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Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade natures polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

BackgroundTraditional hookah smoking has grown quickly to become a global tobacco epidemic. More recently, electronic hookahs (e-hookahs)-vaped through traditional water pipes-were introduced as healthier alternatives to combustible hookah. With combustible tobacco smoking, oxidative stress, inflammation, and vascular stiffness are key components in the development and progression of atherosclerosis. The comparable effects of hookah are unknown.Research questionWhat is the differential acute effect of e-hookah vaping vs combustible hookah smoking on oxidation, inflammation, and arterial stiffness?Study design and methodsIn a randomized crossover design study, among a cohort of 17 healthy young adult chronic hookah smokers, we investigated the effect of e-hookah vaping and hookah smoking on measures of conduit arterial stiffness, including carotid-femoral pulse wave velocity (PWV), augmentation index-corrected for heart rate before and after a 30-min exposure session. We assessed a panel of circulating biomarkers indicative of inflammation and oxidants and measured plasma nicotine and exhaled carbon monoxide (CO) levels before and after the sessions.Resultse-Hookah vaping tended to lead to a larger acute increase in PWV than hookah smoking (mean ± SE: e-hookah, +0.74 ± 0.12 m/s; combustible hookah, +0.57 ± 0.14 m/s [P < .05 for both]), indicative of large artery stiffening. Compared with baseline, only e-hookah vaping induced an acute increase in augmentation index (e-hookah, +5.58 ± 1.54% [P = .004]; combustible hookah, +2.87 ± 2.12% [P = not significant]). These vascular changes were accompanied by elevation of the proinflammatory biomarkers high-sensitivity C-reactive protein, fibrinogen, and tumor necrosis factor α after vaping (all P < .05). No changes in biomarkers of inflammation and oxidants were observed after smoking. Compared with baseline, exhaled CO levels were higher after smoking than after vaping (+36.81 ± 6.70 parts per million vs -0.38 ± 0.22 parts per million; P < .001), whereas plasma nicotine concentrations were comparable (+6.14 ± 1.03 ng/mL vs +5.24 ± 0.96 ng/mL; P = .478).InterpretationAlthough advertised to be "safe," flavored e-hookah vaping exerts injurious effects on the vasculature that are, at least in part, mediated by inflammation.Trial registryClinicalTrials.gov; No.: NCT03690427; URL: www.clinicaltrials.gov.

BackgroundSinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.MethodsWe evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788-6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion.ResultsOverall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/- CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling.ConclusionCollectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

Plants emit high rates of methanol (meOH), generally assumed to derive from pectin demethylation, and this increases during abiotic stress. In contrast, less is known about the emission and source of acetic acid (AA). In this study, Populus trichocarpa (California poplar) leaves in different developmental stages were desiccated and quantified for total meOH and AA emissions together with bulk cell wall acetylation and methylation content. While young leaves showed high emissions of meOH (140 μmol m-2) and AA (42 μmol m-2), emissions were reduced in mature (meOH: 69%, AA: 60%) and old (meOH: 83%, AA: 76%) leaves. In contrast, the ratio of AA/meOH emissions increased with leaf development (young: 35%, mature: 43%, old: 82%), mimicking the pattern of O-acetyl/methyl ester ratios of leaf bulk cell walls (young: 35%, mature: 38%, old: 51%), which is driven by an increase in O-acetyl and decrease in methyl ester content with age. The results are consistent with meOH and AA emission sources from cell wall de-esterification, with young expanding tissues producing highly methylated pectin that is progressively demethyl-esterified. We highlight the quantification of AA/meOH emission ratios as a potential tool for rapid phenotype screening of structural carbohydrate esterification patterns.

Extremozymes from halophiles that thrives in extreme hypersaline habitats have been the subject of extensive and expanding research owing to their value in a variety of industrial applications. Esterases and lipases are the most industrially applied enzymes due to their specific hydrolysis of ester bonds. In the present study, esterase and lipase producing extreme and moderate halophilic strains were isolated from brackish water lake Tso Kar of Ladakh in India. All the fifteen isolates were screened for esterase and lipase production. Among the fifteen isolates, two isolates, TKW1 and TKW12, having extracellular and intracellular esterase activity, and one isolate, TKW3, having intracellular lipase activity, were selected for identification using 16S rRNA gene sequence analysis and media optimization to determine the optimum production of esterase/lipase using a "one variable at a time" strategy. The cumulative effect of all the optimized conditions led to maximu m esterase production of about 82.59 U/ml, 136.9 U/ml after 48 hours and 36 hrs by TKW1 and TKW12, respectively, and maximum lipase of about 199.4 U/ml after 60 hrs by TKW3. These strains had marked hal-ophilic esterase and lipase properties, indicating wide potential applications in industrial biotechnology as salt-adapted haloalkaliphilic enzymes. This is the first report of the isolation and molecular identification of an esterase and lipase producing halo-philic bacterium from the Tso Kar brackish water lake. [ABSTRACT FROM AUTHOR]

ContextIn end-stage renal disease (ESRD), serum high-density lipoprotein cholesterol (HDL-C) level is not an accurate predictor of mortality, partly because it does not necessarily correlate with indices of HDL function. Paraoxonase (PON) is a major enzyme constituent of HDL and a key component of HDL antioxidant activity. Apolipoprotein A-I (Apo A-1) is the core HDL structural protein that plays a major role in various aspects of HDL function.ObjectiveWe sought to examine PON activity and Apo A-I levels in patients with ESRD vs healthy controls.Design and settingPON/arylesterase activity was measured in 499 patients with maintenance hemodialysis (MHD) and 24 healthy controls with similar distributions of age, sex, and race/ethnicity. Serum acrolein-modified Apo A-I was measured in 30 patients with MHD and 10 healthy controls.Main outcome measuresMultilevel Cox models were used to assess associations among PON activity, Apo A-I, and HDL-C levels with 12-month all-cause mortality.ResultsPON activity was significantly lower in patients with MHD vs controls. Furthermore, acrolein-modified Apo A-I levels were higher in patients with MHD vs controls. In fully adjusted models, high PON activity was associated with lower 12-month mortality, whereas no difference of mortality risk was observed across HDL-C levels. The combination of high PON and low Apo A-I compared with low PON and low Apo A-I was associated with lower mortality risk.ConclusionsIn patients with MHD, PON activity had a stronger association with 12-month mortality than HDL-C. Future studies are needed to examine the role of these markers as potential diagnostic and therapeutic tools in ESRD.

Reports of the test accuracy of the urinalysis for diagnosing urinary tract infections (UTIs) in young febrile infants have been variable. We evaluated the test characteristics of the urinalysis for diagnosing UTIs, with and without associated bacteremia, in young febrile infants. We performed a planned secondary analysis of data from a prospective study of febrile infants ≤60 days old at 26 emergency departments in the Pediatric Emergency Care Applied Research Network. We evaluated the test characteristics of the urinalysis for diagnosing UTIs, with and without associated bacteremia, by using 2 definitions of UTI: growth of ≥50 000 or ≥10 000 colony-forming units (CFUs) per mL of a uropathogen. We defined a positive urinalysis by the presence of any leukocyte esterase, nitrite, or pyuria (>5 white blood cells per high-power field). Of 4147 infants analyzed, 289 (7.0%) had UTIs with colony counts ≥50 000 CFUs/mL, including 27 (9.3%) with bacteremia. For these UTIs, a positive urinalysis exhibited sensitivities of 0.94 (95% confidence interval [CI]: 0.91-0.97), regardless of bacteremia; 1.00 (95% CI: 0.87-1.00) with bacteremia; and 0.94 (95% CI: 0.90-0.96) without bacteremia. Specificity was 0.91 (95% CI: 0.90-0.91) in all groups. For UTIs with colony counts ≥10 000 CFUs/mL, the sensitivity of the urinalysis was 0.87 (95% CI: 0.83-0.90), and specificity was 0.91 (95% CI: 0.90-0.92). The urinalysis is highly sensitive and specific for diagnosing UTIs, especially with ≥50 000 CFUs/mL, in febrile infants ≤60 days old, and particularly for UTIs with associated bacteremia.

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

This work describes the use of computational strategies to design megamolecule building blocks for the self-assembly of lattice networks. The megamolecules are prepared by attaching four Cutinase-SnapTag fusion proteins (CS fusions) to a four-armed linker, followed by functionalizing each fusion with a terpyridine linker. This functionality is designed to participate in a metal-mediated self-assembly process to give networks. This article describes a simulation-guided strategy for the design of megamolecules to optimize the peptide linker in the fusion protein to give conformations that are best suited for self-assembly and therefore streamlines the typically time-consuming and labor-intensive experimental process. We designed 11 candidate megamolecules and identified the most promising linker, (EAAAK) 2 , along with the optimal experimental conditions through a combination of all-atom molecular dynamics, enhanced sampling, and larger-scale coarse-grained molecular dynamics simulations. Our simulation findings were validated and found to be consistent with the experimental results. Significantly, this study offers valuable insight into the self-assembly of megamolecule networks and provides a novel and general strategy for large biomolecular material designs by using systematic bottom-up coarse-grained simulations.

To investigate the characteristics of Cd transport and distribution in the wild rice roots, Oryza rufupogon Griff. and Oryza officinalis Wall. were used at research materials. Pot experiment was conducted to study the effects of different concentrations of Cd (0, 1, 5, 15 and 30 mg∙kg -1 ) treatments on the root pectin content, root pectin methyl esterase(PME) activity, Cation/proton exchanger(CAX), heavy metal ATPase(HMA) and ATP-binding cassette protein(ABC) activities of wild rice. The difference in Cd flow rate of wild rice root was investigated through non-invasive micro-test technique (NMT). The results showed that the Cd in roots tended to increase with the increase of Cd treatment concentrations. Compared to the CK treatment (0 mg∙kg -1 Cd), the root pectin content of O. rufupogon and O. officinalis under 30 mg∙kg -1 Cd stress were significantly increased by 126.73 % and 109.69 %, respectively. The distribution of Cd contents in wild rice roots were all greater than those in shoots. Under Cd stress, the translocation factor of O. rufupogon (0.46-1.44) were all higher than those of O. officinalis (0.15-0.37). The Cd transport capacity in O. rufupogon was more stronger than in O. officinalis. The Cd content in the xylem sap of O. officinalis was lower than that of O. rufupogon. Compared to the CK, CAX and ABC activities in O. officinalis root under 30 mg∙kg -1 Cd treatment were significantly increased by 11.03 % and 2.37 %, respectively. HMA activity in O. rufupogon root was significantly increased by 4.95 %. Cd flow rate in the root xylem of O. officinalis was higher than that of O. rufupogon. In root cells, the subcellular distribution of Cd contents showed cell wall > soluble components> organelles. With the Cd treatment concentrations increased, the percentage of Cd content in the soluble fractions of the root cells of wild rice showed an increasing. In general, the result showed that O. officinalis immobilized more Cd in the roots by influencing the pectin methyl esterification reaction through PME activity. Under high Cd stress, Cd was immobilized in root cell vacuoles of O. officinalis by enhancing roots CAX and ABC activities, whereas HMA activities were enhanced of O. rufupogon to reduce the damage caused by Cd., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The influence of four microbial biostimulants containing various strains of Bacillus subtilis and/or Paenibacillus sp. on the quality of raspberries cv. Delniwa, Poemat, and Enrosadira cultivated in two consecutive seasons was investigated. The biostimulants influenced the antioxidant level, antioxidant capacity, phenolic acids and flavonoids profiles, enzymatic activity, and the degree of methylation and acetylation of the pectin in the raspberry fruits. The biostimulants had the greatest effect on the antioxidant content (16% - 20% increase) and capacity in the Delniwa raspberry fruits from the first season. A positive correlation was found between the activity of the β-galactosidase enzyme and ferric reducing power. In the second season, a decrease in the activity of pectin esterase and α-L-arabinofuranosidase and an increase in the degree of methylation of pectin were noted. Our results suggest that the changes in raspberry quality were related to the type of biostimulant applied., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Magdalena Frac reports financial support was provided by National Centre for Research and Development Poland. Magdalena Frac has patent #Method for obtaining a bacterial biopreparation and a bacterial biopreparation for maintenance and/or improving soil microbial biodiversity while controlling pathogens: Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in soft fruit cultivation (in Polish), P.445051 pending to Institute of Agrophysics Polish Academy of Sciences. Magdalena Frac has patent #Method for obtaining a microbial fertilizing product and a microbial fertilizing product for conditioning seedlings, maintaining and/or improving the microbiological quality of the soil, while allowing for the control of phytopathogens in the cultivation of soft fruit (in Polish), P.445052 pending to Institute of Agrophysics Polish Academy of Sciences. Magdalena Frac has patent #A method of obtaining a microbiological fertilizing product and a microbiological fertilizing product for maintaining and/or improving the microbiological quality of the soil, allowing at the same time to control the phytopathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit (in Polish), P.445053 pending to Institute of Agrophysics Polish Academy of Sciences. Magdalena Frac has patent #Method for obtaining a microbial fertilizing product and a microbial fertilizing product for soil conditioning and improving its biological properties while controlling pathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in soft fruit cultivation (in Polish), P.445054 pending to Institute of Agrophysics Polish Academy of Sciences., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lower urinary tract infection (UTI) is common but only rarely complicated by pyelonephritis. However, the mechanisms preventing extension to the kidney are unclear. Here, we identified neutrophil extracellular traps (NETs) in healthy human urine that provide an antibacterial defense strategy within the urinary tract. In both in vivo murine models of UTI where uropathogenic E. coli are inoculated into the bladder and ex vivo human urine models, NETs interacted with uromodulin to form large webs that entrapped the bacteria. Peptidyl arginine deiminase 4 (PADI4) inhibition in mice blocked NETosis and resulted in progression of cystitis into pyelonephritis, suggesting that NETosis of urinary neutrophils acts to prevent bacterial ascent into the kidney. Analysis of UK Biobank data revealed that genetic variants in PADI4 that associated with increased risk of rheumatoid arthritis in multiple genome-wide association studies were consistently associated with reduced susceptibility to UTI. Last, we showed that urine dipstick testing for leukocyte esterase was negative in the presence of intact blood neutrophils but became positive when neutrophils were stimulated to NET, and this could be prevented by selective PADI4 inhibition, demonstrating that this test does not detect absolute neutrophil count, as has long been assumed, but specifically detects neutrophils that have undergone NETosis. These findings highlight the role of NETosis in preventing ascending infections in the urinary tract and improve our understanding of one of the most common clinical tests in medicine.

Background: In adult females, mycoplasma infection is common and challenging to diagnose. This study aimed to use retrospective laboratory data to construct a nomogram for predicting the mycoplasma infection of individuals with probable urogenital tract mycoplasma infection., Methods: A total of 2,859 patients with suspected urogenital tract mycoplasma infection were retrospectively enrolled in this study. Demographics and routine examinations of leucorrhea were used to develop a nomogram for predicting mycoplasma infection. The least absolute shrinkage and selection operator (LASSO) method was applied to filter variables and select predictors, and multivariable logistic regression was used to construct a nomogram. The discriminatory ability of the model was determined by calculating the area under the curve (AUC). The performance and clinical utility of the nomogram were generated by using Harrell's concordance index, calibration curve, and decision curve analysis (DCA)., Results: By using the LASSO regression method, seven variables (age, white blood cell, epithetical cell, cleanliness, candidiasis vaginalis, sialidases, and leukocyte esterase) were chosen, and a nomogram was constructed using these variables. The prediction nomogram (0.676, 95% CI: 0.611 - 0.744) demonstrated a satisfactory performance. The prediction model's AUC was 0.679 (95% CI: 0.660 - 0.691). Furthermore, the DCA showed a good clinical net benefit based on the mycoplasma infection nomogram., Conclusions: A nomogram was created in this study, which included seven demographic and clinical characteristics of female patients. The nomogram could be of great value for the diagnosis of mycoplasma infection.

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO • ) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO • , a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO • . Enzyme mixtures (1:1:1 ratio) were exposed to ROO • produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP + generated ∼175 μM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 μM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An organic solvent-free method based on limited dosing options (biocatalyst and zinc chloride) for the quick and mild recovery of chlorophyll (Chl) from spinach has been proposed. This tailored, custom-made protocol has been designed to produce stable green natural colorants. The kinetics of pigment extraction turned out to be a very useful tool to identify the proper conditions, in terms of biocatalyst dose (0.10-50 U/g), extraction time (1-48h), and ZnCl 2 amount (50-300ppm), both for enhancing the recovery yield and preserving the green color. Considering the extraction kinetics, the recovery yield, and the colorimetric data, the suitable conditions to produce stable green and food-grade colorants are 0.10 U/g of enzyme, 3h, and 150ppm of ZnCl 2 at 25°C. The extraction yield of Chl (4863µg/U) was about 51% greater than control, with a higher extraction rate constant (5.43 × 10 -4  g/(µg min)). Considering the impact of ZnCl 2 amount on Chl, its protective action resulted to be more noticeable toward Chl a: at 150ppm, an increased amount of about 2.5 and 1.5 times was found for Chl a and Chl b, respectively, in comparison to the reference (0ppm ZnCl 2 ). PRACTICAL APPLICATION: This research demonstrates how a suitable kinetic approach helps to provide a tailored protocol, customized for the vegetable matrix, to produce stable green natural colorants from spinach. Lowering enzyme dosage and ZnCl 2 amount during the extraction of chlorophyll at low temperature is crucial for its potential use as a colorant in food industry, providing high economic values through saving time and environmental protection., (© 2024 The Author(s). Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

The present study evaluated the effect of ultrasonication prior to microwave heating applied at 60 °C, 70 °C, and 80 °C on the quality characteristics of verjuice. The sole microwave heating and conventional heating were also performed at the same temperature levels, and effectiveness of three different treatments were evaluated. The required treatment times were decided based on obtaining <10% pectin methylesterase (PME) activity, and ultrasound pretreatment provided the least heating durations. Turbidity, browning index, and viscosity values increased by 3.4 to 14.8-fold, 0.24 to 1.26-fold, and 9.2% to 48.0%, respectively, after all thermal treatments, while Brix values decreased by 1.4% to 15.7%. Ultrasound pretreatment revealed relatively lower browning index in all temperature levels, and almost the highest viscosity values were obtained in sonication pretreated microwave heating as compared with sole microwave and conventional heating. The minimum turbidity value (0.035) was determined in ultrasound-assisted microwave heating at 60 °C. The highest antioxidant capacity (DPPH and ABTS) values were achieved by ultrasound-assisted microwave heating (up to 4.96 and 28.4 mmol Trolox equivalent (TE)/kg, respectively) followed by microwave heating (up to 4.30 and 27.0 mmol TE/kg) and conventional heating (up to 3.72 and 26.8 mmol TE/kg). Furthermore, ultrasonication resulted in better retentions of PME residual activity during 60 days of refrigerated storage (4 °C). Ultrasound pretreatment prior to microwave heating could be a convenient approach for juice processing by reducing the required treatment time and by conserving the quality parameters., Competing Interests: DECLARATION OF CONFLICTING INTERESTSThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.