Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24 cm immobilized pH gradient strips (pH 4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

Speeding up SDS-PAGE: Theory and experiment.

In order to accelerate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), here we propose an optimized version of the technique enabled by experimental tuning reinforced by theoretical description. In the resulting system, the gel buffer was diluted twofold and supplemented with glycine at a low concentration, whereas a higher voltage was applied. This approach reduced runtime from 90 to 18 min. It is important to emphasize that, despite the high voltage applied to the gel, the resolution of the bands did not decrease compared to the original Laemmli method. The proposed acceleration approach can be used in other variants of SDS-PAGE.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Supplementation of Boswellia serrata and Salix alba Extracts during the Early Laying Phase: Effects on Serum and Albumen Proteins, Trace Elements, and Yolk Cholesterol.

Extracts from Boswellia serrata (Bs) and Salix alba (Sa) are used as supplements in poultry feed. The aims of this research were to study the possible effects of dietary supplementation with Bs and Sa extracts on serum and albumen proteins, zinc and iron, and yolk cholesterol content in Leghorn hens during the critical phase of the onset of laying. A total of 120 pullets, 17 weeks of age, were assigned to two groups (control (C) and treated (T), n = 60 each). The T group received a supplement containing Bs (5%) and Sa (5%) for 12 weeks. The study lasted 19 weeks. Serum proteins were fractionated using agarose gel electrophoresis (AGE) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Trace elements were determined in serum using atomic absorption spectrometry, and yolk cholesterol was determined using a colorimetric test. No significant differences were observed between control and supplemented hens for the analyzed biochemical indices. Moreover, the supplementation with phytoextracts did not negatively affect the physiological variations in serum proteins; therefore, it can be safely used as a treatment to prevent inflammatory states at onset and during the early laying phase.

Morphological and structural characterization of chitin as a substrate for the screening, production, and molecular characterization of chitinase by Bacillus velezensis.

The processing of shellfishery industrial wastes is gaining much interest in recent times due to the presence of valuable components. Chitin is one of the valuable components and is insoluble in most common solvents including water. In this study, a novel gram-positive bacterial strain capable of solubilizing chitin was screened from a prawn shell dumping yard. The chitinolytic activity of the isolated strain was observed through the zone of hydrolysis plate assay. The hyper-producing isolate was identified as Bacillus velezensis through the 16S rRNA sequencing technique. The structural and morphological characterization of raw and colloidal chitin preparation was carried out using FTIR, XRD, and SEM analysis. The residual protein and mineral content, degree of polymerization, and degree of acetylation were reported for both raw and colloidal chitin preparations. There was a linear increase in the chitinase activity with an increase in the colloidal chitin concentration. The maximum activity of chitinase was observed as 38.98 U/mL for the initial colloidal chitin concentration of 1.5%. Supplement of additional carbon sources, viz., glucose and maltose, did not improve the production of chitinase and resulted in a diauxic growth pattern. The maximum chitinase activity was observed to be 33.10 and 30.28 U/mL in the colloidal chitin-containing medium with and without glucose as a secondary carbon source, respectively. Interestingly, the addition of complex nitrogen sources has increased the production of chitinase. A 1.95- and 2.14-fold increase in the enzyme activity was observed with peptone and yeast extract, respectively. The chitinase was confirmed using SDS-PAGE, native PAGE, and zymograms. The optimum pH and temperature for chitinase enzyme activity were found to be 7.0 and 44 °C, respectively.

Morphogenesis and Genetic Diversity of Some Virulent Phages Specific for Bacillus velezensis.

<b>Background and Objective:</b> Phosphorus (P) is one of the most limiting nutrients for plant growth. Phosphorus deficiency is limiting crop production in many agricultural soils worldwide. The application of phosphorus solubilizing bacteria (PSB) to soils can replace or partially reduce using of inorganic P fertilizers. A bacteriophage, or phage, is a virus that infects a bacterial cell, taking over the host cell's genetic material. The four phages were propagated, purified, studied for the morphological properties, finally studying the genetic diversity. <b>Materials and Methods:</b> Obtained, examined the efficiency and identification of bacteria for solubilizing phosphorus. Isolation, studying the properties and studying genetic diversity. <b>Results:</b> Four virulent phages (Bv₁, Bv₂, Bv₃ and Bv₄) specific for <i>Bacillus velezensis</i> were isolated from the Egyptian soil. The <i>Bacillus</i> phages were purified by alternative low and high-speed centrifugation methods. Electron micrographs showed that phages appeared to be a member of the <i>Siphoviridae </i>family based on their structure and particle morphology (the particles have a head and long non-contractile tail). Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was performed to determine the properties of viral proteins. It was found that the Bv₁ virus had five structural proteins, while Bv₂ and Bv₃ virus had eight structural proteins and finally, the Bv₄ virus had ten structural proteins. The purity and quantity of isolated DNAs were determined spectrophotometrically. Data showed that the concentration of Bv₁ DNA was 0.75 µg, Bv₂ DNA and Bv₃ DNA was 0.60 µg and finally Bv₄ DNA 0.55 µg µL¹. The analysis of genetic material of <i>B. velezensis</i> phages was determined based on both the ISSR-PCR technique and the effect of restriction enzymes. Data showed different amplification patterns with all phages. <b>Conclusion:</b> The bacteriophages of <i>B. velezensis</i> were isolated from soil, propagated, purified, study some of its properties.

A novel phenolic formulation for treating hepatic and peripheral insulin resistance by regulating GLUT4-mediated glucose uptake.

Chronic insulin resistance suppresses muscle and liver response to insulin, which is partially due to impaired vesicle trafficking. We report here that a formula consisting of resveratrol, ferulic acid and epigallocatechin-3-O-gallate is more effective in ameliorating muscle and hepatic insulin resistance than the anti-diabetic drugs, metformin and AICAR. The formula enhanced glucose transporter-4 (GLUT4) translocation to the plasma membrane in the insulin-resistant muscle cells by regulating both insulin-independent (calcium and AMPK) and insulin-dependent (PI3K) signaling molecules. Particularly, it regulated the subcellular location of GLUT4 through endosomes to increase glucose uptake under insulin-resistant condition. Meanwhile, this phytochemicals combination increased glycogen synthesis and decreased glucose production in the insulin-resistant liver cells. On the other hand, this formula also showed anti-diabetic potential by the reduction of lipid content in the myotubes, hepatocytes, and adipocytes. This study demonstrated that the three phenolic compounds in the formula could work in distinct mechanisms and enhance both insulin-dependent and independent vesicles trafficking and glucose transport mechanisms to improve carbohydrate and lipid metabolism.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

A Comprehensive Workflow for the Analysis of Bio-Macromolecular Supplements: Case Study of 20 Whey Protein Products.

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small-molecule nutritional supplements or protein analysis.

Characterization of urinary protein profile in regular kratom (Mitragyna speciosa korth.) users in Malaysia.

Mitragyna speciosa (Korth.) also known as kratom or ketum has been traditionally used for its diverse medicinal value in Southeast Asia. Despite of its therapeutic value, kratom's safety profile remains deficiently elucidated. Our study aims to characterize the urinary protein profile of regular kratom users to determine its toxic effects on renal functioning. A total of 171 respondents (comprising of n = 88 regular kratom users, and n = 83 healthy controls) were recruited for this study. Urine specimens were collected and analyzed using SDS-PAGE, followed by LC/MS/MS analysis. Our results show albumin is the primary, and most abundant form of protein excreted in kratom user's urine specimens (n = 60/64), indicating that kratom users are predisposed to proteinuria. Kratom users had an elevated urinary protein (with an intensity of 66.7 kDa band), and protein: creatinine ratio (PCR) concentrations relative to healthy controls. However, kratom user's urinary creatinine concentration was found to be in the normal range as the healthy control group. While, kratom users who tested positive for illicit drug use had an elevated urinary albumin concentration. Our preliminary findings indicate that regular consumption of freshly brewed kratom solution over a protracted period (for an average of eleven years) seems to induce proteinuria, suggestive of an early stage of kidney injury. Hence, further studies are urgently needed to confirm our findings, and establish kratom's renal impairing effects.

Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents, and Antioxidant Activity of Micropropagated Lycium schweinfurthii Plants.

Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L-1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L-1 Indole-3-butyric acid (IBA). The Jaccard's similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL-1 of MiP dried leaves' methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.

Potential of Ageratum conyzoides in Inhibiting Nitric Oxide Synthase.

<b>Background and Objective:</b> Inflammation occurs <i>via</i> several mechanisms, one of which includes the production of Nitric Oxide (NO) catalyzed by inducible nitric oxide synthase (iNOS), which is inhibited selectively by isothioureas. <i>Ageratum conyzoides</i> L. has shown activity in reducing pain and inflammation, although the molecular mechanism had not been undertaken. The objectives of this work were (1) to study the mechanism of anti-inflammatory activity of <i>A. conyzoides</i> through inhibition of iNOS, (2) to correlate the iNOS inhibitory activity of the plant with the total flavonoid content of the plants and (3) to identify the flavonol synthase (FLS), an enzyme that catalyzes the production of quercetin. <b>Materials and Methods:</b> The inhibitory activity against iNOS was assayed by <i>in vitro</i> method. The total flavonoids (calculated as quercetin) of <i>A. conyzoides</i> were determined by fluorometry. The protein extraction of the leaves was carried out by employing Laing and Christeller's (2004) method, followed with SDS-PAGE. <b>Results:</b> The inhibitory activity (IC₅₀) of ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> against iNOS was 92.05 and 4.78 µg mL¹, respectively. Pearson correlation analysis resulted in 0.548 (ethanol extract) and 0.696 (ethyl acetate fraction). The total flavonoids (calculated as quercetin) contained in the ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> were 0.71 and 7.65%, respectively. The FLS in <i>A. conyzoides</i> leaves was identified at 31 kDa. <b>Conclusion:</b> <i>A. </i>c<i>onyzoides</i> L. is potential in inhibiting iNOS due to quercetin contained in the leaves. This report will add a scientific insight of <i>A. conyzoides</i> for biological sciences.

Exploring Effects of Protease Choice and Protease Combinations in Enzymatic Protein Hydrolysis of Poultry By-Products.

A study of the effects of single and combined protease hydrolysis on myofibrillar versus collagenous proteins of poultry by-products has been conducted. The aim was to contribute with knowledge for increased value creation of all constituents of these complex by-products. A rational approach was implemented for selecting proteases exhibiting the most different activity towards the major protein-rich constituents of mechanically deboned chicken residue (MDCR). An initial activity screening of 18 proteases on chicken meat, turkey tendons and MDCR was conducted. Based on weight yield, size exclusion chromatography (SEC) and SDS-PAGE, stem Bromelain and Endocut-02 were selected. Studies on hydrolysis of four different poultry by-products at 40 °C, evaluated by protein yield, SEC, and SDS-PAGE, indicate that the proteases' selectivity difference can be utilized in tailor-making hydrolysates, enriched in either meat- and collagen-derived peptides or gelatin. Three modes of stem Bromelain and Endocut-02 combinations during hydrolysis of MDCR were performed and compared with single protease hydrolysis. All modes of the protease combinations resulted in a similar approximately 15% increase in product yield, with products exhibiting similar SEC and SDS-PAGE profiles. This shows that irrespective of the modes of combination, the use of more than one enzyme in hydrolysis of collagen-rich material can provide means to increase the total protein yield and ultimately contribute to increased value creation of poultry by-products.

Metabolism of Tracer 75Se Selenium From Inorganic and Organic Selenocompounds Into Selenoproteins in Rats, and the Missing 75Se Metabolites.

We now know much about selenium (Se) incorporation into selenoproteins, and there is considerable interest in the optimum form of Se for supplementation and prevention of cancer. To study the flux of 75Se into selenoprotein, rats were fed 0 to 5 µg Se/g diet as selenite for 50-80 d and injected iv with 50 µCi of 75Se-labeled selenite, selenate, selenodiglutathione, selenomethionine, or selenobetaine at tracer levels (~0.5 µg Se). The rats were killed at various times and 75Se incorporation into selenoproteins was assessed by SDS/PAGE. These studies found that there is very rapid Se metabolism from this diverse set of selenocompounds to the common intermediate used for synthesis and incorporation of 75Se into the major selenoproteins in a variety of tissues. No selenocompound was uniquely or preferentially metabolized to provide Se for selenoprotein incorporation. Examination of the SDS/PAGE selenoprotein profiles, however, reveals that synthesis of selenoproteins is only part of the full Se metabolism story. The 75Se missing from the selenoprotein profiles, especially at early timepoints, is likely to be both low-MW and high-MW selenosugars and related precursors, as we recently found in livers of turkeys fed Se-adequate and high-Se diets. Differential metabolism of different selenocompounds into different selenosugar species may occur; these species may be involved in prevention of cancer or other diseases linked to Se status and may be associated with Se toxicity. Additional studies using HPLC-mass spectroscopy will likely be needed to fully flesh out the complete metabolism of selenium.

Selenium biofortification of different varieties of apples (Malus domestica) - Influence on protein content and the allergenic proteins Mal d 1 and Mal d 3.

As allergy towards apples is widespread, the evaluation of various cultivation and postharvest influences on the allergenic potential is of great importance. Therefore, the analysis of the Mal d 1 content was the focus of this study, originally dealing with investigating the influence of a selenium biofortification on apple quality. The Mal d 1 content of apples was in most cases reduced when the fruits were biofortified with selenium. Apple variety and climatic conditions were identified as further influencing factors for the Mal d 1 content of the fruits. The separate analysis of the peel and the fruit flesh showed that the content of Mal d 1 in the fruit flesh was significantly lower in the biofortified samples than in the controls. In conclusion, the results indicate that the selenium biofortification of apples and biochemical mechanism behind can reduce the allergenic potential regarding the content of Mal d 1.

Chemical and Antioxidant Characteristics of Skin-Derived Collagen Obtained by Acid-Enzymatic Hydrolysis of Bigeye Tuna (Thunnus obesus).

The utilization of bigeye tuna skin as a source of collagen has been increasing the value of these skins. In this study, the quality of the skin was studied first. The skin after 14 h freeze-drying showed a high protein level (65.42% ± 0.06%, db), no histamine and a lack of heavy metals. The collagens were extracted through acid and acid-enzymatic methods. The enzymes used were bromelain, papain, pepsin, and trypsin. The two highest-yield collagens were pepsin-soluble collagen (PSC) and bromelain-soluble collagen (BSC). Both were type I collagen, based on SDS-PAGE and FTIR analysis. They dissolved very well in dimethyl sulfoxide and distilled water. The pH ranges were 4.60-4.70 and 4.30-4.40 for PSC and BSC, respectively. PSC and BSC were free from As, Cd, Co, Cr, Cu, and Pb. They showed antioxidant activities, as determined by the DPPH method and the reducing power method. In conclusion, bigeye tuna skin shows good potential as an alternative source of mammalian collagen. Although further work is still required, PSC and BSC showed the potential to be further used as antioxidant compounds in food applications. Other biological tests of these collagens might also lead to other health applications.

Changes in qualitative and quantitative traits of birch (Betula pendula) pollen allergenic proteins in relation to the pollution contamination.

Birch (Betula pendula) pollen causes inhalant allergy in about 20% of human population in Europe, most of which is sensitive to the main birch allergen, Bet v1. The aim of the study was to find out (i) whether and how the analysed birch individuals differ in regard to composition of individual subunits of pollen proteins and to protein content in these subunits; (ii) whether the level of particulate matter relates to concentration of Bet v1 allergen. Study was performed in Southern Poland, in 2017-2019. Pollen material was collected at 20 sites, of highly or less polluted areas. Protein composition was analysed by SDS-PAGE, while the concentration of Bet v1 was evaluated by ELISA. The obtained results were estimated at the background of the particulate matter (PM10) level and the birch pollen seasons in Kraków. The electrophoregrams of pollen samples collected at different sites showed huge differences in staining intensities of individual protein subunits, also among important birch allergens: Bet v1, Bet v2, Bet v6 and Bet v7. The level of Bet v1 was significantly higher in the pollen samples collected at the more polluted sites. While the birch pollen allergenic potential is determined, the both pollen exposure and the content of the main allergenic components should be considered, as factors causing immunological response and clinical symptoms manifestation in sensitive individuals.

A simple, economical and environmental-friendly method for staining protein gels using an extract from walnut-husk.

We wish to present a simple, rapid, cost-effective and environmentally safe method for staining proteins in polyacrylamide gels, using aqueous-based natural extracts from fresh green walnut (Juglans regia) hulls/husks. The technique takes not more than 10 min for staining and is comparable in sensitivity to the most commonly used Coomassie R-250 staining method when applied to different concentrations of Bovine Serum Albumin (BSA) and various amounts of E. coli extracts. The protein (BSA) band (~0.5 µg) and E. coli extract comprising ~25 µg total protein can be visualized on polyacrylamide gels. Compared to both Coomassie and Ponceau S staining, the current method displayed more intense bands when proteins are transferred to polyvinylidene fluoride (PVDF) membrane. Although the walnut-dye (WD) method does not require a time-consuming destaining step, excess background stain can simply be removed by washing in water. Extract from old dried black husks and extract from fresh green husks kept for a year was also effective. Using LC-MS, Myricetin and/or Kaempferol were found to be active compounds responsible for staining proteins. Compared to traditional Coomassie method, the inclusion of expensive and toxic solvents (methanol and acetic acid) is completely avoided resulting in positive health, environmental and economic benefits. In view of all these advantages, the WD method has immense potential to replace currently used protein staining techniques.

Extraction, Partial Purification and Characterization of Bromelain from Pineapple (Ananas Comosus) Crown, Core and Peel Waste

Abstract Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.