) (10 and 30 min) of proteins were 2.75 g water/g protein, 2.59 g oil/g protein, 29.00%, 93.10% - 69.05%, 6.25 m2/g and 29.27-87.81 min, respectively. Bands regarding CSPC in the 45 kDa molecular weight were detected by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns. Fourier-transform infrared spectroscopy (FTIR) was used to verify the protein-specific structures. Sheet structures in the surface morphology of CSPC were dominant when scanning electron microscopy (SEM) images were investigated. Thermal gravimetric analyzer (TGA) results showed that the protein concentrate exhibited excellent stability to temperature.

Combined Effects of Cold and Hot Air Drying on Physicochemical Properties of Semi-Dried Takifugu obscurus Fillets.

The physicochemical properties of semi-dried Takifugu obscurus fillets in cold air drying (CAD), hot air drying (HAD), and cold and hot air combined drying (CHACD) were analyzed based on pH, water state, lipid oxidation, protein degradation, and microstructure, using a texture analyzer, low-field nuclear magnetic resonance, thiobarbituric acid, frozen sections, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and differential scanning calorimetry. Water binding to the samples was enhanced by all three drying methods, and the immobilized water content of CHACD was between that of HAD and CAD. The pH of the semi-dried fillets was improved by CHACD. When compared to HAD and CAD, CHACD improved the springiness and chewiness of the fillets, especially cold air drying for 90 min (CAD-90), with values of 0.97 and 59.79 g, respectively. The muscle fibers were arranged compactly and clearly in CAD-90, having higher muscle toughness. CHACD reduced the drying time and degree of lipid oxidation compared to HAD and CAD. CAD better preserved protein composition, whereas HAD and CHACD promoted actin production; CHACD had a higher protein denaturation temperature (74.08-74.57 °C). CHACD results in better physicochemical properties than HAD or CAD, including shortened drying time, reduced lipid oxidation, enhanced protein stability, and denser tissue structure. These results provide a theoretical basis for selecting the appropriate drying method for T. obscurus in industrial applications.

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

BackgroundEimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear.MethodsWFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy.ResultsPurified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive.ConclusionsEnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development.Graphical

Grain Characteristics, Moisture, and Specific Peptides Produced by Ustilaginoidea virens Contribute to False Smut Disease in Rice (Oryza sativa L.).

The fungus Ustilaginoidea virens, the causative agent of false smut in rice (Oryza sativa L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing U. virens infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that U. virens produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi U. virens play a vital role in the formation of false smut.

Inhibitory effect of baicalein against glycation in HSA: an in vitro approach

Hyperglycaemia accelerates the aging process significantly. Diabetes problems can be mitigated by inhibiting glycation. To learn more about glycation and antiglycation mediated by methyl glyoxal and baicalein, we studied human serum albumin as a model protein. A Methylglyoxal (MGO) incubation period of seven days at 37 degrees Celsius induced glycation of Human Serum Albumin.s Hyperchromicity, decreased tryptophan and intrinsic fluorescence, increased AGE-specific fluorescence, and reduced mobility were all seen in glycated human serum albumin (MGO-HSA) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FT-IR) and then far ultraviolet dichroism were used to detect secondary and tertiary structural perturbations (CD). The Congo red assay (CR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) all verified the presence of amyloid-like clumps. Structure (carbonyl groups on ketoamine moieties) (CO), physiological problems including diabetes mellitus, and cardiovascular disease, etc. are linked to the structural and functional changes in glycated HSA, as proven by these studies. Communicated by Ramaswamy H. Sarma

A Mouse Monoclonal Antibody Against Human IFN-γ and its Characters

Background and Aims: A monoclonal antibody (mAb) can unambiguously identify, quantify, and purify an antigen or particular epitope at a large scale. The superiority of these antibodies lies in their specificity for the antigenic determinant. So, this study aims to prepare mouse mAb-secreting hybridoma against human gamma interferon (IFN-γ) and determine the produced antibody's characters.&#x0D; Materials and Methods: Mouse splenic B lymphocytes immunized with recombinant human IFN-γ were fused with mouse SP2/0 cells. The hybridized cells were selected by hypoxanthine-aminopterin-thymidine and hypoxanthine-thymidine media to obtain monoclonal antibody-producing hybridoma cells. Finally, indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot were used to confirm the creation of antibody-secreting hybridoma cells.&#x0D; Results: mAb against IFN-γ were produced by fusing SP2/0 mouse non-secretory myeloma cell line with the spleen cells of immunized mice. This antibody's indirect ELISA optical density was 2.055 on average, and the desired antibody bands were confirmed in SDS-PAGE compared to Septicol® (commercial antibody). Also, in the western blot, the desired antibody could bind to the antigen. IFN-γ transferred on nitrocellulose membrane. In ELISA and western blot tests, anti-mouse IgG conjugated antibodies were used; therefore, the mAb IgG isotype was taken into consideration.&#x0D; Conclusion: In this study, a mouse mAb was obtained by immunization of Balb/C mice and fusion of spleen cells of these mice with the SP2/0 cells, which can specifically bind to recombinant human IFN-γ and can be used to detect IFN-γ secretion in all types of intracellular infections, including latent tuberculosis.

Purified cellulase-mediated simultaneous sugar utilization by Bacillus albus isolated from Similipal, Odisha, India.

Among 24 isolated cellulolytic bacteria from Similipal Biosphere Reserve, the most efficient isolate was recognized as a strain of Bacillus albus. This strain of B. albus was evaluated for cellulase production and the cellulase activity was measured in submerged fermentation using substrate carboxymethyl cellulose (CMC). Different nutritional (carbon, nitrogen, and metal-ion sources) and physical variables (pH, temperature, substrate concentration, and incubation time) during the growth of B. albus were optimized to obtain maximum cellulase activity. The highest cellulase activity of 5.79 U/mL for B. albus was observed at pH 6.75, temperature 37.5°C, CMC concentration 8.5 g/L, and 42 h incubation time. Further, supplementation of glucose as a subsidiary carbon source, yeast extract, peptone as nitrogen sources, and MgSO4 and MnSO4 as metal-ion sources enhance the cellulase activity of B. albus. The purified enzyme was reported to have a molecular weight of ∼54 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A zymogram analysis evidenced the cellulase activity of the purified enzyme fractions obtained from diethylaminoethyl cellulose chromatography. The purified cellulase was reported to have an optimum pH and temperature of 7.0°C and 50°C, respectively with a capacity of retaining its 60% residual activity within pH 6.0-8.0 and temperature 30-40°C, respectively. The metal ions, K+ and Na+ were the activators, while Pb2+ and Hg2+ were the inhibitors for the purified cellulase. The purified cellulase showed Km and Vmax values of 0.38 M and 8.19 U/mL, respectively, in presence of the substrate CMC and also simultaneous consumption of both hexose and pentose sugars.

Ultrasound-assisted free radical modification on the structural and functional properties of ovalbumin-epigallocatechin gallate (EGCG) conjugates

The influence of ultrasound-assisted free radical modification on the structure and functional properties of ovalbumin-epigallocatechin gallate (OVA-EGCG) conjugates was investigated by experimental measurements and computer simulations. Compared with the traditional free radical condition, the ultrasonic-assisted processing significantly increased the conjugating efficiency of OVA and EGCG and shortened the conjugating from 24 h to 1 h without affecting the equivalent amount of EGCG conjugating. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and multi-spectroscopy analysis (Fourier transform infrared spectroscopy, intrinsic fluorescence spectroscopy, and UV spectroscopy) indicated that the covalent conjugates could be formed between OVA and EGCG. And modification in the conformation of OVA was induced by EGCG. Furthermore, molecular docking results demonstrated the possession of high-affinity EGCG binding location on OVA, supporting and clarifying the experimental results. In addition, the functional properties of OVA including emulsification (emulsifying activity and emulsion stability) and antioxidant properties (DPPH scavenging capacity and ABTS scavenging capacity) were significantly improved after conjugation with EGCG, especially in ultrasound-assisted conditions. Overall, OVA-EGCG conjugates produced by ultrasound-assisted free radical treatment could be applied as a potential emulsifier and antioxidant, thereby expanding the application of OVA as a dual-functional ingredient.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens

Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. We investigated the unexpected cross-reactivity between peanut major allergens. Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n= 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Changes in wheat protein digestibility and allergenicity: Role of Pediococcus acidilactici XZ31 and yeast during dough fermentation

Wheat flour, as the most important source of food globally, is one of the most common causative agents of food allergy. This study aimed to investigate the effects of fermentation on wheat protein digestibility and allergenicity. Protein digestibility were evaluated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and enzyme-linked immunosorbent assay. The effect of protein on intestinal permeability was investigated by Caco-2 cell monolayers. Co-culture fermentation with Pediococcus acidilactici XZ31 and yeast leads to improvement in digestibility of wheat protein compared to single strain fermentation. Fermentation leads to a decrease in albumin/globulin antigenicity and an increase in gluten R5 reactivity, with the most significant changes in the co-culture group. Digestion strengthen the decrease of protein antigenicity and counteracts the difference in antigenicity induced by fermentation between groups. However, pretreatment with P. acidilactici XZ31 reduces the amount of allergens across Caco-2 monolayer and attenuates the gluten-induced increase in permeability of Caco-2 cell monolayer by reducing actin polymerization and villous atrophy. Co-culture fermentation reduces gluten-induced cell monolayer damage to a greater extent than P. acidilactici XZ31 monoculture. These results gives valuable insight into the effects of P. acidilactici XZ31 fermentation on the allergenicity and toxicity of wheat proteins, which contribute to promoting the application of multi-strain leavening agent in hypoallergenic and gluten-free wheat products.