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Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with ß-lactamase activity, it is important to identify and characterize ß-lactamases with clinical significance. In this study, the protein sequence of the metallo-ß-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, ß-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against ß-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with kcat/Km values ranging from 6.23 × 102 to 1.02 × 104 M-1 s-1. The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.
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Vibrio vulnificus , beta-Lactamases , Antibacterianos/farmacologia , Cefalosporinas , Monobactamas , Vibrio vulnificus/genética , Inibidores de beta-Lactamases , beta-Lactamases/genéticaRESUMO
This research focuses on the proteolytic capacity of sea bass byproduct (SB) and their hypocholesterolemic activity via the cholesterol micelle formation (CMF) inhibition. SB was fermented with seven mixed lactic acid bacteria for 5 h at 42 °C. The lactic fermented SB was hydrolyzed with Protease N for 6 h under HHP to obtain the SB hydrolysates (HHP-assisted Protease N hydrolysis after fermentation, F-HHP-PN6). The supernatant was separated from the SB hydrolysate and freeze-dried. As the hydrolysis time extended to 6 h, soluble protein content increased from 187.1 to 565.8 mg/g, and peptide content increased from 112.8 to 421.9 mg/g, while inhibition of CMF increased from 75.0% to 88.4%. Decreasing the CMF inhibitory activity from 88.4% to 42.1% by simulated gastrointestinal digestion (FHHP-PN6 was further hydrolyzed by gastrointestinal enzymes, F-HHP-PN6-PP) reduced the CMF inhibitory activity of F-HHP-PN6. Using gel filtration chromatography, the F-HHP-PN6-PP was fractioned into six fractions. The molecular weight of the fifth fraction from F-HHP-PN6-PP was between 340 and 290 Da, and the highest inhibitory efficiency ratio (IER) on CMF was 238.9%/mg/mL. Further purification and identification of new peptides with CMF inhibitory activity presented the peptide sequences in Ser-Ala-Gln, Pro-Trp, and Val-Gly-Gly-Thr; the IERs were 361.7, 3230.0, and 302.9%/mg/mL, respectively.
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Bass/metabolismo , Colesterol/química , Hidrolisados de Proteína/farmacologia , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Antioxidantes/farmacologia , Fermentação , Hidrólise , Pressão Hidrostática , Micelas , Peso Molecular , Oligopeptídeos , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Hidrolisados de Proteína/metabolismo , Proteínas/química , Proteínas/metabolismo , ProteóliseRESUMO
In this study, the low-molecular-weight (LMW) fucoidan, rich in fucose and sulfate, was extracted and purified from the edible brown seaweed, Laminaria japonica. In this study, we orally administered LMW fucoidan to mice for 6 weeks. We then examined fucoidan's effects on innate immunity, adaptive immunity, and Mycoplasma pneumoniae (MP)-antigen-stimulated immune responses. Our data showed that LMW fucoidan stimulated the innate immune system by increasing splenocyte proliferation, natural killer (NK) cell activity, and phagocytic activity. LMW fucoidan also increased interleukin (IL)-2, IL-4, and interferon (IFN)-γ secretion by splenocytes and immunoglobulin (Ig)-G and IgA content in serum, which help regulate adaptive immune cell functions, and decreased allergen-specific IgE. In MP-antigen-stimulated immune responses, the IgM and IgG content in the serum were significantly higher in the LMW fucoidan group after MP-antigen stimulation. Our study provides further information about the immunomodulatory effects of LMW fucoidan and highlights a potential role in preventing M. pneumoniae infection.
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Suplementos Nutricionais , Pneumonia por Mycoplasma/prevenção & controle , Polissacarídeos/administração & dosagem , Substâncias Protetoras/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Laminaria/química , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/microbiologia , Polissacarídeos/química , Substâncias Protetoras/químicaRESUMO
Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Vibrio cholerae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/química , Clonagem Molecular , Citosol/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Espaço Extracelular/metabolismo , Técnicas de Inativação de Genes , Hidrólise , Fosfatos/metabolismo , Vanadatos/farmacologia , Vibrio cholerae/genéticaRESUMO
The combined effects of low-molecular-weight fucoidan (LMF) and fucoxanthin (Fx) in terms of antihyperglycemic, antihyperlipidemic, and hepatoprotective activities were investigated in a mouse model of type II diabetes. The intake of LMF, Fx, and LMF + Fx lowered the blood sugar and fasting blood sugar levels, and increased serum adiponectin levels. The significant decrease in urinary sugar was only observed in LMF + Fx supplementation. LMF and Fx had ameliorating effects on the hepatic tissue of db/db mice by increasing hepatic glycogen and antioxidative enzymes, and LMF was more effective than Fx at improving hepatic glucose metabolism. As for glucose and lipid metabolism in the adipose tissue, the expression of insulin receptor substrate (IRS)-1, glucose transporter (GLUT), peroxisome proliferator-activated receptor gamma (PPARγ), and uncoupling protein (UCP)-1 mRNAs in the adipose tissue of diabetic mice was significantly upregulated by Fx and LMF + Fx, and levels of inflammatory adipocytokines, such as adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were significantly modulated only by LMF + Fx supplementation. The efficacy of LMF + Fx supplementation on the decrease in urinary sugar and on glucose and lipid metabolism in the white adipose tissue of db/db mice was better than that of Fx or LMF alone, indicating the occurrence of a synergistic effect of LMF and Fx.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Polissacarídeos/farmacologia , Xantofilas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Insulina/metabolismo , Interleucina-6/metabolismo , Fígado/metabolismo , Camundongos , Peso Molecular , PPAR gama/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
For a long time, fucoidan has been well known for its pharmacological activities, and recently low molecular weight fucoidan (LMF) has been used in food supplements and pharmaceutical products. In the present study, LMF was extracted from Laminaria japonica by enzyme hydrolysis. The toxicity of LMF in mouse and rat models was determined by many methods, such as total arsenic content, bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus assay. The present findings showed that LMF at 5000 µg/mL exhibited no mutagenicity. It also produced no formatting disruption of red blood cells in vivo. At 2000 mg/kg BW/day there were no toxicological indications. LMF is expected to be used as a safe food supplement.
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Polissacarídeos/efeitos adversos , Animais , Células CHO , Linhagem Celular , Cricetulus , Suplementos Nutricionais/efeitos adversos , Feminino , Laminaria/química , Masculino , Camundongos , Peso Molecular , Ratos , Ratos Sprague-DawleyRESUMO
The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type ß-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.
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Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Pseudomonas/enzimologia , Sefarose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Galactosídeos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Oligossacarídeos/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Pseudomonas/genéticaRESUMO
The fermented marine alga Porphyra dentata suspension was tested for its fibrinolytic and immune-enhancing activities. An isolated Bacillus subtilis N2 strain was selected for its fibrinolytic activity on fibrin plates. After investigating the effects of biomass amounts of P. dentata powder in water, various additives including sugars, nitrogen-containing substances, lipids and minerals, and cultural conditions of temperature and agitation in flask, the highest fibrinolytic activity in the cultural filtrate was obtained by cultivating N2 strain in 3% (w/v) P. dentata powder suspension containing 1% peanut oil at 37 °C, 150 rpm for 48 h. A fermentor system was further established using the same medium with controlled pH value of 7.0 at 37 °C, 150 rpm, 2.0 vvm for 48 h for the best fibrinolytic activity. The fermented product also showed its immune-enhancing activity by increasing cell proliferation and stimulating the secretion of IL-1ß, IL-6, and TNF-α in J774.1 cells.
Assuntos
Bacillus subtilis/metabolismo , Fermentação , Fibrinolíticos/metabolismo , Imunomodulação/efeitos dos fármacos , Porphyra/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Manipulação de Alimentos , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Suspensões , TemperaturaRESUMO
Traditionally, the processing of Gelidium seaweed into Gelidium jelly was very complicated, and involved repeated washing with water and sun drying for seven rounds. The seaweed, which is originally reddish-purple in color, turns yellow in color after the repeated washing and sun drying cycles. However, the sun drying process can only be used on sunny days. Therefore, this study evaluated an alternative method, halogen lamp drying, and compared the qualities of the product, Gelidium jelly, made using the halogen lamp drying and traditional sun drying methods. The properties investigated included the agar yield, gelling temperature, hardness, springiness, rheological parameters, sensory attributes, color, and volatile compounds. The results demonstrated that the halogen lamp drying method required 12 washing and drying cycles to achieve similar jelly properties to seven rounds of sun drying in the experimental conditions. Volatiles including heptanal, ß-ionone, and (E)-2-decenal could be used as indicators to monitor the washing and drying processes. Halogen lamp drying could be an alternative processing method for seaweed drying, especially on rainy days.
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The interactions among agar, gellan gum, gelatin, and modified waxy corn starch in the formation of mixed gels were examined in five different ratios. Binary hydrocolloid gels were prepared using three ingredients: two hydrocolloids (total hydrocolloid concentration: 0.5 wt%, ratios of mixture: 0/0.5, 0.1/0.4, 0.2/0.3, 0.3/0.2, 0.4/0.1, and 0.5/0) and water. The textural properties of the hydrocolloid gels were studied by measuring the gel strength, rigidity, breaking force, breaking point, and syneresis as functions of the mixing ratio. The higher syneresis percentage of binary modified waxy corn starch and gum gels than that of mixed gum gels after cold storage was mainly due to the retrogradation of amylopectin. Agar was shown be the most influential with regards to increasing the gel strength, breaking force, and rigidity among the three kinds of gum, while gellan gum was more effective against syneresis than agar and gelatin for storage periods of 7 and 14 days. In the mixed gels, a dramatic increase in the breaking point from 0 to 0.5% was only exhibited for gellan gum. The results provided useful information, including gel strength, rigidity, breaking force, breaking point, and syneresis, for gum and modified corn starch ingredients selected from refrigerated binary gum gels such as pudding for food product development.
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This study examined the physicochemical effects of the fortification of noodles with 0.25-1.00% (w/w) calcium salts, viz. calcium acetate, calcium carbonate, calcium citrate, and calcium lactate. Fortification with calcium citrate, calcium acetate, and calcium carbonate increased the pH and breaking force of the dried noodles. However, the fortification of noodles with any concentration of calcium did not increase the extent of elongation of the control raw noodles. The L* and b* values of the raw and dried noodle color increased with increasing concentrations of calcium salts, except for noodles with added calcium citrate. Fortification with calcium citrate yielded no significant influence on color, texture, adhesiveness, springiness, flavor, and overall scores for cooked noodles. Noodles fortified with 0.5% calcium citrate made from oyster shells were compared with a control sample of noodles and noodles fortified with commercially available calcium citrate. The particle size of the calcium citrate made from oyster shells (258 nm) was smaller than that of the purchased calcium citrate (2631 nm). Noodles fortified with calcium citrate made from oyster shells showed no significantly difference compared to noodles fortified with commercially available calcium citrate. These results suggest that calcium citrate made from oyster shells may be used as the additive of choice for the manufacture of calcium-fortified noodles.
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The objectives of this study were to evaluate the effect of 0.5% chitosan incorporation on acrylamide development in a food model solution containing 0.5% fructose and asparagine after heating for 30 min at 180 °C. All the solutions were investigated for the following characteristics: acrylamide, asparagine, reducing sugar content, color, kinematic viscosity, Maillard reaction products (MRPs), and pH every 10 min. After heating for 10 min, the viscosity of chitosan-containing solutions reduced significantly. The investigational data confirmed that chitosan may have decomposed into lower molecular structures, as demonstrated by the reduced viscosity of the solution at pH < 6 and a decrease in the acrylamide content during 30 min of heating in a fructose−asparagine system. This study also confirms that the formation of ultraviolet-absorbing intermediates and browning intensity of MRPs containing acrylamide prepared by fructose−asparagine was more than those of MRPs prepared by glucose−asparagine solution system. MRPs containing acrylamide resulted from the reaction of asparagine with fructose (ketose) rather than glucose (aldose). Acrylamide formation could be significantly mitigated in the fructose−asparagine−chitosan model system as compared to the fructose−asparagine model system for possible beverage and food application.
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ß-lactam-resistant Vibrio strains are a significant clinical problem, and ß-lactamase inhibitors are generally coadministered with ß-lactam drugs to control drug-resistant bacteria. Seaweed is a rich source of natural bioactive compounds; however, their potential as ß-lactamase inhibitors against bacterial pathogens remains unknown. Herein, we evaluated the potential ß-lactamase inhibitory effect of the ethanolic extracts of the red seaweed Gracilaria sp. (GE) against four Vibrio strains. The minimum inhibitory concentration, half-maximal inhibitory concentration, checkerboard assay results, and time-kill study results indicate that GE has limited antibacterial activity but can potentiate the activity of the ß-lactam antibiotic carbenicillin against Vibrio parahemolyticus and V. cholerae. We overexpressed and purified recombinant metallo-ß-lactamase, VarG, from V. cholerae for in vitro studies and observed that adding GE reduced the carbenicillin and nitrocefin degradation by VarG by 20% and 60%, respectively. Angiotensin I-converting enzyme inhibition studies demonstrated that GE did not inhibit VarG via metal chelation. Toxicity assays indicated that GE exhibited mild toxicity against human cells. Through gas chromatography and mass spectrometry, we showed that GE comprises alkaloids, phenolic compounds, terpenoids, terpenes, and halogenated aromatic compounds. This study revealed that extracts of the red seaweed Gracillaria sp. can potentially inhibit ß-lactamase activity.
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The World Health Organization indicated that antibiotic resistance is one of the greatest threats to health, food security, and development in the world. Drug resistance efflux pumps are essential for antibiotic resistance in bacteria. Here, we evaluated the plant phenolic compound ethyl 3,4-dihydroxybenzoate (EDHB) for its efflux pump inhibitory (EPI) activity against drug-resistant Escherichia coli. The half-maximal inhibitory concentration, modulation assays, and time-kill studies indicated that EDHB has limited antibacterial activity but can potentiate the activity of antibiotics for drug-resistant E. coli. Dye accumulation/efflux and MALDI-TOF studies showed that EDHB not only significantly increases dye accumulation and reduces dye efflux but also increases the extracellular amount of antibiotics in the drug-resistant E. coli, indicating its interference with substrate translocation via a bacterial efflux pump. Molecular docking analysis using AutoDock Vina indicated that EDHB putatively posed within the distal binding pocket of AcrB and in close interaction with the residues by H-bonds and hydrophobic contacts. Additionally, EDHB showed an elevated postantibiotic effect on drug-resistant E. coli. Our toxicity assays showed that EDHB did not change the bacterial membrane permeability and exhibited mild human cell toxicity. In summary, these findings indicate that EDHB could serve as a potential EPI for drug-resistant E. coli.
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We have investigated different properties (thickness, moisture loss, oil uptake, breaking force, color, puffing ratio during 0.5-5 min frying, microstructure, and sensory evaluation) of raw pork skins with varying thickness (2, 3, and 4 mm) after drying, intended as deep-fried snacks. We have found that the oil content, breaking force, and puffing ratio of fried pork skin with different raw skin thickness have no significant difference under similar water content (1.68-1.98 g/100 g wet weight basis, wb) after 3-5 min of deep-frying at 180 °C. Additionally, sensory score results have shown that fried pork skins with 4 mm raw skin thickness had lower flavor, texture, and overall acceptability than those with 2 mm and 3 mm raw skin thickness. Scanning electron micrographs (SEM) have revealed less holes and irregular and crack microstructure in fried pork skins with 4 mm raw skin thickness than in other groups. Different thickness of raw pork skins resulted in different effects in microstructure and influenced water evaporation and oil uptake of fried pork skin. Finally, we have proposed the kinetic equations of water loss and oil uptake of fried pork skins. Fried pork skin from raw skin thicker than 4 mm need frying at temperature higher than 180 °C to improve their puffing ratio and sensory acceptability.
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The aim of this research was to investigate the effects of the addition of 0.5% hydroxymethylfurfural (HMF) and low molecular chitosan on acrylamide and HMF formation in a food model system, which contains 0.5% glucose, asparagine, and HMF within 30 min of heating at 180 °C. At an interval of 10 min, all solutions were evaluated in the following aspects: reducing sugar, asparagine, acrylamide, HMF content, pH, Maillard reaction products, kinematic viscosity, and color. After heating for 10 min, the kinematic viscosity of solutions containing chitosan reduced significantly. The values of the acrylamide, HMF, and absorbance increased at OD294 and OD420 (optical density measured at 294 nm and 420 nm) of solutions. Experimental results showed that low-molecular-weight chitosan might be hydrolyzed into much lower molecular weight, followed by the decrease in kinematic viscosity of the solution at pH lower than 6 and the increase in the formation of acrylamide after heating for 30 min.
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The fat content of fried pork rinds is high, and alternative frying helps reduce the oil content and maintain their texture and taste. Different frying methods such as microwave-assisted, traditional deep frying and vacuum frying on the breaking force, color, microstructure, water loss and oil absorption attributes of fried pork rinds were evaluated in this study. The fat content of microwave-assisted and vacuum-fried pork rinds was lower (24.2 g/100 g dry weight basis (db) and 17.1 g/100 g db, respectively) than that (35.6 g/100 g db) of traditional deep-fat frying. Non-uniform, holy and irregular surface microstructures were obtained by vacuum frying due to rapid mass transfer at low pressure. The first-order kinetic models of water loss and oil absorption of traditional and microwave-assisted frying of pork rinds were established. Microwave frying caused a faster moisture loss rate, shorter frying time and lower pork rind oil content, makes it an attractive substitute for traditional deep-fat frying.
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Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.
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Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB's efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB's substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.
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Periodontitis is an inflammatory disease that can lead to the periodontal pocket formation and tooth loss. This study was aimed to develop antimicrobials loaded hydrogels composed of cellulose nanofibers (CNF) and κ-carrageenan oligosaccharides (CO) nanoparticles for the treatment of periodontitis. Two antimicrobial agents such as surfactin and Herbmedotcin were selected as the therapeutic agents and the hydrogels were formulated based on the increasing concentration of surfactin. The proposed material has high thermal stability, controlled release, and water absorption capacity. This study was proceeded by investigating the in vitro antibacterial and anti-inflammatory properties of the hydrogels. This material has strong antibacterial activity against periodontal pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, and Pseudomonas aeruginosa. Moreover, a significant increase in malondialdehyde (MDA) production and a decrease in biofilm formation and metabolic activity of the bacteria was observed in the presence of hydrogel. Besides, it reduced the reactive oxygen species (ROS) generation, transcription factor, and cytokines production in human gingival fibroblast cells (HGF) under inflammatory conditions. In conclusion, the hydrogels were successfully developed and proven to have antibacterial and anti-inflammatory properties for the treatment of periodontitis. Thus, it can be used as an excellent candidate for periodontitis treatment.