Bradford Assay Research Articles

Evaluating the efficacy of protein quantification methods on membrane proteins.

Protein quantification is an important tool for a wide range of biological applications. The most common methods include the Lowry, bicinchoninic acid (BCA) and Coomassie Bradford assays. Despite their wide applicability, the mechanisms of action imply that these methods may not be ideal for large transmembrane proteins due to the proteins' integration in the plasma membrane. Here, we investigate this problem by assessing the efficacy and applicability of these three common protein quantification methods on a candidate transmembrane protein: Na, K-ATPase (NKA). We compared these methods with an ELISA, which we newly developed and describe here for the quantification of NKA. The use of a relative standard curve allows this ELISA to be easily adapted to other proteins and across the animal kingdom. Our results revealed that the three conventional methods significantly overestimate the concentration of NKA compared with the ELISA. This is due to the samples containing a heterogeneous mix of proteins, including a significant amount of non-target proteins. Further, by applying the protein concentrations determined by the different methods to in vitro assays, we found that variation in the resulting data was consistently low when the assay reactions were prepared based on concentrations determined from the ELISA.

Impact of Nano-Pomegranate Seed Oil on the Expression of TLR2 and TLR4 Genes in A549 Cells Sensitized with Alternaria alternata Cellular Extract

Background: Allergies impact nearly 30% of the world's population, with fungi being remarkable contributors to allergic sensitivity. Exposure to fungi allergens can trigger allergic reactions and severe asthma has been linked to hypersensitivity to fungi such as Aspergillus and Alternaria spp. Alternaria alternata, a common allergen in respiratory allergic diseases, releases proteases that initiate Th2 responses, causing inflammatory cytokine production. Given the anti-inflammatory properties of Pomegranate Seed Oil (PSO) and the potential of Nano-emulsions (NEs) to enhance drug delivery, this study investigates the impact of PSO-loaded NEs on TLR2 and TLR4 gene expression in A549 cells sensitized with A. alternata extract (ALT). Methods: A. alternata (ATCC 6663) was cultured and processed to obtain a cytosolic extract, with protein content measured using the Bradford method. Using a Soxhlet apparatus, PSO was extracted from cleaned, dried seeds and analyzed by gas chromatography. Alginate nanospheres containing PSO were prepared through a modified water-in-oil emulsification method and characterized for particle size, polydispersity index, and zeta potential. A549 cells were cultured and treated with various ALT, PSO, and PSO-loaded NEs combinations. Following treatment, RNA was extracted, and real-time RT- PCR was conducted to analyze TLR2 and TLR4 gene expression. Results: All treatment groups showed an increase in TLR2 gene expression compared to the control, with the ALT combined with PSO (P+ALT) causing the highest increase at 4.82-fold. Free PSO (P) and free NEs (NP) resulted in 3.92-fold and 2.93-fold increases, respectively, while the ALT and PSO- loaded NEs (NP+ALT) led to 2.26-fold and 2.50-fold increases. For TLR4 gene expression, the ALT treatment increased expression by 2.29-fold, but treatments containing PSO (P, P+ALT, NP+ALT) reduced TLR4 expression, with P+ALT and NP+ALT causing 0.45-fold and 0.61-fold decreases. Conclusion: The study confirms that herbal extracts like PSO selectively upregulate TLR2 and downregulate TLR4, suggesting targeted therapeutic potential in inflammation and immune modulation. PSO-loaded NEs demonstrated superior anti-inflammatory effects, supporting their development for treating inflammatory diseases and warranting further research into their molecular mechanisms and therapeutic applications.

Biological importance and environmental quality of the Laguna Santa Ana in Zacatecas, Mexico

Objective: To determine the environmental quality and diversity of the waterfowl species of the Laguna Santa Ana, located in the municipality of Fresnillo, Zacatecas. Design/Methodology/Approach: A physicochemical analysis of the water was carried out to determine its organic matter pollution. In addition, the Bradford method was applied to establish protein levels, and an optical emission spectrometry was conducted to detect heavy metals. The Shannon-Weiner diversity index (H’) was carried out to identify and count the populations of the lagoon. Results: The physicochemical analysis of water recorded 55 and 230.6 mg/L BOD5 and COD, respectively. High protein levels (0.27-2.95 mL/mL) indicated organic pollution and high arsenic levels. The Shannon-Weiner index (2.7) recorded a high waterfowl diversity. Study Limitations/Implications: Biological abundance was not determined, as a result of the sample size. The changes regarding waterfowl diversity may be the consequence of the seasonal conditions. Findings/Conclusions: The Laguna de Santa Ana has a high waterfowl diversity, including species of international interest; however, organic pollution has caused a a significant environmental deterioration.

Maternal BMI During Lactation Is Associated with Major Protein Compositions in Early Mature Milk.

The present study identified multiple proteins in early mature milk and explored the correlation between protein compositions in HM and maternal BMI during lactation. A total of 70 mothers giving birth to single-term infants from four representative cites were enrolled in this research. Milk samples were collected between 9 and 11 a.m. to avoid the influence of circadian rhythms. The concentration of total protein in the milk samples was determined using the Bradford method, and the concentrations of α-lactalbumin, lactoferrin, osteopontin, αs-1 casein, β-casein, and κ-casein, butyrophilin, periodic acid Schiff 6/7, fatty acid-binding protein, and xanthine oxidoreductase in the milk samples were measured through a previously published method using ultra-performance liquid chromatography coupled with mass spectrometry. A semi-structured questionnaire investigation and body measurements were carried out by trained investigators to collect the information of subjects. In the univariate models, the concentrations of TP (r = 0.306), α-La (r = 0.260), LF (r = 0.371), OPN (r = 0.286), and αS1-CN (r = 0.324) were all positively and significantly correlated with maternal BMI. In the models' adjusted covariates, the concentrations of TP (Lg β = 7.4 × 10-3), LF (Lg β = 19.2 × 10-3), αS1-CN (Lg β = 8.2 × 10-3) and the proportion of LF (β = 0.20%) were positively correlated with continuous maternal BMI changes. TP concentrations in the HM of obese mothers were higher than in the other three groups (Lg β: 66.7 × 10-3~140.5 × 10-3), α-La concentrations were higher than in the underweight and normal groups (Lg β: 94.4 × 10-3~145.7 × 10-3), and OPN concentrations were higher than in the overweight groups (Lg β = 103.6 × 10-3). The concentrations of LF (Lg β: -298.2 × 10-3~-191.0 × 10-3), OPN (Lg β: -248.9 × 10-3~-145.3 × 10-3), and αS1-CN (Lg β: -160.7 × 10-3~-108.3 × 10-3) in the HM of underweight mothers were lower than those in the other three groups. β-CN concentrations were lower than normal (Lg β = -125.1 × 10-3) and obese groups (Lg β = -165.7 × 10-3), κ-CN concentrations were lower than the overweight (Lg β = -132.5 × 10-3) and obese groups (Lg β = -147.9 × 10-3), and the proportion of LF was lower than that of the overweight (β = -2.80%) and obese groups (β = -2.52%). The proportion of LF in normal mothers was lower than that in the overweight group (β = -1.15%). No statistically significant associations between four MFGM proteins and maternal BMI were determined as the equation models could not be fitted (p for F-test < 0.05). Obese mothers had higher concentrations of multiple protein components than other groups, while underweight mothers had lower concentrations. The association between BMI and protein compositions may be more pronounced for certain protein types.

Isolation, identification and optimization for tyrosinase production by banana peel waste for industrial application

The study aimed to isolate, purify and optimise tyrosinase from banana peel waste for industrial use. Tyrosinase was extracted from banana peel using phosphate buffer. Purification initiated with centrifugation followed by ammonium sulphate precipitation. Further purification and characterisation was done through gel filtration. Bradford assay was used to determine the protein concentration. Enzyme activity of each sample was measured using tyrosinase activity assay. The pH and temperature optimisation of tyrosinase were also obtained. Results indicated that fractions obtained through ammonium sulphate precipitation at 70% saturation showed more activity than that of crude extract and 35% saturated fractions. Gel filtration column results showed that fraction number 17 &18 have maximum activity. Tyrosinase showed maximum activity at pH 7.0; 37 °C. Comparison between industrially purified and lab-isolated enzyme showed that both have similar optimum pH and temperature. It can be concluded that banana peel can be a source for synthesis of tyrosinase.

Surface Metabolite Amino Acids and Collagen Profiling in Refrigerated Tilapia Fish (Oreochromis niloticus): Implications for Identification and Quality Assessment

Nile tilapia (Oreochromis niloticus) by-products, including the scales, skin, and mucus, are rich sources of protein with a balanced amino acid profile and high collagen content. Cold storage at 4 ℃ is commonly used to maintain the freshness of tilapia fish in retail settings. This study aims to investigate the changes in surface metabolites and collagen content in tilapia fish during cold storage at 4 ℃. Fresh tilapia fish was stored at 4 ℃ for 7 days to monitor physicochemical and metabolite changes. Fish collagen content was extracted using acetic acid and pepsin, pH values were measured using a calibrated pH meter, protein content was determined via the Bradford method, and creatine and phenylalanine levels were assessed using High-Performance Liquid Chromatography (HPLC). Analysis was conducted on days 0, 3, and 7 of cold storage. Significant reductions (p&lt;0.05) in collagen content from fish scales were observed on the 3rd day of storage, declining from 72.60 ± 12.40% to 30.17 ± 17.62%. pH levels of fish scales and mucus showed a slight alkaline shift, while the skin turned acidic due to bacterial and enzymatic activities. Protein content in the scales, skin, and mucus showed a substantial loss exceeding 50% after 7 days of cold storage. Changes in creatine and phenylalanine concentrations in the mucus further indicated a decline in tilapia fish freshness due to biochemical reactions post-mortem, compromising overall quality. In conclusion, the duration of cold storage significantly affects the composition of tilapia fish scales, skin, and mucus, with a 7-day storage period identified as a suitable freshness indicator in compliance with FDA guidelines permitting fresh fish to be stored at 4 °C for up to two days.

Total protein concentration and stability of Amb a 1 in glycerinated ragweed sublingual immunotherapy stored at room temperature and refrigerated cold temperature.

Few studies have investigated optimal storage conditions or expiration dates for sublingual immunotherapy (SLIT) formulations prepared from glycerinated allergen extracts. The objective of this study was to compare concentrations of short ragweed major allergen (Amb a 1) and total protein in SLIT formulations stored at two different temperatures. It was hypothesised that protein concentrations would show greater decline over time in a formulation stored at room temperature (RT) than in one stored under refrigeration. Two SLIT samples containing equal volumes of 20,000 PNU Amb a 1 extract were prepared and stored at refrigerated cold (CT) (2-8°C) or RT (20-24°C) for 140 days. Changes in total protein and major allergen concentration and composition were measured by Bradford assay, two-site enzyme-linked immunosorbent assay and SDS-PAGE. Presence of Amb a 1 was confirmed with Western immunoblot. Data were analysed using an analysis of covariance, with p < 0.05 considered significant. SDS-PAGE showed compositional changes in a ~26-30 kDa protein band under RT and not CT storage. The Amb a 1 concentration of the RT SLIT sample declined significantly over time, compared to that of the CT SLIT sample (F(1,8) = 47.69, p < 0.0001). There was no significant difference in total protein concentration over time between groups (F(1,8) = 1.79, p = 0.22). These results demonstrate that storage of glycerinated SLIT formulations in refrigerated CT preserved the highest concentration of the specific allergen Amb a 1, suggesting that SLIT formulations containing short ragweed should be stored under refrigeration.

Steroid-based hydrazones as anabolic agents: Design, synthesis, computational studies, and biological evaluation in human skeletal muscle cell line

Hydrazone derivatives were synthesized in a three-step process starting from diosgenin and then tested for their anabolic activity. Through in silico analysis, we identified proteins associated with muscle growth to elucidate the plausible mechanisms and interactions between these compounds and the active site amino acids, thereby providing a rationale for the observed biological results. The results of the in vitro assay on the human skeletal muscle cell line showed an increase in the number of viable cells, which was determined using increasing concentrations of each compound through the XTT assay. Additionally, the increase in protein concentration was measured using the Bradford method. The experimental evaluation revealed that all compounds have differences in increased protein/cell percentage compared to the control and testosterone, which suggests a hyperplastic/hypertrophic effect with a normal morphology.

Common structural features in some of the sequentially distant neurotransmitter transporters N-termini

The N-terminal regions of SLC6 transporters are sequentially unrelated, and the majority of such transporters contain only relatively short peptide N-terminal extensions. Currently, it is not clear if a diversity of N-terminal sequences represents diverse functions among the transporters or if there are common functions hidden behind similar, as yet unidentified, structures. Using alignment of amino acid sequences with the hydropathy plot, disorder prediction, and calpain recognition sites, we show that common structural features among the N-termini of some transporters might exist.We previously showed that polymeric neurotransmitter transporter N-termini exhibit very similar profiles of dynamic, time-dependent 465-595-350-750 nm absorbance metachromasia in the Bradford assay. Here we report that under certain mild denaturing conditions, filamentous aggregation of glutathione S-transferase (GST) protein results in similar near-infrared metachromasia. This effect was eliminated by further GST protein denaturation and solubilization. The results suggest that aggregation of partially denatured GST stabilizes Coomassie dye docking sites, producing a near-infrared absorbance shift similar to that observed in the polymeric unstructured N-termini of transporters.

Antifungal Efficacy of Ultrashort β-Peptides against Candida Species: Mechanistic Understanding and Therapeutic Implications.

Candidiasis, a condition spurred by the unchecked proliferation of Candida species, poses a formidable global health threat, particularly in immunocompromised individuals. The emergence of drug-resistant strains complicates management strategies, necessitating novel therapeutic avenues. Antimicrobial peptides (AMPs) have garnered attention for their potent antifungal properties and broad-spectrum activity against Candida species. This study assessed the antifungal effectiveness of ultrashort β-peptides against Candida strains, with a specific focus on peptide P3 (LAU-β3,3-Pip-β2,2-Ac6c-PEA). Our findings showed P3's remarkable fungistatic and fungicidal activities against Candida albicans, exhibiting an MIC of 4 μg/mL, comparable to those of standard antifungal drugs. The MIC value remained unchanged in the presence of ADC and BSA, indicating that serum albumin does not diminish the activity of P3. P3 demonstrates synergistic effects when combined with Fluconazole (FLU), Itraconazole (ITR), and Nystatin (NYS) to the extent that it becomes effective at 0.125, 0.125, and 0.03125 μg/mL, respectively. Concentration versus time-kill kinetics showed its time-dependent activity up to the first 12 h against C. albicans, and later concentration also played a role; indeed, at 24 h the whole culture was sterilized at 8× MIC. Post-antifungal effect assays confirmed prolonged suppression of pathogen growth after the removal of P3 from the media for significant durations. More importantly, P3 inhibits hyphae formation and biofilm development of Candida, outperforming Fluconazole with respect to these properties. Mechanistic insights display P3's potential to disrupt fungal cell membrane integrity and dose-dependent inhibition of ergosterol biosynthesis, essential for fungal cell wall integrity. Using the Bradford assay, it was observed that extracellular protein concentrations increased with higher doses of the compound, thereby validating the effect of P3 on membrane integrity. A comparative gene analysis using RT-PCR showed that P3 downregulates ERG3, ERG11, and HWP1, which are crucial for the survival and pathogenicity of C. albicans. The impact of P3 on ERG11 and ERG3 is more effective than that of Fluconazole. Molecular docking studies revealed strong binding of P3 to various isoforms of lanosterol 14-α-demethylase, a key enzyme in ergosterol synthesis. Furthermore, molecular dynamic simulations validated the stability of the most promising docking complex. Overall, our findings underscore P3's potential as a leading candidate for the development of innovative antifungal therapies, warranting further investigation and optimization.

Effects of Bacteriocin Extracted from Lactobacillus plantarum on Treatment-Resistant Escherichia coli Bacteria Isolated from Humans and Livestock

Background: Lactic acid bacteria (LAB) are among the most common probiotics and play a significant role in promoting health within the human intestinal microbiota. Their protective functions in the intestine include producing antimicrobial compounds, regulating the balance of the intestinal microbiome, and more. Objectives: This study aims to investigate the antimicrobial effects of bacteriocin extracted from Lactobacillus plantarum (L. plantarum) on Escherichia coli (E. coli) strains that cause treatment-resistant urinary tract infections (UTIs) in humans and mastitis in livestock. Methods: Twenty E. coli isolates were obtained from patients with UTIs and cows with mastitis. After culturing the samples on Eosin-methylene blue, McConkey agar, and blood agar, the strains were identified using biochemical tests. Bacteriocin produced by L. plantarum ATCC8014 was extracted, and its concentration was measured using the Bradford method. The antimicrobial effect of the bacteriocin was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, the bacteriocin's antimicrobial activity was evaluated through three repetitions of disk diffusion and agar well diffusion methods. Results: The findings revealed that the MIC and MBC concentrations for all human and animal strains were 13.38 μg/mL and 26.76 μg/mL, respectively. Notably, two animal samples (No. 3 and No. 10) and one human sample (No. 6) showed lower MIC and MBC concentrations compared to the other samples and the standard E. coli sample. However, all strains were resistant to bacteriocin in the disk diffusion and agar well diffusion methods. Conclusions: The data indicate a high risk of increasing treatment-resistant E. coli strains in UTIs and livestock, which could pose a significant threat to public and animal health. Therefore, it is essential to further investigate the effects of different bacteriocins on these resistant strains, in conjunction with antibiotic therapies.

Is there an association between salivary immune and microbial profile with dental health in systematically healthy children?

ObjectiveThis study aimed to characterize the inflammatory profile of systemically healthy children’s saliva and its association with clinical diagnoses of caries and gingival inflammation.Materials and methodsUnstimulated saliva was collected from 100 children before clinical dental examinations. The saliva samples were analyzed for total protein and specific inflammatory cytokines (IL-10, IL-8, IL-6, and TNFα) with Bradford and ELISA assays, respectively. Salivary bacteria were quantified using a quantitative real-time polymerase chain assay. The salivary values were then correlated with age, DMFT index, plaque index (PI), and gingival index (GI).ResultsThe mean age of the cohort was 8.08 ± 0.23 years with 49% females, the mean DMF of the cohort was 2.64 ± 0.31, the mean GI was 0.51 ± 0.06, and the mean PI was 1.33 ± 0.07. Significant correlations were found between PI with DMFT and GI. Children with DMFT > 2 had significantly higher levels of IL-8 compared with children with DMFT ≤ 2. IL-6 and TNFα were significantly higher among children with PI > 1 than among children with PI ≤ 1.ConclusionsSalivary cytokine were found to be associate with clinical parameters as DMFT and PI, thus may be a potential tool that reflects dental health status.Clinical relevanceThe presence of salivary cytokines in children may reflect evaluation of dental caries and oral inflammation.

Comparative analysis of different methods for protein quantification in donated human milk.

Human milk is the best option for feeding newborns, especially premature infants. In the absence of breast milk, milk from a human milk bank can be a suitable alternative. However, the nutritional content of human milk may be insufficient to meet these high requirements and milk fortification is needed. To facilitate the implementation of simpler and faster analyzers in neonatal healthcare facilities, this study focuses on the concordance analysis of two different analyzers, one based on mid-infrared and the other on ultrasound, in comparison to the Bradford method for determining protein concentration in human milk. Mature milk samples from donor mothers were collected and pasteurized at the Human Milk Bank of Barcelona and protein quantification was performed using mid-infrared (MIRIS-HMA), ultrasound (MilkoScope Julie27), and the classical Bradford reference methods. The intraclass correlation coefficient (ICC) with 95% confidence interval and Bland-Altman plots were used to assess the agreement between methods. The mean protein concentration of 142 milk samples calculated using MIRIS-HMA, MilkoScope, and the Bradford assay were 1.38, 1.15, and 1.19 g/100 ml, respectively. The ICC was 0.70 for MIRIS-HMA vs. Bradford and 0.37 for MilkoScope vs. Bradford. MIRIS-HMA obtained a better agreement with the Bradford technique and is a promising method for developing new devices based on MIR transmission spectroscopy principles. This study confirms how MIRIS-HMA can be used to accurately calculate the protein concentration of human milk.

Long-term effects of transition milk feeding on feed intake, growth performance, feeding behavior, and oxidative status of Holstein calves

This study investigated the long-term effects of feeding 5-d transition milk (TRANS) compared with milk replacer (MR) on parameters, including intake, growth, feeding behavior and oxidative stress. Fifty Holstein calves (30 females and 20 males) were fed either 12 L/day TRANS or MR for the first 5 d after an initial colostrum feeding of 3.5 L. Thereafter, all calves were fed with 12 L of MR/d (140 g/L) and were gradually weaned starting in wk 8 until wk 14. Throughout the 14 wk the calves had unrestricted access to concentrate (up to 9.8 kg/calf/day), hay, and water. After weaning all heifers were fed a total mixed ration for young cows. Oxidative status was assessed in blood samples from birth to first insemination. Parameters assessed included the ferric reducing ability of plasma (FRAP) for antioxidant capacity and the concentration of reactive oxygen metabolites by the dROM (detection of reactive oxygen metabolites) assay. In addition, the activity of glutathione peroxidase (GSH-Px), oxidative damage in the form of lipid peroxidation as thiobarbituric acid reactive substances (TBARS) and as advanced oxidation protein products (AOPP) were measured. An oxidative stress index (OSi) was calculated: dROM/FRAP x 100. Total protein (TP) concentration was also quantified via the Bradford assay. The only significant difference in feeding behavior between the 2 treatment groups was a higher concentrate intake by the TRANS calves during the weaning phase. Body weight and ADG did not differ significantly between the TRANS and MR groups. TRANS calves showed a trend for fewer cases of health disorders. Markers of oxidative status, including TBARS, AOPP, GSHPx, FRAP and ROM, showed no treatment effects but varied significantly over time. Of note, the oxidative stress index as ratio between pro- and antioxidants in both groups peaked during weaning and then returned to baseline, suggesting an effective response to this transition phase Overall, the results indicate that feeding TRANS during the first 5 d of life had no long-term effect on the parameters studied as compared with MR feeding under the present rearing conditions. These results provide insight into the changes of oxidative status with age and confirm that the relatively high milk feeding level, slow and late weaning enables calves to adapt well to solely solid feed.

Evaluation of antioxidant status of lens epithelial cells in cataract patients.

The main factor that causes cataracts is the increased oxidative stress and imbalance of an antioxidant defense mechanism, which leads to significant changes in the lens microarchitecture. Senile cataract is the most common type of acquired cataracts due to aging. We carried out a case-control study in the biochemistry department to examine the antioxidant status (catalase and total antioxidant capacity [TAC]) and lipid peroxidation marker, that is, malondialdehyde (MDA) in human lens epithelial cells (HLECs) of different grades of senile cortical, nuclear, and posterior subcapsular cataracts. We collected 150 samples from patients aged 50-90 years. These included 50 samples of cortical cataracts, 50 of nuclear cataracts, and 50 samples of posterior subcapsular cataracts. We measured catalase activity by the Beer method, TAC by the Benzie and Strain method, and protein by the Bradford method. We also estimated TAC in the aqueous extract of HLECs by the ferric reducing ability of plasma (FRAP) method and MDA by the thiobarbituric acid assay method. The results of this study showed that the level of catalase enzyme was higher in the first grade of nuclear, posterior subcapsular, and cortical cataracts than in other grades. This suggests that the catalase enzyme activity drops sharply in the second and third grades of these types of cataracts. The same pattern was observed for TAC, which was higher in the first grade of nuclear, posterior subcapsular, and cortical cataracts than in other grades. There were significant differences between catalase and TAC in different grades of cataracts, indicating that as the grading increases, both catalase and TAC decrease. The results of this study showed that the levels of MDA were higher and the levels of catalase and TAC were lower in patients with more severe cataracts compared to the healthy controls.

Potential antimicrobial and fruit juice clarification activity of amylase enzyme from Bacillus strains

The hydrolytic enzyme, amylase possesses wide industrial applications and its production from bacterial sources by submerged fermentation is much simplified and economical. The research aimed to characterize amylase-producing bacteria and evaluate their potential for amylase activity regarding antimicrobial and fruit juice clarification. In current study, Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus subtilis and Bacillus paramycoides was identified by 16S rRNA sequencing. After submerged fermentation, amylase activity of bacteria was measured by 3, 5-dinitro salicylic acid (DNS) assay. A substantial amount of amylase (423.47 mg/ml) in crude extract was measured by Bradford protein assay. Later, ammonium sulfate (80 %) precipitated partially purified amylase showed 1.6 times enhanced amylase activity (1484.94 U/ml) compared to crude amylase (973.23 U/ml). For highest amylase production, 72 h of optimum fermentation period was recorded at pH 7 with 2 % starch as substrate. Potent thermophilic amylase activity was observed at 65 °C. In apple juice clarification activity of amylase, turbidity of juice was reduced to 54.18 %. Potential antimicrobial property of amylase was detected with largest zone of inhibition against Escherichia coli ATCC 25922 (22.36 mm) and Mucor sp. ATCC 48559 (22.45 mm). Considering promising amylase properties, amylase-producing Bacillus strains from rice mill soil can be fermented for large scale amylase production providing application for industrial purposes including fruit juice clarification and antimicrobial activities. It will also overthrow the requirement of employing expensive and harmful chemicals in fruit juice clarification and combating pathogens.

COMPARATIVE ANALYSIS OF METHODS FOR DETERMINING THE CONTENT OF THE TOTAL PROTEIN SARS-COV-2 OF THE COVID-19 VIRUS

One of the main requirements for the new vaccines being created is their safety in use, including the total protein content, which are one of the main indicators of the quality and safety of vaccines. This article presents the results of work on the comparative analysis of methods for determining the total protein in the active pharmaceutical substance (AFS) and also the bulk product of the vaccine "QazCovid-in ® .Determination of the total protein concentration was carried out by comparing the methods of Lowry, Bradford, commercial sets of Clini Test-BL and BSA Gold Protein. As a result of the determination of the total protein in the composition of the AFS by the classical Lowry method and the commercial CliniTest-BL kit, the highest values were obtained on average 616.67 and 589 μl/ml, and in the bulk product 51.11 and 47.38 μl/ml, respectively. The most minimal indicator of the total protein content of 38.39±0.000059 is shown by the method using the commercial BSA Gold Protein kit. The average values of total protein content showed the results obtained by the Bradford method and the determination of total protein by a commercial set of Clini Test-BL. The data obtained allow us to recommend the use of a set of Clini Test-BL for use in quality control, since this set is characterized by the ease and speed of the determination method, which is not inferior to the classical Lowry method. When determining the total protein content in the bulk product used to prepare the QazVac vaccine against COVID-19, the classical Lowry method showed the best result with a high degree of sensitivity.

Co-production of hemagglutinin H9N2 influenza virus and fusion protein Newcastle virus in insect cell using baculovirus expression system.

Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

Improvement of bioanalytical parameters through automation: suitability of a hand-like robotic system.

Commercial automation systems for small- and medium-sized laboratories, including research environments, are often complex to use. For liquid handling systems (LHS), development is required not only for the robot's movements but also for adapting the bioanalytical method to the automated system. This study investigates whether a more human-like automation strategy-using a robotic system (RS)-is more suitable for research laboratories than a professional automation approach utilizing a commercial automated LHS. We conducted a series of measurements for protein determination using a Bradford assay manually, with a fully automated LHS, and with our human-like RS. Although the hand-like RS approach requires more than twice the time of the LHS, it achieved the best standard deviation in this setup (RS = 0.5, manual = 0.71, LHS = 0.86). Due to the low limit of detection (LOD) and limit of quantification (LOQ), most protein samples could be quantified with the RS (samples below LOQ = 9.7%, LOD = 0.23; LOQ = 0.25) compared to manual (samples below LOQ = 28.8%, LOD = 0.24; LOQ = 0.26) and the LHS (samples below LOQ = 36.1%, LOD = 0.27; LOQ = 0.31). In another time-dependent enzymatic assay test, the RS achieved results comparable to the manual method and the LHS, although the required time could be a constraint for short incubation times. Our results demonstrate that a more hand-like automation system closely models the manual process, leading easier to accurate bioanalytical results. We conclude that such a system could be more suitable for typical research environments than a complex LHS.

Comparison of Colorimetric Methods for Measuring the Solubility of Legume Proteins.

Increasing the use of plant proteins in foods requires improving their physical and chemical properties, such as emulsification, gelation capacity, and thermal stability. These properties determine the acceptability and functionality of food products. Higher protein solubility significantly impacts these properties by affecting denaturation and the stability of emulsifiers or gels. Therefore, developing plant-based protein ingredients requires accurately and conveniently measuring their solubility. Colorimetric solubility methods overcome many issues of more robust combustion and titration methods, but complicated chemical mechanisms limit their applicability for certain proteins. This study aims to compare the effectiveness of four common colorimetric solubility measurement methods for pulse and non-pulse legume proteins and hydrolysates. Pea, chickpea, lentil, and soy protein isolates were made from defatted flour and their solubility at a range of pHs was measured using the Bradford, Lowry, bicinchoninic acid (BCA), and biuret methods. Solubility was also measured for chickpea and soy protein hydrolysates made using Alcalase and Flavourzyme. A comparison of the methods for solubility quantification revealed that the Bradford and Lowry methods most closely match the expected results for the unhydrolyzed protein, with the BCA and biuret methods underestimating solubility by 30%. The Lowry method was the preferred method for hydrolysate solubility measurement, with the Bradford method measuring 0% solubility at the isoelectric point due to an inability to interact with peptides that are soluble at this pH. This study identifies reliable methods for measuring plant protein solubility that establish uniform outcomes and enable a better comparison across studies, giving a consensus for key functional properties in food applications.