Bioactive Properties of Enzymatic Gelatin Hydrolysates Based on In Silico, In Vitro, and In Vivo Studies.

This current study aims to analyze the potential bioactivities possessed by the enzymatic hydrolysates of commercial bovine, porcine, and tilapia gelatins using bioinformatics in combination with in vitro and in vivo studies. The hydrolysate with superior inhibition of angiotensin converting enzyme (ACE) activity was used to treat the D-galactose (DG)-induced amnesic mice. In silico digestion of the gelatins led to the identification of peptide sequences with potential antioxidant, ACE-inhibitory, and anti-amnestic properties. The results of in vitro digestion revealed that the <1 kDa peptide fraction of porcine gelatin hydrolysate obtained after 1 h digestion with papain (PP) (PP1, <1 kDa) potently inhibited ACE, acetylcholinesterase, and prolyl endopeptidase activities at 87.42%, 21.24%, and 48.07%, respectively. Administering the PP1 to DG-induced amnesic mice ameliorated the spatial cognitive impairment and Morris water maze learning abilities. The dentate area morphology in the PP1-treated mice was relatively similar to the control group. In addition, PP1 enhanced the antioxidant capacity in the DG-induced amnesic mice. This study suggests that PP1 could serve as a potential treatment tool against oxidative stress, hypertension, and neurodegenerative diseases.

Impact of adding papain to sous vide cooking on texture and sensorial traits of marinated semitendinosus beefsteaks.

Sous vide meat is an emerging food category, the consumption of which has increased owing to greater convenience, sensory traits, elderly consumers acceptance, and low-cost cuts use. However, required prolonged thermal treatment to achieve desired tenderness, impact energy-consumption besides triggering lipid oxidation, undesired off-flavors, and cooked meat profiles. Using a response surface methodology (RSM), this study evaluated the effects of the vegetal proteolytic papain (0 to 20 mg/kg) and low-temperature sous vide cooking (SVC) time (1 to 8 h at 65°C) in low-value marinated M. semitendinosus beefsteaks on technological characteristics associated with tenderness, and lipid oxidation. Additionally, the sensory profile traits of the pre-selected treatments were described using check-all-that-apply (CATA) and preference mapping. Shear force (WBsSF) was reduced with greater papain addition, whereas higher cooking losses (CL) were observed with longer SVC cooking times. Both the released total collagen and TBARS values increased with increasing papain concentrations and SVC times. Combining high levels of papain (>10 mg/kg) and SVC time (>6 h) resulted in lower WBsSF values (<20 N) but higher CL (>27%) and the CATA descriptors "aftertaste" and "mushy." The optimized conditions (14 mg/kg papain; 2 h SVC) also reduced WBsSF values (<26 N) with lower CL (<20%) and were most preferred and described as "juicy" and "tender" by consumers. Observed results suggest that combined mild SVC and papain may potentiate tenderness, conjointly favor juiciness and oxidation, further representing a promising tool for reducing SVC time without compromising valued sous vide sensory traits.

An in-vitro evaluation of residual dentin retained after using novel enzymatic-based chemomechanical caries removal agents.

To assess the biochemical, mechanical and structural characteristics of retained dentin after applying three novel bromelain-contained chemomechanical caries removal (CMCR) formulations in comparison to the conventional excavation methods (hand and rotary) and a commercial papain-contained gel (Brix 3000). Seventy-two extracted permanent molars with natural occlusal carious lesions (score > 4 following the International Caries Detection and Assessment System (ICDAS-II)) were randomly allocated into six groups (n = 12) according to the excavation methods: hand excavation, rotary excavation, Brix 3000, bromelain-contained gel (F1), bromelain-chloramine-T (F2), and bromelain-chlorhexidine gel (F3). The superficial and deeper layers of residual dentin were examined by Raman microspectroscopy and Vickers microhardness, while the surface morphology was assessed by the scanning electron microscope (SEM). A multivariate analysis of variance followed by Tukey's test (p > 0.05) was performed for data analysis. The novel formulations showed an ability to preserve the partially demineralized dentin that showed a reduced phosphate content with a higher organic matrix. This was associated with lower Vickers microhardness values in comparison to sound dentin and rotary excavation. The collagen integration ratio in all methods was close to sound dentin (0.9-1.0) at the deeper dentin layer. The bromelain-chloramine-T gel (F2) produced the smoothest smear-free dentin surface with a higher number of opened dentinal tubules. In contrast, dense smearing covering the remaining dentin was observed in the manual and rotary methods with obstructed dentin tubule orifices. The bromelain-contained formulations can be considered a new minimally invasive approach for selectively removing caries in deep cavitated dentin lesions.

MDM2 Is Essential to Maintain the Homeostasis of Epithelial Cells by Targeting p53.

INTRODUCTION: MDM2 is known as the primary negative regulator of p53, and MDM2 promotes lung cancer fibrosis and lung injury through p53-dependent and p53-independent pathways. However, the mechanism by which MDM2 influences the pathogenesis of asthma is unknown. In this study, we investigated the function of MDM2 in lung epithelial cells in type 2 lung inflammation. METHODS: We used type II alveolar epithelial cell-specific heterozygous knockout of Mdm2 mice to validate its function. Then papain-induced asthma model was established, and changes in inflammation were observed by measuring immunohistochemistry and flow cytometry analysis. RESULTS: In this study, we knockdown the mouse Mdm2 gene in type 2 alveolar epithelial cells. We demonstrated that heterozygous Mdm2 gene-deleted mice were highly susceptible to protease allergen papain-induced pulmonary inflammation characterized by increased ILC2 numbers, IL-5 and IL-13 cytokine levels, and lung pathology. A mechanistic study showed that following the decreased expression of Mdm2 in lung epithelial cells and A549 cell line, p53 was overactivated, and the expression of its downstream genes p21, Puma, and Noxa was elevated, which resulted in apoptosis. After Mdm2 knockdown, the mRNA expression of inflammation-related gene IL-25, HMGB1, and TNF-α were increased, which further amplified the downstream ILC2 response and lung inflammation. CONCLUSION: These results indicate that Mdm2 maintains the homeostasis of lung epithelial cells by targeting P53 and regulates the function of lung epithelial cells under type 2 lung inflammation.

An Improved Diabatization Scheme for Computing the Electronic Circular Dichroism of Proteins.

We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a ß-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for ß-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (ß strand), concanavalin A (ß type I), elastase (ß type II), papain (α + ß), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.

Novel mode of kafirin modification using combination of enzyme and thermal treatment to expand its food application.

Kafirin in sorghum inhibits starch digestion and exhibits antioxidant properties, however its potential in food industry remains unexplored. Therefore, the study was aimed to explore and improve the potential of kafirin as natural carbohydrate blocker using papain (6 NFU/mL) and/or infrared treatment (220 °C/3 min). Results indicated that the combined treatment, PIR (infrared + papain) is the most efficient treatment to modify kafirin. PIR generated a new ∼37 kDa high molecular weight moiety in kafirin with a crystal size of 157.44 Å. All samples showed superior antioxidant activity post-treatments, with PIR exhibiting highest scavenging activity from 31.09 to 82.97%, 15.09 to 42.82%, and 25.92 to 38.58% for DPPH, FRAP, and ABTS, respectively. PIR-modified kafirin limited malondialdehyde production, and increased α-amylase and α-glucosidase inhibition. Incorporation of 7.5% kafirin in corn starch increased resistant starch from 5.09 to 21.04% after cooking, which suggests potential of kafirin in development of diabetic-friendly food formulations.

Liensinine inhibits IL-1ß-stimulated inflammatory response in chondrocytes and attenuates papain-induced osteoarthritis in rats.

Osteoarthritis (OA) is a joint disease caused by inflammation of cartilage and synovial tissue. Suppressing the process of inflammatory reaction and the generation of oxidative stress is an effective strategy to alleviate the progression of OA. Liensinine is one of the main components of lotus seeds, which has anti-hypertensive and anti-arrhythmia activities. In this study, we aimed to determine the anti-inflammatory effect of liensinine in an OA. Here, we found that liensinine significantly inhibited the inflammatory response of SW1353 cells and primary chondrocytes by inhibiting the release of inflammatory cytokines and oxidative stress. Moreover, we showed that liensinine was able to inhibit the activation of the NF-κB signaling pathway in IL-1ß-induced SW1353 cells. Lastly, we found that liensinine significantly ameliorated cartilage damage and inflammatory response in papain-induced rats. Our study demonstrated a significant protective effect of liensinine against OA, which might be by inhibiting the activation of the NF-κB signaling pathway, and provide a new insight for the treatment of OA using liensinine.

Papain-Mediated Conjugation of Peptide Nucleic Acids to Delivery Peptides: A Density Functional Theory/Molecular Mechanics Metadynamics Study in Aqueous and Organic Solvent.

Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol-1, although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.

A composite enzyme derived from papain and chymotrypsin reduces the Allergenicity of Cow's Milk allergen casein by targeting T and B cell epitopes.

Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.

Liposome-Papain Conjugates for Catalytic Digestion of Antibody Producing Fab Fragments.

Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.

Controlled Primary Amine-Enriched SG-Bonded Papain Surface: Synthesis, Characterization, and Extraction of Protonated Dichromate.

The single-step synthesis of nitro-derivatized SG using dimethyldichlorosilane in an aprotic solvent dichloromethane at 300 K is efficient and straightforward. Reduction and diazotization effectively functionalize the material for enzyme coupling at the O-carbon of the enzyme's tyrosine. The high extraction efficiency of protonated dichromate ions with a breakthrough capacity of 480 µmol·g-1 is notable. Eco-friendly elution using distilled water achieves a significant enrichment factor of 23.2. Excellent reusability (up to 900 cycles) and stable sorption efficiency (ζ ≥ 0.9) highlight the material's potential for practical applications and future research.

Anti-biofilm effect of enzymatic hydrolysates of ovomucin in Listeria monocytogenes and Staphylococcus aureus.

Despite modern advances in food hygiene, food poisoning due to microbial contamination remains a global problem, and poses a great threat to human health. Especially, Listeria monocytogenes and Staphylococcus aureus are gram-positive bacteria found on food-contact surfaces with biofilms. These foodborne pathogens cause a considerable number of food poisoning and infections annually. Ovomucin (OM) is a water-insoluble gel-type glycoprotein in egg whites. Enzymatic hydrolysis can be used to improve the bioactive properties of OM. This study aimed to investigate whether ovomucin hydrolysates (OMHs) produced using five commercial enzymes (Alcalase®, Bromelain, α-Chymotrypsin, Papain, and Pancreatin) can inhibit the biofilm formation of L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7. Particularly, OMH prepared with papain (OMPP; 500 µg/mL) significantly inhibited biofilm formation in L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7 by 85.56 %, 80.28 %, 91.70 %, and 79.00 %, respectively. In addition, OMPP reduced the metabolic activity, exopolysaccharide production (EPS), adhesion ability, and gene expression associated with the biofilm formation of these bacterial strains. These results suggest that OMH, especially OMPP, exerts anti-biofilm effects against L. monocytogenes and S. aureus. Therefore, OMPP can be used as a natural anti-biofilm agent to control food poisoning in the food industry.

Thermal Inactivation, Denaturation and Aggregation Processes of Papain-Like Proteases.

The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate.

Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.

Impact of papain on the treatment of raw diluted dromedary semen.

A variety of parameters, including liquefaction and semen viscosity, affect the sperm's ability to travel and reach the egg for fertilization and conception. Given that the details behind the viscosity of the semen in male camels have not yet been fully clarified, the purpose of this study was to ascertain how the addition of papain affected the viscosity of fresh diluted camel semen. The study examined semen samples derived from camels that had distinct viscosities. Sperm motility, viability, abnormal sperm percentage, concentration, viscosity, morphometry, acrosome integrity and liquefaction were among the evaluations following 0, 5, 10, 20 or 30 min of incubation at 37°C with papain (0.004 mg/mL, 0.04 mg/mL or 0.4 mg/mL; a semen sample without papain was used as a control). A statistically significant interaction between the effects of papain concentrations and incubation time was found (F = 41.68, p = .0001). Papain concentrations (p = .0001) and incubation times (p = .0001) both had a statistically significant impact on viscosity, according to a simple main effects analysis. A lower viscosity was found (p < .05) at 0.04 mg/mL (0.1 ± 0.0) after 10 min of incubation. A simple main effects analysis showed that papain concentrations and incubation time have a statistically significant effect on sperm motility (p = .0001). At 0.04 mg/mL papain, the sperm motility % was higher (p < .05) after 10 min (64.4 ± 4.8), 20 min (68.4 ± 6.2), and 30 min incubation (72.2 ± 6.6) compared to 0, 5 min (38.3 ± 4.1 and 51.6 ± 5.0, respectively). In conclusion, the fresh diluted camel semen had the lowest viscosity properties after 10 min of incubation with 0.04 mg/mL papain, without compromising sperm motility, viability, acrosome integrity and sperm morphology.

SLAM-family receptors promote resolution of ILC2-mediated inflammation.

Type 2 innate lymphoid cells (ILC2) initiate early allergic inflammation in the lung, but the factors that promote subsequent resolution of type 2 inflammation and prevent prolonged ILC2 activation are not fully known. Here we show that SLAM-family receptors (SFR) play essential roles in this process. We demonstrate dynamic expression of several SFRs on ILC2s during papain-induced type 2 immunity in mice. SFR deficiency exacerbates ILC2-driven eosinophil infiltration in the lung, and results in a significant increase in IL-13 production by ILC2s exclusively in mediastinal lymph nodes (MLN), leading to increased dendritic cell (DC) and TH2 cell numbers. In MLNs, we observe more frequent interaction between ILC2s and bystander T cells, with T cell-expressed SFRs (especially SLAMF3 and SLAMF5) acting as self-ligands to suppress IL-13 production by ILC2s. Mechanistically, homotypic engagement of SFRs at the interface between ILC2s and T cells delivers inhibitory signaling primarily mediated by SHIP-1. This prevents activation of NF-κB, driven by IL-7 and IL-33, two major drivers of ILC2-mediated type 2 immunity. Thus, our study shows that an ILC2-DC-TH2 regulatory axis may promote the resolution of pulmonary type 2 immune responses, and highlights SLAMF3/SLAMF5 as potential therapeutic targets for ameliorating type 2 immunity.

Eco-friendly shrink-resist finishing of wool through synergistic effect of disulfide bond reducing agent and papain.

The environmental benefits of utilizing protease as a biocatalyst for wool shrink-resist finishing have been widely recognized. However, the efficacy of individual protease treatment is unsatisfactory due to its incapability towards the outermost cuticle layer of wool fibers that contains hydrophobic fatty acids. In order to weaken the structural integrity of the highly cross-linked scales and promote the enzymatic anti-felting, sodium sulfite and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were employed in combination with papain, respectively, aiming at obtaining a low shrinkage without unacceptable fiber damages. Based on the synergistic effect of papain and TCEP, the edges of wool scales were slightly destroyed by the reduction of disulfide bonds, accompanied by enzymatic hydrolysis of the keratin component. Through the controlled reduction and hydrolysis of wool scales, satisfactory anti-felting result was achieved without causing severe damage to the fiber interiors. In the presence of 0.25 g/L TCEP and 25 U/mL papain, the area shrinkage of wool fabric decreased to approximately 6 %, with a low strength loss of less than 8 %. Meanwhile, the dyeing behavior of the wool fabric under low-temperature conditions was dramatically improved, leading to decreased energy consumption during production. The present work provides an alternative for eco-friendly finishing of wool fabrics, which can be applied commercially.

Interfacial Protein Fibril Polymorphisms Regulate In Vivo Adipose Expansion for Control of Obesity.

Obesity is becoming a worldwide pandemic. Interfacial engineering of food lipid is expected to inhibit diet-induced obesity without damage to the eating enjoyment brought by high-fat diets. Unfortunately, this strategy has not been achieved yet. After screening different plant proteins, bromelain and papain were found to form wormlike and long-straight protein fibrils, respectively. The conversion of long-straight amyloid-like fibrils to wormlike fibrils was demonstrated in the fibrillation of bromelain. Using oil-in-water high internal phase emulsions (HIPEs) as a proof of concept, bromelain fibrils showed dramatically stronger interfacial stabilization capabilities than papain fibrils with high application potentials in the real-world formulation of high-fat food products such as mayonnaise. Compared with papain fibrils, oral administration of HIPEs stabilized by bromelain fibrils resulted in substantially higher fecal lipid contents and significantly decreased expression levels of the genes related to lipid absorption and transport in the intestine, including CD36, FATP-2, FATP-4, and APOA-4, without a difference in intervening gut microbiota. Consequently, dramatically less lipid absorption in the small intestine, markedly smaller chylomicron particles in the plasma, lower serum triglycerides, and controlled energy and lipid metabolism, as well as the inhibition of adipose expansion and overweight, were observed in the group with gavage of HIPEs stabilized by the bromelain fibrils rather than the papain fibrils. Furthermore, with the same calorie, substitution of all the fat in the standard high-fat feed of mice with the HIPEs emulsified by the bromelain fibrils showed a significantly stronger effect than the ones prepared by the papain fibrils on preventing high-fat-diet (HFD)-induced obesity including alleviation of adipose expansion and inflammation as well as fatty liver, also via inhibiting the absorption and transport of lipid in the intestine. The effect is ascribed to the suppressed lipolysis caused by a more compact and elastic interfacial layer formed by the wormlike fibrils than that of the long-straight fibrils, which are resistant to gastric environments and replacement by bile acids in digestion. Therefore, we provide an appealing and general strategy for controlling obesity by reducing the supply of free fatty acids (FAs) for absorption in the enteric lumen through protein fibril polymorphisms at the interface.

Microhardness and elemental analysis of ion-releasing restoration/ dentin interface following enzymatic chemomechanical caries excavation.

BACKGROUND: This study was conducted to compare chemical, elemental and surface properties of sound and carious dentin after application of two restorative materials resin-modified glassionomer claimed to be bioactive and glass hybrid restorative material after enzymatic chemomechanical caries removal (CMCR) agent. METHODS: Forty carious and twenty non-carious human permanent molars were used. Molars were randomly distributed into three main groups: Group 1 (negative control) - sound molars, Group 2 (positive control) - molars were left without caries removal and Group 3 (Test Group) caries excavated with enzymatic based CMCR agent. After caries excavation and restoration application, all specimens were prepared Vickers microhardness test (VHN), for elemental analysis using Energy Dispersive Xray (EDX) mapping and finally chemical analysis using Micro-Raman microscopy. RESULTS: Vickers microhardness values of dentin with the claimed bioactive GIC specimens was statistically higher than with glass hybrid GIC specimens. EDX analysis at the junction estimated: Calcium and Phosphorus of the glass hybrid GIC showed insignificantly higher mean valued than that of the bioactive GIC. Silica and Aluminum mean values at the junction were significantly higher with bioactive GIC specimens than glass hybrid GIC specimen. Micro-raman spectroscopy revealed that bioactive GIC specimens showed higher frequencies of v 1 PO 4, which indicated high level of remineralization. CONCLUSIONS: It was concluded that ion-releasing bioactive resin-based restorative material had increased the microhardness and remineralization rate of carries affected and sound dentin. In addition, enzymatic caries excavation with papain-based CMCR agent has no adverse effect on dentin substrate.

Simultaneous extraction of five heavy metal ions from root vegetables via dual-frequency ultrasound-assisted enzymatic digestion.

The dual-frequency ultrasound-assisted enzymatic digestion (DUED) technique was developed for synchronous green extraction of five heavy metal ions in root vegetables. The combination of α-amylase, cellulase, and papain showed significant advantageous in extracting heavy metal ions. Under optimized dual-frequency ultrasonic conditions, the extraction rates of Cr, As, Cd, Pb, and Hg in carrots reached 99.04%, 105.88%, 104.65%, 104.10%, and 103.13% respectively. And the extraction process is highly efficient, completing in just 15 min. Compared to conventional microwave-assisted acid hydrolysis method, this technique eliminates the need for high-temperature concentrated acid, enhancing its environmental sustainability while maintaining mild reaction conditions, making it ideal for biosensors application. Additionally, simultaneous extraction and detection of four heavy metals in lotus roots were successfully achieved by using DUED and a fluorescent paper-based microfluidic chip. The obtained results are consistent with those obtained using conventional methods.