Hydrolysates with emulsifying properties prepared from protein wastes using microbial protease.

Plant-based protein hydrolysates have found applications in food industry for emulsification, foaming, and increasing shelf life of food products. The objective of this study is to isolate protease-secreting bacteria hydrolyzing protein waste, and subjecting the resultant hydrolysates for the characterization for application in the food industry. Peanut cake hydrolysates were prepared using proteases from two microorganisms selected for the purpose, viz., Aneurinibacillus migulanus, VITPM11 and Aneurinibacillus aneurinilyticus, VITPS07. The cleavage specificity of the proteases from VITPM11 and VITPS07 were found to be like plasmin and elastase respectively. The cleaving sites of proteases for peanut proteins were predicted using expasy tool. The protease of VITPM11 had maximal activity of 325.8 ± 0.1 U/mL in peanut-cake media. The degree of hydrolysis (32.03 ± 0.89%), solubility (88.5 ± 1.18%), emulsion stability index (89.76 ± 2.80) and foaming stability (68.67 ± 1.53%) properties of VITPM11 protease correlated well with results from bioinformatic studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01490-z.

Casting Machines in Dentistry - A Review Part I.

Dental casting machine is an electrical device used to extrude molten materials to fabricate dental prostheses such as crowns, bridges, intracoronal and extracoronal restorations, and removable partial dentures. The casting process basically include melting and casting. Firstly, the solid material is heated in a crucible in temperature-controlled conditions to melt the material to its smelled form. The dental casting process is a complex one with multifaceted steps and equipment. Different types of casting machines are available to produce heat using different sources and techniques. It includes Arc melting, Open flame casting, and Electrical resistance. Arc melting involves the application of an electric or gas discharge on tungsten electrodes, causing the metal base to melt utilizing the heat produced by arcing. Open flame casting or induction melting employs water-cooled alternate current induction coils to induce heat. However, resistance heating uses electric current to melt precious metals.

Comparative Evaluation of the Rigidity and Accuracy of Different Elastomeric Impression Materials with Open Tray Implant Level Impression Technique-An In vitro Study.

Introduction: An implant-supported prosthesis requires a precise imprint to provide a good fit. It has been suggested that for implant/abutment level impressions, both the indirect (closed tray) and direct (open tray) methods are effective in achieving a passively fitting prosthesis. Aim: With an open plate embed level impression technique, this in vitro investigation set out to evaluate three different elastomeric imprint materials for their relative rigidity and precision. Materials and Methods: Sixty bespoke trays were created. Part I (rotational opposition assessment) of the investigation included selecting 30 uniquely shaped plates at random and using them to create embed-level open plate engraves. The second part of this investigation (evaluation of rotational discrepancy and vertical inconsistency) used open plate embed level impressions generated with the remaining thirty individualized plates to evaluate how well these materials recreated the patient's impressions. Result: Within the bounds of the current investigation, vinylpolysiloxane had the highest degree of stiffness among the impression materials examined in comparison to polyether and vinylsiloxanether, as seen by its much greater rotational resistance to torquing. A rotational discrepancy was shown to be much lower in polyether open tray implant level impressions compared to vinylpolysiloxane and comparable to vinylsiloxane. Conclusion: In this investigation, the firmest impression medium was vinylpolysiloxane, followed by polyether and vinylsiloxanether for taking imprints of implants. Polyether material, however, was more precise than vinylpolysiloxane and vinylsiloxanether.

Purified fish skin collagen hydrolysate attenuates TNF-α induced barrier dysfunction in-vitro and DSS induced colitis in-vivo model.

Inflammatory mediators are key components in establishing pathogenesis in inflammatory bowel disease. Balanced expression of anti-inflammatory and pro-inflammatory cytokines is an important cue in maintaining gut native and adaptive immunity. In the present study, purified hydrolysate fraction of fish skin collagen from Clarias batrachus and Pangasius pangasius was evaluated as a treatment agent against TNF-α induced barrier dysfunction in Caco-2 cell line model and DSS induced colitis in mice model. Cell adhesion on purified hydrolysate fraction coated surfaces was found to be enhanced with increasing concentration in both Clarias batrachus and Pangasius pangasius. Alkaline phosphatase activity was enhanced in a concentration-dependent manner. The paracellular permeability assay demonstrated that Pangasius pangasius purified hydrolysate fraction had countered TNF-α induced barrier dysfunction. Analysis of the tight junction proteins (occludin, zonulae occluden, and claudin) by RT PCR, immunofluorescence, and western blot, further confirmed the effectiveness of Pangasius pangasius purified hydrolysate fraction against TNF-α. The Pangasius pangasius purified hydrolysate fraction was further evaluated for efficacy in DSS-induced colitis mice model. Two concentration of Pangasius pangasius purified hydrolysate was chosen based on in-vitro experiments, 80 µg/kg and 200 µg/kg BW of Balb/C male mice administered through intra-rectal route along with fish skin collagen 80 µg/kg BW. Pangasius pangasius purified hydrolysate fraction treatment improved the clinical signs of colitis such as body weight, rectal bleeding, colon length, and stool consistency caused by DSS administration. Immunofluorescence of colon tissue section showed that Pangasius pangasius purified hydrolysate fraction enhanced the expression of occludin protein. This study hints at the use of Pangasius pangasius purified hydrolysate fraction as a potential nutraceutical or treatment agent in healing ulcers of the mucosa.

Production of dehairing protease by Bacillus cereus VITSN04: a model cradle-to-cradle approach for sustainable greener production of leathers.

Despite several attempts over decades, process scalability and sustainability remain a challenge to produce an environmental-friendly enzyme to gain industrial attention. In the present study, microbial degradation of chrome shavings (chromium-collagen leather waste) and the resulting collagen hydrolysate for producing the dehairing protease by Bacillus cereus VITSN04 were investigated in a lab-scale fermentor. Scale-up degradation of shavings resulted in higher recovery of collagen hydrolysate (76%) within 72 h compared to shake flasks (68% in 120 h). Earlier achieved medium composition of collagen hydrolysate (12 g L-1) and molasses (15 g L-1) appeared to induce amylase at the high rate, despite the maximal production of protease (203.8 ± 0.18 U mL-1), which was analysed by ANS fluorescence spectroscopy. Optimization of the media containing collagen hydrolysate (12 g L-1) and molasses (5 g L-1) was effective in producing protease (170.6 ± 0.1 U mL-1) and reduced the co-synthesis of amylase (48.2 ± 0.09 U mL-1). The controlled fermentation process by feeding molasses during the exponential growth phase had enhanced the dehairing protease production (∼2.96 fold). The produced protease then partitioned through the biphasic system and showed significant dehairing of goat skins on the pilot scale. Thus, the scalability of the process to produce dehairing enzymes using waste, generated at the site of its use, offers hope for sustainable greener production of leathers.

Biocompatible Films of Collagen-Procyanidin for Wound Healing Applications.

The study investigated the effect of polyphenols present in Cassia auriculata (CA) leaves in enhancing the stability of the collagen protein and the wound healing potential of collagen films. The crude ethanol extract of CA was analyzed for the presence of phytochemicals and purified by column chromatography using solvents with increasing polarity. The ethanol eluted active fractions (EEAF) that precipitated gelatin was characterized using HP-TLC, FTIR spectroscopy, ESI-FT-MS/MS, and 1H NMR spectroscopy. The active compound was identified to be procyanidin B belonging to the proanthocyanidins group. The wound healing property of EEAF and collagen type I extracted from Clarias batrachus fish skin and the bovine tendon was assessed by in vitro scratch assay on L929 mice fibroblast cell lines. The EEAF-treated collagen coating enhanced in vitro wound closure in comparison with the uncoated dish. It was observed that EEAF treatment improved the physical strength of collagen films. The in vivo wound healing of the EEAF-treated collagen film was examined in male Wister rats and the wound site tissues were assessed. In vivo wound examination showed enhanced healing with EEAF incorporated collagen films. Comparatively, the EEAF-treated bovine tendon collagen films showed improved physical properties and better wound healing property than fish collagen films.

Adhesion and proliferation properties of type I collagen-derived peptide for possible use in skin tissue engineering application.

The surface properties of three-dimensional scaffolds are improved by coating or covalently linking certain adhesion-promoting proteins or peptides. In the present study, the effect of type I collagen-derived peptide (GKNGDDGEA) on adhesion and proliferation of HaCaT keratinocytes and NIH3T3 murine fibroblast cell lines was studied to assess its suitability for possible skin tissue engineering applications. Cell adhesion and proliferation of HaCaT and NIH3T3 were found to be enhanced by peptide coating. The optimum peptide coating densities to obtain the best cell adhesion and proliferation were found to be 0.827 µmoles/cm2 and 0.62 µmoles/cm2 for HaCaT and NIH3T3, respectively. Cell adhesion, in the presence of anti-integrin α1 antibody, inhibited attachment of NIH3T3 cells indicating the involvement of integrin α1 receptor. However, the attachment of HaCaT cells was not affected by anti-integrin treatment. The higher expression of paxillin confirmed the effect of the peptide in mediating focal adhesion kinases (FAKs) in cell adhesion and proliferation. Gene expression analysis was performed on cell migration proteins like Rho, Rac, Cdc42, integrin receptor α1, and ß1, and the extracellular matrix modulating proteins like MMP2, TIMP, and COL1A1 to validate their role on the peptide-mediated cell proliferation. Immunofluorescence analysis showed the distribution and localisation of phospho-FAK on cells cultured on the peptide-coated surfaces. Results support the role of peptides in enhancing cell adhesion and proliferation properties.

Role of fish collagen hydrolysate in attenuating inflammation-An in vitro study.

Collagen hydrolysate, an extensively used protein obtained from different sources, has various beneficial effects on human health and diseases. The benefits of collagen hydrolysate are well known and presently varied sources for the preparation of hydrolysate are being investigated. Food as a therapy to combat inflammation is presently a much-focused field of research. The present study aims at screening the anti-inflammatory property of collagen hydrolysate from the skin of Cypselurus melanurus, Catla catla, Indian mackerel, Clarias batrachus (Cb), and Pangasius pangasius (Pp) in activated RAW 264.7 macrophage cells. The fractions, Cb (C2) and Pp (P2) with anti-inflammatory property obtained after two-step chromatographic purification contained peptides in the range of 1-3 kDa molecular weight. The active fractions C2 and P2 showed a reduction in gene expression of TNF-α to 1.6- and 1-fold difference, whereas IL6 expression to 30- and 40-fold difference, respectively, in comparison to LPS treatment. The suppression of inflammatory proteins (TNF-α, IL6, NFκB, and p-IκB) by fractions C2 and P2 confirmed the anti-inflammatory activity. PRACTICAL APPLICATIONS: Collagen hydrolysate and its derived low molecular weight peptides are of great interest in the field of nutraceuticals and biomedical applications. The purified peptide fraction of fish skin hydrolysate displayed a promising anti-inflammatory property. The collagen hydrolysate of Cb and Pp can be a functional food or its purified fraction used as a nutraceutical supplementation due to their anti-inflammatory property in the cellular microenvironment.

Insights into substrate specificity of proteases for screening efficient dehairing enzymes.

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Chromium binding Bacillus cereus VITSH1-a promising candidate for heavy metal clean up.

Bacteria survive metal stress by several mechanisms and metal binding is one such mechanism which has been screened in the present study to investigate the survival strategies of metal resistant bacteria. The production of siderophores, a metal chelating agent, was detected by chrome azurol S agar assay. The changes in cell wall studied by analysing the peptidoglycan and teichoic acid content indicated an increase in the cell wall content. Evaluation of morphological and physiological alterations like cell size, granularity analysed by SEM and flow cytometry analysis revealed an increase in cell size and granularity respectively. The transformation of phosphates monitored by 31 P NMR analysis indicated the presence of inorganic phosphate. Based on the cell wall changes and the 31 P NMR analysis, the surface charge of the organism was studied by zeta potential which displayed a difference at pH7.

Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase.

Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.

A potential bioactive peptide candidate for biomaterial and tissue engineering applications.

The surface modification of biomaterials with matrikines for tissue engineering application is one of the recent approaches to improve their biocompatibility. In an earlier study, a peptide containing 21 amino acid isolated from bovine tendon collagen was shown to promote good cell adhesion in HeLa cell, and a smaller region in the peptide was identified using bioinformatics tool to mediate cell-peptide interaction. Hence, the present study was undertaken to validate the cell adhesion property of the smaller region of the peptide and elucidate probable peptide-cell interaction pathway. Cell adhesion and proliferation properties of the peptide were studied on cells cultured on surfaces coated with varying concentrations of peptide. Expression of focal adhesion related proteins like paxillin and pFAK Tyr397 was confirmed by immunoblotting and immunofluorescence microscopy respectively. The anti-pFAK Tyr 397 stained confocal micrographs and mRNA transcription levels of Cdc42 and Rho further confirmed peptide mediated cell spreading. The change in the expression levels of integrin α1 and ß1 indicates an integrin mediated cell-peptide interaction for cell survival and proliferation. Integrin mediated adhesion was further confirmed by anti-integrin blocking assay. The modulation of ECM components by the peptide was assessed by expression of COL1A1, TIMP mRNA levels and gelatin zymography for MMPs. The results of the study confirm the role of the small region of the larger collagen peptide in cell adhesion and proliferation and hint at the possible use of such small peptides as biocompatible surface modifiers for tissue scaffolds.

Protein profiling of metal-resistant Bacillus cereus VITSH1.

AIMS: The aim of the study was to investigate the proteomic changes and antioxidant enzyme activity in chromium-resistant Bacillus cereus VITSH1 in response to heavy metal toxicity. METHODS AND RESULTS: The variation in protein expression and antioxidant enzyme activity in the presence and absence of metal was studied in B. cereus VITSH1. The differentially expressed proteins in chromium-treated conditions were determined by SDS PAGE. The proteins involved in metal binding, protein biosynthesis, protein folding, energy metabolism and motility were identified by mass spectrometry coupled with bioinformatics search tools. The in gel assays for antioxidant enzymes indicated a change in their activity under metal stress conditions. CONCLUSIONS: The findings of this study suggest that the organism combats metal stress probably by restricting the entry of metal inside the cell. The role of the differentially expressed proteins clearly indicates that the first line of defence is to avoid the entry of metal into the cell either by possessing a modified outer membrane or by moving away from the toxicant. Further work on the identification of other proteins playing a role in resistance would help in integrating the available knowledge on the resistance mechanisms the organisms employ to combat toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The proteomic changes in the metal-exposed bacteria would give an insight into the proteins involved in metal resistance mechanisms and thereby aid in the development of biosensor-based technology for heavy metal detection.

Matrikines for therapeutic and biomedical applications.

Matrikines, peptides originating from the fragmentation of extracellular matrix proteins are identified to play important role in both health and disease. They possess biological activities, much different from their parent protein. Identification of such bioactive cryptic regions in the extracellular matrix proteins has attracted the researchers all over the world in the recent decade. These bioactive peptides could find use in preparation of biomaterials and tissue engineering applications. Matrikines identified in major extracellular matrix (ECM) proteins like collagen, elastin, fibronectin, and laminin are being extensively studied for use in tissue engineering and regenerative medicine. They are identified to modulate cellular activity like cell growth, proliferation, migration and may induce apoptosis. RGD, a well-known peptide identified in fibronectin with cell adhesive property is being investigated in designing biomaterials. Collagen hexapeptide GFOGER was found to promote cell adhesion and differentiation. Laminin also possesses regions with strong cell adhesion property. Recently, cell-penetrating peptides from elastin are used as a targeted delivery system for therapeutic drugs. The continued search for cryptic sequences in the extracellular matrix proteins along with advanced peptide coupling chemistries would lead to biomaterials with improved surface properties. This review article outlines the peptides derived from extracellular matrix and some of the possible applications of these peptides in therapeutics and tissue engineering applications.

Specificity studies on proteases for dehairing in leather processing using decorin as model conjugated protein.

Enzyme based dehairing of animal skins in leather processing is considered a greener alternative to conventional chemical based dehairing process. The specificity based screening of proteases would be an important criterion as it would help in removing hairs by acting particularly on the proteoglycans holding the hair to the bulb without damaging the other proteins. The present paper describes a comparative study of the specificity of a dehairing protease with non-dehairing one isolated from Bacillus sps. Dehairing protease was found to be trypsin-like, non-collagenolytic and non-keratinolytic enzyme whereas non-dehairing one was a metalloprotease with non-collagenolytic activity but specificity for soluble keratin. Substrate specificity was studied using decorin as model proteoglycan. The dehairing enzyme was effective in digesting the core protein of the decorin as confirmed by high performance liquid chromatography analysis and SDS-polyacrylamide gel electrophoresis. The histochemical studies on dehaired skins also confirmed preservation of skin matrix with no visible damage to collagen. This study emphasizes the importance of screening proteases based on their specificity characteristics for effective and safer enzyme-only dehairing process. The acquired knowledge can be employed in formulating bacterial growth media to produce dehairing proteases that would help in commercial exploitation of this greener option.

An artificial intelligence based improved classification of two-phase flow patterns with feature extracted from acquired images.

Flow pattern recognition is necessary to select design equations for finding operating details of the process and to perform computational simulations. Visual image processing can be used to automate the interpretation of patterns in two-phase flow. In this paper, an attempt has been made to improve the classification accuracy of the flow pattern of gas/ liquid two- phase flow using fuzzy logic and Support Vector Machine (SVM) with Principal Component Analysis (PCA). The videos of six different types of flow patterns namely, annular flow, bubble flow, churn flow, plug flow, slug flow and stratified flow are recorded for a period and converted to 2D images for processing. The textural and shape features extracted using image processing are applied as inputs to various classification schemes namely fuzzy logic, SVM and SVM with PCA in order to identify the type of flow pattern. The results obtained are compared and it is observed that SVM with features reduced using PCA gives the better classification accuracy and computationally less intensive than other two existing schemes. This study results cover industrial application needs including oil and gas and any other gas-liquid two-phase flows.

Cryptic Peptides from Collagen: A Critical Review.

Collagen, a predominant structural protein in extracellular matrix (ECM), is now considered to have probable roles in many biological activities and hence, in different forms have found application as nutraceutical or pharmaceutical therapy option. Many of the biological properties are believed to be due to small hidden peptide residues in the collagen molecules, which come into play after the biodegradation or biosorption of the parent molecule. These peptide regions are called cryptic peptides or by some, as cryptides. The proteolytic hydrolysis of the ECM protein releases the cryptic peptides with many novel biological activities not exhibited directly by the parental protein which include angiogenic, antimicrobial, mitogenic and chemotactic properties. The research for understanding the role of these cryptic peptide regions and making use of them in medical field is very active. Such an understanding could lead to the development of peptide supplements for many biomedical applications. The prolific research in this area is reviewed in this paper.

Bio-mimetic mineralization potential of collagen hydrolysate obtained from chromium tanned leather waste.

Hydroxyapatite (HA) ceramics serve as an alternative to autogenous-free bone grafting by virtue of their excellent biocompatibility. However, chemically synthesized HA lacks the strong load-bearing capacity as required by bone. The bio-mimetic growth of HA crystals on collagen surface provides a feasible solution for synthesizing bone substitutes with the desired properties. This study deals with the utilization of the collagen hydrolysate recovered from leather waste as a substrate for promoting HA crystal growth. Bio-mimetic growth of HA was induced by subjecting the hydrolysate to various mineralization conditions. Parameters that would have a direct effect on crystal growth were varied to determine the optimal conditions necessary. Maximum mineralization was achieved with a combination of 10mM of CaCl2, 5mM of Na2HPO4, 100mM of NaCl and 0.575% glutaraldehyde at a pH of 7.4. The metal-protein interactions leading to formation of HA were identified through Fourier-transform infrared (FTIR) spectroscopy and x-ray diffraction (XRD) studies. The crystal dimensions were determined to be in the nanoscale range by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The size and crystallinity of bio-mimetically grown HA indicate that hydrolysate from leather waste can be used as an ideal alternative substrate for bone growth.

Synthesis, growth and spectral, optical and thermal characterization studies on L-Tryptophan p-nitrophenol (LTPN) single crystals for NLO applications.

The novel nonlinear optical single crystal of L-Tryptophan p-nitrophenol (LTPN) has been successfully synthesized by taking the appropriate amount of L-Tryptophan and p-nitrophenol. The single crystals have been grown by slow evaporation solution growth technique. The single crystal XRD studies confirmed that the grown crystal belongs to the monoclinic system. The various functional groups presented in the crystal were confirmed by FT-IR and (1)H NMR spectroscopic studies. The absorptions of the grown crystals were analyzed using UV-Vis-NIR spectral studies. The thermal analysis was performed to study the thermal stability of the grown crystals. The second harmonic generation behavior of L-Tryptophan p-nitrophenol crystal was tested by Kurtz-Perry powder technique.

Wound healing activity of a collagen-derived cryptic peptide.

Wound healing involves a well-controlled series of interactions among cells and several mediators leading to the restoration of damaged tissue. Degradation of the extracellular matrix (ECM) protein collagen during remodelling of wound tissue leads to the release of bioactive peptides that can possibly influence the healing process. The RGD-containing, antioxidative collagen peptide E1 isolated in an earlier work was screened in this study for its ability to influence multiple steps of the wound healing process. E1 was assayed for and found to be chemotactic. Excision and incision wounds were created on separate groups of rats and E1 was administered topically. The wound tissues were isolated on the 4th and 8th days post-wound and subjected to biochemical and biophysical analysis. A significant decrease in lipid peroxides in the treatment group confirmed the in vivo antioxidant capacity of E1. The treatment group also displayed significant increase in total protein, collagen and amino sugar synthesis indicating faster ECM formation. The significantly increased rate of wound contraction and reepithelialisation along with higher tensile strength of the wound tissue corroborated the results of biochemical analysis. The results confirm the significant role played by collagen peptides in accelerating the healing process and justify their possible use as a pharmaceutical agent.