SDS-PAGE Research Articles

Contribution of haptoglobin phenotypic variation to the presence of hyperhomocysteinemia in type 2 diabetics with and without angiopathy.

The genetic polymorphism of haptoglobin (Hp) has been associated with several cardiovascular risk factors, but a possible relationship between Hp phenotypic variation and increased levels of homocysteine (Hcy) and cysteine (Cy) is still unknown. The objective of this study is to evaluate the relationship between the Hp polymorphism and hyperhomocysteinemia (HHcy) and hypercysteinemia (HCy) in type 2 diabetics (T2D) with and without angiopathy (AGP). A case-control study was carried out on 293 adults: Group I (GI) - 75 subjects with T2D and AGP; Group II (GII) - 75 subjects with T2D without AGP; Group III (GIII) - 143 controls. Plasma levels of Hcy, Cy and vitamin B6 were measured by high performance liquid chromatography (HPLC) and vitamins B9 and B12 determined by electrochemiluminescence (ECL). The Hp polymorphism was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peroxidase staining. The results were analyzed in SPSS®, version 26.0 with a significance of 95%. Mean Hcy concentrations were significantly lower in carriers of the Hp2-2 phenotype (6.14 µM; p = 0.046) compared to the other genotypes. The presence of Hp2-1 is associated with an approximately 3.3 times greater probability of occurrence of HHcy (p = 0.015) and 3.7 times greater probability occurrence of HCy (p = 0.021) in T2D with AGP. The presence of the Hp2-1 phenotype is associated with the predisposition of HHcy and HCy in individuals with T2D and AGP, possibly through a positive heterosis mechanism. Carriers of the Hp2-2 phenotype appear to have a greater activation of the transsulfuration pathway in the Hcy cycle and consequent protection for its accumulation.

Rheological properties and in vitro digestibility of chicken breast myofibrillar protein containing various psyllium (Plantago ovata) husk concentrations

SummaryMost dietary fibres improved rheological properties but negatively impacted digestibility. However, the effect of psyllium husk powder (PHP) on digestibility has not been yet reported. Thus, this study aimed to evaluate the rheological properties and digestibility of chicken myofibrillar protein (MP) containing different concentrations of PHP (0%, 0.5%, 1.0%, 1.5%) at 0.45 m NaCl. To assess the rheological properties of MP, viscosity, hydrophobicity, disulphide bond, SDS–PAGE, cooking yield and gel strength were evaluated. The addition of PHP enhanced water retention capacity and protein–protein interactions in the MP system. To evaluate the effect of PHP on digestibility, protein digestibility, SDS–PAGE and microstructure observations were performed. It was confirmed that the addition of PHP improved the protein digestibility of MP by measuring the differences in protein content (%). Therefore, the addition of PHP into MP gel improved not only the rheological properties but also the protein digestibility during in vitro digestion.

Comparative Study on Protein Composition and Foam Characteristics of Barley and Wheat Beer

Protein is an important component of beer, and its type, content and molecular weight directly affect the quality of beer, especially the foam quality of beer. Different brands of wheat beer and barley beer available in the market were used for this analysis. The differences in protein composition and foam performance between multi-sample barley and wheat beer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure size exclusion chromatography. Protein significantly influences beer quality, particularly its foam properties. Wheat beer (WB) has 9.52–84.10% more total protein content than barley beer (BB). The primary proteins in both beers are 6.9–20.1 kDa, with WB having 1.04 g/L more of this protein, 60.11% higher than that of BB. It is one of the main different proteins between WB and BB. WB also contains 66.67% more 20.1–32.4 kDa protein compared to BB. This is one of the main differences between WB and BB proteins. Both 6.9–20.1 kDa and 20.1–32.4 kDa proteins enhance beer viscosity and foam properties. Additionally, WB’s > 32.4 kDa protein content is 246.67% higher than BB’s, significantly improving beer hydrophobicity and foam performance. These protein differences are key factors in the superior foam quality of WB.

Sonication-Assisted Decellularization of Waste Tilapia (Oreochromis niloticus) Heads for Extracellular Matrix Extraction

Tilapia (Oreochromis niloticus), which is extensively farmed globally and ranks as the second most cultivated fish in the Philippines, generates significant amounts of waste that are often underutilized. One specific type of waste material consists of fish heads, which contain a valuable source of extracellular matrix (ECM). This study aims to evaluate the effects of sonication as a viable decellularization method for the extraction of ECM from tilapia fish heads. Particularly, two treatments were tested on the head samples: sonication-assisted decellularization (dWS) using a water bath sonicator, and decellularization without sonication (dNS), each with different contact times (5 min and 10 min). Histological analysis with H and E staining and DNA quantification revealed that sonication-assisted samples (dWS) showed a greater reduction in basophilic components and DNA content, achieving a 93.7% removal rate. These dWS samples also had the highest protein loss, retaining only 33.86% of the original protein. SDS–PAGE analysis indicated that both dWS and dNS samples maintained similar collagen structures, as evidenced by identical subunit bands. ATR–FTIR spectra confirmed the presence of collagen type I in all samples, detecting characteristic amides A, B, I, II, and III. The results revealed that varying treatments and contact times had significant effects on the physical and mechanical properties of the decellularized extracellular matrix (ECM). These findings highlight the effectiveness of sonication in the decellularization process, particularly for utilizing waste tilapia heads.

Evaluation of Protective Efficacy of Recombinant Toxoplasma gondii DDX39 Protein Vaccine against Acute and Chronic T. gondii Infection in Mice

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+ T cells rising by 21.9% and CD8+ T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response.Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15-20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

Advancing Albumin Isolation from Human Serum with Graphene Oxide and Derivatives: A Novel Approach for Clinical Applications.

This study introduces a novel, environmentally friendly albumin isolation method using graphene oxide (GO). GO selectively extracts albumin from serum samples, leveraging the unique interactions between GO's oxygen-containing functional groups and serum proteins. This method achieves high purification efficiency without the need for hazardous chemicals. Comprehensive characterization of GO and reduced graphene oxide (rGO) through techniques such as X-ray diffraction (XRD) analysis, Raman spectroscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) confirmed the structural and functional group transformations crucial for protein binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses demonstrated over 95% purity of isolated albumin, with minimal contamination from other serum proteins. The developed method, optimized for pH and incubation conditions, showcases a green, cost-effective, and simple alternative for albumin purification, promising broad applicability in biomedical research and clinical applications.

Antioxidant analysis of protein from fresh and dry leaf of Orthosiphon aristatus (Blume) Miq.

Orthosiphon aristatus (Blume) Miq. is a folklore plant consumed as brewed tea for various health benefits. The white variety of the plants leaf and stem are predominantly sold in the market in dried form, blooming as a natural herbal product. To date, no proteomics and antioxidant studies are available on the fresh and dried leaf protein extract of O. aristatus since most studies take an interest in the crude extracts of plants. Thereupon, this study focuses on the One-dimensional (1D) electrophoretic pattern of the fresh and dry leaf determined via sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, antioxidant proteins are currently being focused on for their link to controlling disease through their potential to destroy free radicals that are present in excess. Hence, an antioxidant assay was conducted for the fresh and dry leaf protein extract using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical assay and ferric reducing antioxidant power (FRAP) assay. The electrophoretic pattern obtained resulted in approximately 16 and 10 bands for the fresh and dry leaves respectively. As for the antioxidant activity, the dry leaf had higher radical scavenging activity and higher reducing power compared to the fresh leaf. The findings obtained provides insight in to the protein content and antioxidant activity found in the protein extract of fresh and dry leaves of O. aristatus. Hence, further detailed study of the protein extracts could further unveil their therapeutic potential.

Selection of antibacterial aptamers against Pseudomonas plecoglossicida and analysis on their potential binding proteins

Pseudomonas plecoglossicida is one of the main pathogens causing visceral white spot disease of the large yellow croaker (Pseudosciaena crocea). Although antibiotics are used to control P. plecoglossicida infections, the long-term and large-scale use of antibiotics can lead to the development of drug-resistant bacteria as well as environmental pollution. Therefore, it is necessary to develop novel antibacterial agents to treat P. plecoglossicida infections. In this study, we first developed a two-step-centrifugation method to isolate live and dead P. plecoglossicida cells. Subsequently, we used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screening based on inhibition rate to directly isolate antibacterial aptamers from the random library without post-processing. We isolated five antibacterial aptamers, namely B1, B2, B4, B8, and B109, which showed good inhibitory effects against P. plecoglossicida. Among these, B4 showed the highest inhibitory effect (62.40 ± 4.17 %). Further analysis revealed no positive correlation between the inhibitory effect of aptamers and their affinities with the target bacterium. The B4- and B109-binding proteins were isolated from P. plecoglossicida by magnetic separation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry analysis revealed that the 26 kDa ribosomal protein L2 was the probable B4-binding protein, while the 26 kDa ribosomal protein S3 and 75 kDa ribosomal protein S1 or succinate dehydrogenase flavoprotein subunit were the probable B109-binding proteins. Structural and subcellular localization analyses of these potential B4- and B109-binding proteins were also conducted. Our findings suggest that the inhibitory activity of the antibacterial aptamers against P. plecoglossicida may be mediated via their interaction with the ribosomal proteins, which can interfere with the protein synthesis process in the bacterium, affecting its growth. These findings provide the scientific basis for the development of functional antibacterial aptamers and the elucidation of their mechanisms of action.

Effect of pH-shifting and ultrasound on soy/potato protein structure and gelation

The substitution of meat proteins with plant-based proteins from various sources is often motivated by nutritional considerations. However, the inherent limited solubility of plant proteins, which results in suboptimal techno-functional properties, remains a persistent challenge in food formulation. The purpose of this study was to investigate the impact of pH-shifting (P), high-intensity ultrasound (U), and ultrasound-assisted pH-shifting on the structure and solubility of soy protein isolate (SPI), potato protein isolate (PPI), and the soy/potato protein complex (SPI/PPI complex). Additionally, a detailed examination of the gel properties of SPI, PPI, and the SPI/PPI complex was conducted to assess the changes that occurred before and after modification. The results of particle size, zeta potential, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), free sulfhydryl (R-SH), surface hydrophobicity (H0), Fourier transform infrared spectrum (FTIR) and fluorescence intensity showed that P and U treatment promoted the unfolding of proteins structures and decreased particle size, and augmented protein-water interactions, leading to enhanced solubility. The pH-shift and ultrasound (PU) treatment further amplified these structural alterations in proteins. Consequently, the gelling characteristics of the modified proteins were substantially improved, with notable increases in hardness, elasticity, and water-holding capacity (WHC). The findings facilitate developing more targeted plant protein-based gel products that specific techno-functional attributes, concurrently allowing for modifications to their nutritional profiles.

Comprehensive Analysis of Phycoerythrin 545 Stability and the Apoptotic Impact of Its Degradation Products on HT29 Cells.

This study investigates the properties and potential applications of phycoerythrin 545, a naturally occurring light-harvesting pigment protein from Rhodomonas salina. Phycoerythrin 545, characterized by its bright red color and maximum absorption wavelength at 545 nm, was extracted using freeze-thawing methods, further purified, and analyzed using chromatographic, spectroscopic techniques, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phycoerythrin 545 consists of two subunits, primarily α and β, but lacks the γ subunit, and is stable at 4 °C within a pH range of 3-10. To further characterize it, its susceptibility to degradation by trypsin was assessed. The biological activity of phycoerythrin 545 and its degradation products were investigated in HT29 human colon cancer cells. The results showed that the degradation products, particularly those within 3-10 kDa, significantly decreased the viability of HT29 cells by inducing apoptosis. Mechanistic studies indicated these effects were mediated through the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases and MAPK/c-Jun N-terminal Kinase signaling pathways and involved the regulation of key apoptotic proteins such as p53, Bim, Bad, Bak, and Bax, leading to the activation of the Caspase-3 apoptotic pathway. These findings contribute to understanding the structural and functional properties of phycoerythrin 545, laying a foundation for its exploration in food industry applications and cancer therapy supplementation.

Simultaneous and sequential combination of techniques for the sustainable and extensive extraction of proteins and polyphenols from malt rootlets

The emergence of tecnhiques such as microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), ultrasounds-assisted extraction (UAE), and pressurized liquid extraction (PLE) has brough many benefits but their single use does not guarantee the hollistic extraction of target compounds from complex samples. Taking into account their different principles and that one technique may favour the extraction of certain compounds over others, this work proposes the combination of these techniques for improving extraction yields in the recovery of proteins, as example of large molecule, and phenolic compounds, as example of small molecule, from a recalcitrant sample. No single extraction technique recovered all proteins or phenolic compounds, even after a proper optimization. Among them, PLE was the most advantageous, enabling the extraction of 67 % proteins and 1.20 g gallic acid equivalent (GAE)/100 g sample. Analysis of samples by scanning electron microscopy (SEM) and separation of extracts by reversed-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that PLE showed the most distinct efficiency among EAE, MAE, and UAE, whereas the latter three exhibited more similar results. Based in these results, ten different combinations (sequential and/or simultaneous) of these techniques were proposed. PLE in combination with another technique showed a great potential for the extraction of both large and small molecules. Particularly, the combination of EAE and PLE recovered 100 % of proteins and more than three times the phenolic compounds extracted by other methods reported in the literature, even revealing significant synergistic effects.

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Generation of Anti-Epidermal Growth Factor Receptor-2 (HER2) Immunoliposomes Using Microbial Transglutaminase (mTG)-Mediated Site-Specific Conjugated Antibodies.

Nanocarriers have found their interests in many fields including drug delivery and labeling of cells with the aim to target and eradicate tumor cells. One of the approaches to specifically address nanocarriers, such as liposomes, to their target is to attach antibodies of interest to their surface. To date, the development of immunoliposomes has been widely explored but has mainly involved chemical and unspecific reactions that could impair antibody stability, integrity, and orientation, thus reducing optimized immunoliposomes generation. In this study, we report the use of the patented COVISOLINK technology and the strain-promoted alkyne-azide cycloaddition (SPAAC) to generate immunoliposomes that target HER2 positive breast cancer with Trastuzumab as the antibody to be coupled. The efficacy of our two-step functionalization strategy and the successful specific coupling of the antibodies were validated by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC), which allowed a precise quantification of antibodies conjugated to liposomes and confirmed by cryo-TEM and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. We also demonstrate by flow cytometry and epifluorescence microscopy that the produced anti-HER2 immunoliposomes were able to interact specifically with their target cells (SK-BR-3) while remaining negative with cells that express HER2 at a low level (MDA-MB-231). Hence, for the first time, our COVISOLINK strategy using microbial transglutaminase (mTG) enables the preparation and production of well-characterized immunoliposomes that could be used in different applications, including therapies.

Modifying physicochemical properties, rheology, and creaming stability of milk fat globule and membrane through ultrasound treatment

The healthy benefits of milk fat globules and membrane (MFGs/MFGM) ingredients are increasingly recognized in the dairy industry. In this research, we examined the effects of ultrasonic treatment on the physicochemical and rheological properties, as well as the emulsions stability of MFGs/MFGM derived from bovine raw milk. Fresh milk was subjected to sonication at frequencies of 20 kHz and 40 kHz, either individually or simultaneously, for durations of 5 min or 15 min, using work/rest cycles of 5 s on and 3 s off. Bovine milk, without any treatment, served as the control. Regardless of the intensity difference, ultrasonic treatment for 5 min resulted in more pronounced changes in the regions of Amide Ⅱ (1600–1500 cm−1), Amide Ⅲ (1500–1200 cm−1), and fingerprint region (1200–1900 cm−1) compared to both the 15 min treatments and control MFGs/MFGM. Principal component analysis (PCA) conducted on the entire spectra, as well as in the regions of Amide Ⅰ, Amide Ⅱ, and the fingerprint spectra, clustered the 5 min treatment distinctly from the control and MFGs/MFGM ultrasonically treated for 15 min. MFGs/MFGM samples following 20 kHz and 40 kHz synchronous treatment for 15 min exhibited lower absorbance bands at 1727–1726 cm−1, whereas a higher content at 1740 cm−1 was observed compared to control MFGs/MFGM. Additionally, a more significant reduction in the intramolecular β-sheet content in 20 + 40 kHz/ 15 min treatment was observed. According to the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns, a diminished intensity of Periodic Acid Schiff 6/7 (PAS 6/7) bands was observed across all the MFGs/MFGM. Ultrasonic treatment retained more caseins while reducing the β –LG levels compared to the controls, enhancing the stability of MFGs/MFGM, except in MFGs/ MFGM subjected to 20 and 40 kHz simultaneously treated for 15 min. The irregular sphericity of fat globules was noted particularly in MFGs/MFGM treated at 20 kHz independently or in combination with 40 kHz for 15 min. According to the confocal laser scanning microscopy (CLSM), ultrasonic treatment facilitated the binding of caseins or whey proteins to the MFGs surface and induced flocculation of membrane proteins. Hierarchical cluster analysis (HCA) heat map further underscored the impact of ultrasonic treatments on the structural and compositional changes, as well as rheology and emulsions stability, of MFGs/MFGM.

Changes in the structural, aggregation behavior and gel properties of pork myofibrillar protein induced by theaflavins

This study explores the effect of different theaflavins (TFs) concentrations (0, 100, 300, 600 and 900 mg/L) on the structure, aggregation behavior and gelation properties of pork myofibrillar protein (MP). The protein structure and aggregation behavior were characterized by free sulfhydryl groups, surface hydrophobicity, fluorescence emission spectra, particle size and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel properties of samples were characterized by gel strength, cooking loss, microstructure and gel supernatant SDS-PAGE. The results showed a significant decrease in free thiol content with increasing TFs concentration, suggesting thiol-quinone covalent interaction between TFs and thiol group of MP. Intrinsic fluorescence spectroscopy confirmed a static quenching between TFs and MP. And TFs reduced the particle size of MP suspension and caused no protein aggregation bond in SDS-PAGE. For gel properties, TFs caused a decrease of gel strength from 96.77 g to 21.91 g and an increase in cooking loss from 40.34 % to 71.15 %. The bond of protein aggregates in gel supernatants SDS-PAGE revealed that some protein aggregates formed by disulfide bonding were not involve in gel formation with TFs addition. In conclusion, TFs cause thiol loss of MP and impaired MP gelling ability by interfering with disulfide bond formation during gelation.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.

Mechanistic insights into the interaction of Lycium barbarum polysaccharide with whey protein isolate: Functional and structural characterization

Protein-polysaccharide interactions are crucial for food system structure and stability. This study investigates the interaction of Lycium barbarum polysaccharide (LBP) at 0–2.00 % concentrations with whey protein isolate (WPI), focusing on functionality and structural changes. LBP covalently grafted onto WPI, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), forming WPI-LBP complexes with a maximum degree of grafting (DG) of 44.58 % at 2.00 % LBP. This grafting reduced WPI's surface hydrophobicity (H0) and improved solubility, emulsifying properties, and digestibility under certain conditions, with optimal antioxidant activity at 1.00 % LBP. Multispectral analysis and microscopy showed LBP grafting alters WPI's secondary, tertiary, crystalline, and micro/nanostructures. The comprehensive analysis indicates that the interaction between LBP and WPI involves covalent bonding, hydrogen bonding, hydrophobic interactions, and electrostatic forces, as supported by zeta potential and chemical forces results. These findings suggest LBP-protein complexes as promising food materials for enhancing functionality and stability in the food industry.

Insights into the Structural and Thermal Properties of Pepsin-Soluble Collagen from Daggertooth pike conger (Muraenesox cinereus)

Pepsin soluble collagen (PSC) is a type-1 collagen which is abundant in skin. The pepsin soluble collagen was successfully extracted from the skin of Daggertooth pike conger (Muraenesox cinereus) by using the conventional method of salt precipitation followed by dialysis. Pepsin-soluble collagen showed a yield of 10.12% on wet matter basis and 25.5% on dry matter basis. The triple helical structure of collagen was confirmed by Fourier Transform Infrared Spectroscopy (FTIR) and UV-Visible spectrophotometry. Isolated PSC was confirmed as type 1 collagen by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). FTIR spectra of PSC suggested that the secondary structure of the triple helical collagen was intact even after pepsin digestion. Pepsin-soluble collagen was soluble in acidic pH with maximum solubility at pH 4. The solubility was found to be decreased with an increase in the concentration of sodium chloride with a minimum solubility at 4%. The present PSC isolate from eel fish exhibited comparatively high thermal stability of 36 ͦ C. Morphological analysis by Scanning electron microscopy (SEM) revealed a porous structure and comparatively high thermal stability makes it a promising biomaterial.

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Maillard conjugates of whey protein and prebiotic dietary fibers: Emulsion properties, encapsulation efficiency and chemical stability after in vitro gastrointestinal digestion

The incorporation of dietary fibers into food formulations is a promising approach to developing new functional ingredients. In this study, we investigated the formation of Maillard conjugates using whey protein concentrate (WPC) combined with fructooligosaccharides (FOS) and xylooligosaccharides (XOS). These conjugates, innovatively utilized as emulsifiers for annatto seed oil, were subjected to in vitro gastrointestinal digestion to assess their chemical stability and effectiveness as encapsulating systems. Our study confirmed the occurrence of the Maillard reaction through different methods, including grafting degree (GD) assessment and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate molecular weight variations in the proteins. The key innovation of our research lies in demonstrating the conjugation process through carbohydrate mass balance analysis using High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). The GD for the WPC-XOS and WPC-FOS was 46 ± 3 % and 56 ± 3 %, respectively. Our findings suggest that sugar quantification could serve as a novel approach to confirming the formation of Maillard conjugates. The emulsions stabilized by these conjugates exhibited larger oil droplet sizes compared to those stabilized by WPC alone yet maintained high encapsulation efficiency (≥ 90 %). Notably, the WPC-XOS emulsion demonstrated kinetic stability after 30 days of storage at 4°C, while the WPC-FOS emulsion showed signs of creaming after just 16 days. In vitro gastrointestinal digestion revealed that while the conjugates were susceptible to enzymatic degradation, the FOS and XOS sugars displayed gastrointestinal resistance of up to 82 % and 83 %, respectively. This study highlights the potential of WPC-XOS conjugates not only as effective emulsifiers but also as functional ingredients that enhance the nutritional and antioxidant properties of food products, presenting a novel approach to functional food development.