Optimization Of Conditions Research Articles

Optimizing Conditions to Combat Antibiotic Resistance in Pathogenic Bacteria

Background: Antimicrobial resistance (AMR) is a significant adaptive trait that allows pathogenic bacterial subpopulations to out-compete and out-survive their microbial neighbors and overcome host defenses. Specific Background: Despite extensive research, the influence of various environmental parameters on antibiotic sensitivity in pathogenic bacteria remains underexplored. Knowledge Gap: There is limited understanding of how temperature, pH, bacterial inoculum volume, and culture medium amount affect the antibiotic resistance of both gram-negative and gram-positive bacteria. Aims: To investigate the effects of these parameters on the antibiotic sensitivity of four standard laboratory strains: Escherichia coli, Proteus spp., Klebsiella spp., and Staphylococcus aureus. Results: Our findings indicate imipenem exhibited the highest sensitivity, with percentages varying significantly based on temperature (92% at 35-39°C), pH (83% at pH 6-8), inoculum volume (42% at 0.1-1.0 μL), and medium volume (67% at 15-35 ml). Conversely, antibiotics such as Piperacillin, Amoxicillin, Erythromycin, Tetracycline 30, and Cephalexin showed high resistance, with Tetracycline 10 being the most resistant. Novelty: This study highlights the significant impact of environmental conditions on bacterial antibiotic resistance, emphasizing the need for tailored antibiotic use based on specific bacterial characteristics and growth conditions. Implications: The results suggest that optimizing environmental parameters can enhance antibiotic efficacy and inform better clinical practices to combat AMR, thus improving treatment outcomes for bacterial infections. Highlights: Parameter Influence: Temperature, pH, inoculum, medium amount affect antibiotic sensitivity. Highest Sensitivity: Imipenem most effective across conditions. Tailored Use: Optimize conditions for better antibiotic efficacy. Keywords: Antimicrobial resistance, bacterial sensitivity, environmental parameters, Imipenem, pathogenic bacteria

Characterization of protease-producing bacteria from garden soil and antagonistic activity against pathogenic bacteria

Bacteria play an essential role in various industrial processes, including food, medicine, and detergents, due to their ability to produce protease, a hydrolytic enzyme. This study aimed to identify and characterize protease-producing bacterial strains isolated from garden soil, evaluate their antibiotic susceptibility, and optimize conditions for enhanced protease production. Using skimmed milk agar medium and the gelatin hydrolysis method, 7 bacterial strains (S1, S2, S3, S4, S5, S6, and S7, respectively) were identified for protease production in garden soil. The bacteria were identified based on morphological studies, biochemical characterization, and the BIOLOG™ system, and the isolates were determined to be Bacillus cereus (S1), B. thuringiensis (S2), B. subtilis (S3), B. amyloliquefaciens (S4), Pseudomonas aeruginosa (S5), Macrococcus brunensis (S6), and B. schlegelii (S7). The antibiotic susceptibility test, performed using the Kirby-Bauer agar disc diffusion method, revealed that all isolates were resistant to penicillin, nitrofurantoin, and metronidazole. Additionally, B. subtilis and P. aeruginosa demonstrated antagonistic activity against pathogens. Optimal conditions for protease production were found to be a temperature range of 30°C to 40°C and a pH range of 6.5–7.5. Under these conditions, B. cereus (2.19 U/ml) and B. thuringiensis (2.12 U/ml) exhibited the highest protease activity. Further characterization of protease activity in B. cereus, P. aeruginosa, and M. brunensis under different physiological parameters revealed that maximum activity occurred within a pH range of 5.0–6.0 and temperatures between 30°C and 40°C. Protease activity increased in the presence of calcium (CaCl2) but decreased with the addition of urea. These findings underscore the significant industrial applications of proteases produced by these bacterial strains.

Construction of Immobilized Laccase System Based on ZnO and Degradation of Mesotrione.

Mesotrione (MES) is a new environmental pollutant. Some reports have indicated that microbial enzymes could be utilized for MES degradation. Laccase is a green biocatalyst whose potential use in environmental pollutant detoxification has been considered limited due to its poor stability and reusability. However, these issues may be addressed using enzyme immobilization. In the present study, we sought to optimize conditions for laccase immobilization, to analyze and characterize the characteristics of the immobilized laccase, and to compare its enzymatic properties to those of free laccase. In addition, we studied the ability of laccase to degrade MES, and analyzed the metabolic pathway of MES degradation by immobilized laccase. The results demonstrated that granular zinc oxide material (G-ZnO) was successfully used as the carrier for immobilization. G-ZnO@Lac demonstrated the highest recovery of enzyme activity and exhibited significantly improved stability compared with free laccase. Storage stability was also significantly improved, with the relative enzyme activity of G-ZnO@Lac remaining at about 54% after 28 days of storage (compared with only 12% for free laccase). The optimal conditions for the degradation of MES by G-ZnO@Lac were found to be 10 mg, 6 h, 30 °C, and pH 4; under these conditions, a degradation rate of 73.25% was attained. The findings of this study provide a theoretical reference for the laccase treatment of 4-hy-droxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide contamination.

Predictive modeling of garlic quality in hybrid infrared-convective drying using artificial neural networks

Artificial Neural Networks (ANN) application for enhancing food drying processes has been gaining traction within the food industry, particularly because of its potential to optimize conditions while preserving quality. This study delves into the effects of varying convective temperatures (40, 50, 60 °C), air velocities (0.7, 1.0, and 1.5 m/s), and infrared radiation intensities (1500, 2000, 3000 W/m²) on the drying efficiency and quality of garlic slices. Additionally, it explores the capability of ANN to predict optimal drying conditions to balance time efficiency and quality retention. Experimental observations revealed that a combination of a 60°C air temperature, 1.0 m/s air velocity, and 3000 W/m² infrared radiation minimized the drying time to 4.5 h. However, this setting also resulted in a 13.4 % reduction in Allicin content and a decrease in flavor intensity to 4.09 mg/g, underscoring the complexity of optimizing drying conditions without compromising key quality attributes. The study highlights the intricate relationships between drying parameters and garlic quality through precise ANN modeling, which achieved an exceptional fit. Principal Component Analysis (PCA) further elucidated the inverse correlation between drying time and critical quality factors of color, Allicin content, and flavor. Utilizing the Self-Organizing Map (SOM) technique, the study identified five distinct optimization zones, suggesting that lower temperatures, intermediate infrared levels, and higher air velocities present an optimal balance for enhancing garlic slice quality while reducing drying time. This research underscores the potential of advanced computational tools in refining food drying processes, offering valuable insights for the food industry in its quest to improve efficiency without sacrificing product quality.

Optimization of physical and strength performance of cellulose-based fiber additives stabilized expansive soil

Expansive soils are known as geotechnically problematic soils and represent a significant challenge for both civil engineering and geotechnical applications. The primary issue with expansive soils is their susceptibility to moisture-induced volume changes, resulting in both shrinkage and swelling behaviors. This study presents a comprehensive investigation into optimizing the physical and strength properties of expansive soil through the use of cellulose-based fiber additives, namely bamboo fiber (BF), rice husk fiber (RHF), and wheat straw fiber (WSF). Various fiber dosages (5 %, 10 %, and 15 %) and sizes (75 µm, 150 µm, and 300 µm) were employed in combinations to identify the optimal conditions and analyze the soil-fiber reinforcement mechanisms. The experimental design was leveraged by the Taguchi method to optimize conditions, focusing on key response factors such as the Atterberg limit test (PI, LL), free swell ratio (FSR), linear shrinkage (LS), and unconfined compressive strength (UCS) and statistical analysis for results were validated by Analysis of Variance (ANOVA). Additionally, cellulose content and water absorption capacity were assessed to confirm the suitability of cellulose-based fibers as soil stabilizers. Hence, the results demonstrate a substantial enhancement in both the physical and mechanical properties of the stabilized soil with the incorporation of cellulose-based fiber additives. Specifically, the Plastic Index (PI) improved by 85 % when using RHF fibers at a dosage and size of 15 % and 300 µm, respectively. The Free Swell Ratio (FSR) witnessed improvement with WSF fibers at a dosage of 15 % and a size of 150 µm. Linear shrinkage exhibited remarkable improvement, exceeding 95 %, with a combination of 15 % and 75 µm fibers. Furthermore, the Unconfined Compressive Strength (UCS) values were improved by more than 100 % when using 15 % BF fibers with a size of 300 µm. Therefore, the findings of the study highlight that cellulose-based additives as highly effective and sustainable alternatives for soil stabilization, surpassing the engineering performance of traditional soil stabilizers

Optimization of enzyme-assisted microwave extraction of Zanthoxylum limonella essential oil using response surface methodology

Zanthoxylum limonella essential oil possesses potential antimicrobial activity and is of considerable interest as food flavouring and traditional herb. In this study, an enzymolysis-pretreatment-microwave-assisted extraction (EP-MAE) method was used to extract Z. limonella essential oil. The response surface methodology (RSM) with Plackett–Burman design (PBD) and Box-Behnken design (BBD) models were employed to optimize conditions in the EP-MAE method. Seven variables including water to plant ratio, enzyme amount, incubation temperature, incubation time, shaking speed, microwave time, and microwave power were selected to determine the optimal values for extracting Z. limonella essential oil. As the results, four variables including water to plant ratio, enzyme amount, microwave time and power were evaluated as significant variables affecting on yield and volatile compounds of Z. limonella essential oil from both PBD and BBD experiments. The optimum conditions of EP-MAE was obtained as follows: water to plant ratio (11.16 mL/g), enzyme amount (0.68%), microwave time (36.73 min), and power (1665 W). The Z. limonella essential oil composition and its yield from EP-MAE was compared to those extracted from MAE and hydrodistillation. The optimal extraction conditions in the EP-MAE method enhanced significantly higher essential oil yield (7.89 ± 0.08 mg/g) compared to those found by MAE (7.26 ± 0.04 mg/g) and hydrodistillation (7.04 ± 0.03 mg/g), respectively. Fifty-one volatile components were identified among these methods, with similar major compounds of limonene, β-pinene, and α-phellandrene, showing percentage ranging between 34.59–35.78%, 19.91–22.67%, 8.47–8.75%, respectively. However, an extremely higher content of compounds was detected using the EP-MAE method. This study demonstrates the significance of EP-MAE, which may be applied as a more potent extraction method for essential oils in aromatic plants compared to MAE and hydrodistillation.

Optimization of stretching process for improve the textural characteristics of Kashar cheese

This study was aimed to optimize the stretching process (temperature, titration acidity, and NaCl concentration) and to determine the subsequent effects over the quality characteristics of Kashar cheese. The textural and meltability properties were studied in the optimization. Whey was used to set the acidity of stretching water. Textural, meltability, and sensory evaluation scores of matured Kashar cheese obtained from the marketplaces were used for target values ​​to optimize the stretching process. The response surface methodology was used to optimize conditions for stretching process, the arrangement of experimental design and evaluation of analysis results. The optimum conditions were found as follows: 80.20 °C for stretching temperature; 0.61 °SH for stretching water acidity; and 12.00 % for NaCl concentration. Under these conditions: 79.53 N for hardness, -292.24 g.sec for adhesiveness, 0.74 for cohesiveness, 51.20 N for gumminess, and 19.05 mm for meltability values were found in Kashar cheese that was produced for validation. The validation production results were within 95 % confidence intervals for each response obtained at optimal conditions. Thus, this model is successful to predict the textural and meltability values ​​of sample.

Optimization through response surface methodology of 3D printed membrane preparation conditions for use in vanadium redox flow battery: A vanadium/proton selectivity study

AbstractSPEEK membrane applicable in Vanadium Redox Flow Battery (VRFB) have been 3D printed via Fused Deposition Modeling (FDM). Response Surface Methodology (RSM) has been used to optimize conditions for 3D printing membrane. Good membranes characteristics were obtained by manipulating three parameters, namely the printing temperature, the orientation of the printing layer and the sulfonation time. We evaluated the effects of these three variables on the permeability of membranes to protons and vanadium ions. Results have shown that sulfonation is the most significant variable influencing vanadium and proton permeability. The optimal printing conditions were found at 408°C temperature with a horizontal layer orientation, while the best sulfonation is obtained at 6 min. The optimal membrane has an ion exchange capacity of 0.86 mmolg−1, a water uptake ratio of 22%, a VO2+ permeability of 6.1 × 10−8 cm2 min−1, a H+ permeability of 7.0 × 10−6 cm2 min−1, excellent mechanical stability and better H+/VO2+ perm selectivity than conventional sulfonated polyether ether ketone (SPEEK), Fap450 and Nafion211 membranes. The above results show that the 3D‐SPEEK membrane has great advantages and broad prospects for VRFB applications.

Optimization Conditions to Obtain Cationic Polyacrylamide Emulsion Copolymers with Desired Cationic Degree for Different Wastewater Treatments.

The synthesis of cationic polyacrylamides (CPAMs) with the desired cationic degree and molecular weight is essential for various industries, including wastewater treatment, mining, paper, cosmetic chemistry, and others. Previous studies have already demonstrated methods to optimize synthesis conditions to obtain high-molecular-weight CPAM emulsions and the effects of cationic degrees on flocculation processes. However, the optimization of input parameters to obtain CPAMs with the desired cationic degrees has not been discussed. Traditional optimization methods are time-consuming and costly when it comes to on-site CPAM production because the input parameters of CPAM synthesis are optimized using single-factor experiments. In this study, we utilized the response surface methodology to optimize the synthesis conditions, specifically the monomer concentration, the content of the cationic monomer, and the content of the initiator, to obtain CPAMs with the desired cationic degrees. This approach overcomes the drawbacks of traditional optimization methods. We successfully synthesized three CPAM emulsions with a wide range of cationic degrees: low (21.85%), medium (40.25%), and high (71.17%) levels of cationic degree. The optimized conditions for these CPAMs were as follows: monomer concentration of 25%, content of monomer cation of 22.5%, 44.41%, and 77.61%, respectively, and initiator content of 0.475%, 0.48%, and 0.59%, respectively. The developed models can be utilized to quickly optimize conditions for synthesizing CPAM emulsions with different cationic degrees to meet the demands of wastewater treatment applications. The synthesized CPAM products performed effectively in wastewater treatment, with the treated wastewater meeting the technical regulation parameters. 1H-NMR, FTIR, SEM, BET, dynamic light scattering, and gel permeation chromatography were employed to confirm the structure and surface of the polymers.

Electrocoagulation process for oily wastewater treatment and optimization using response surface methodology

AbstractElectrocoagulation technique using aluminum electrodes in a batch bi-polar system was investigated to determine the efficiency of removing oil from oily wastewater. The Box–Behnken design was utilized to optimize conditions and the effects of four independent factors, including oil volume (X1), temperature (X2), initial pH (X3), and treatment time (X4),—were examined to investigate turbidity recovery and conductivity changes. The significant independent variables and their interactions were assessed using ANOVA. The optimal operating conditions for turbidity removal were obtained at an oil volume of 10 ml L−1, a temperature of 28 °C, an initial pH of 4, and a coagulation time of 90 min. The results revealed that turbidity removal and conductivity changes are enhanced significantly with increasing treatment time and decreasing oil volume. The application of the treatment process under optimal operating conditions allows promising removal efficiencies of 97.3%, and 73.4% for turbidity and conductivity, respectively. Also, the treated wastewater showed remarkable changes in removal efficiencies of the main oily wastewater pollution loads for COD, NO2, PO4, DO, and BOD. The economic study indicated that oily wastewater treatment by electrocoagulation is a very cost-effective technique.

Optimization of Solid-Phase Lactobacillus Fermentation Conditions to Increase γ-Aminobutyric Acid (GABA) Content in Selected Substrates

The purpose of this study was to optimize conditions of solid-phase fermentation of lactic acid bacteria to enhance GABA contents in grains. Optimal solid-phase fermentation conditions that could enhance the GABA content after fermenting Oryza sativa (brown rice) were investigated by changing the Lactobacillus strain, fermentation temperature, fermentation time, and inoculated bacteria number. Avena sativa, Cicer arietinum, and red and brown Lens culinaris were then fermented using the optimal solid-phase fermentation conditions to measure changes in GABA content and antioxidant activity. As a result of the experiment, the optimal solid-phase fermentation conditions to enhance the GABA contents in grains were: fermentation time, 48 h; amounts of bacteria, inoculating 5% of 1 × 107 CFU/mL of lactic acid bacteria; and fermentation temperature, 36 °C. When fermented under this condition, the GABA content increased from 4.64 mg/g to 6.93 mg/g (49.0%) compared to unfermented raw material. The results of the DPPH and ABTS radical scavenging activity assays confirmed that both the GABA content and radical scavenging activity were increased after fermentation. Such solid fermentation conditions developed in this study can be used to support the development of health functional food materials with enhanced GABA content and antioxidant activity.

Optimization of Methyl Anthranilate Synthesis Process by Response Surface Methodology and Its Reaction Mechanism

AbstractIn this paper, a unique process for the production of methyl anthranilate (MA) was investigated. The factors of the phthalimide/sodium hypochlorite/methanol molar ratio, reaction temperature, hydrolysis temperature, and water consumption on the yield and purity of MA were analyzed. Response surface methodology (RSM) was used to optimize conditions for the semi-batch synthesis process of MA. The best synthetic conditions for the formation of MA were reaction temperature 0.5 °C, hydrolysis temperature 70 °C and n(phthalimide)/n(sodium hypochlorite)/n(methanol) = 1:2.03:5.87, and water consumption m(H2O)/m(phthalimide) = 7.16:1. The yield of MA could reach 90% under the optimal conditions, which is more than 10% higher than that of the previous semi-batch process. Furthermore, the reaction mechanism was investigated by infrared spectroscopy analysis, and the mechanism of ester group formation and the structure of intermediate products are proposed. The byproduct of the reaction was studied by GC-MS analysis, a byproduct called 2-cyanobenzoic acid has been discovered. Therefore, an unprecedented reaction mechanism of the whole synthesis process is proposed.

Synthesis of MAX-phases, structure and phase composition of modified layers on titanium alloy VT-1 as a result of electron-beam treatment

The article presents the results of research and identification of optimal conditions of formation of MAX-phases during treatment of titanium alloy VT-1 by an electron beam in a vacuum. The study of strength characteristics, thermal properties of composite layers was carried out. Thermodynamic study of phase equilibrium in Ti-Si-C and Ti-B-C systems under high vacuum conditions was carried out in order to optimize conditions of formation of functional layers. A mathematical model of the thermal impact of a powerful fast-moving electron beam on the surface of a titanium alloy has been developed VT-1 under the conditions of electron beam processing in the framework of the theory of thermal conductivity using the COMSOL Multiphysics software complex. The obtained numerical results made it possible to investigate the regularity of the distribution of temperatures and their rates of change depending on the action of the electron beam.

Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency

BackgroundAlthough quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma.MethodsThe annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol.ResultsAn acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction.ConclusionThis is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Effects of ploidy and nutritional conditions on muscle morphology, proliferation and myogenic proteins expression in Rhamdia quelen larvae

Although Rhamdia quelen is a promising species for farming in South America, many aspects of its development and optimal conditions of culture remain unknown. In this sense, we explore muscle development and some proteins related to myogenic process in diploid and triploid larvae submitted to fasting-refeeding. Regarding muscle morphology, within diploid groups, fasted larvae (FD) showed a significant decrease in white muscle fiber area compared with control (CD) and refed (RD-1) groups. Then, when food was provided, area values restored nearly to control. Based on these results, it is probable that temporary muscle fiber atrophy takes place in fasted diploid fish. Conversely, no significant morphological changes were observed among triploid groups. When the effects of ploidy on somatic growth were assessed, FD and RT-1 groups registered a significantly higher percentage of fibers with an area smaller than 500 μm2 compared with fasted triploid larvae (FT) and RD-1 groups, respectively. Additionally, immunolocalization of the proliferating cell nuclear antigen (PCNA) decreased during starvation in fish of both ploidies and only recovered to normal after refeeding in triploid fish. An increase in PCNA related to ploidy was detected in CD and RD-1 compared with their triploid counterparts. In relation to myogenic proteins, Myog showed a significant increase expression in diploid larvae during starvation. Mstn was not affected by ploidy or alimentary variations. Our results show that diploid fish are more affected by short-term starvation than triploid fish, which could be indicative of differential physiological responses of diploid and triploid larvae to alimentary changes in culture. This might be relevant to optimize conditions of culture for both diploid and triploid fish.

Experimental Design and Response Surface Methodologies Use for the Treatment of Leachates by Electrocoagulation Process

In order to optimize conditions for the treatment of leachates by electrocoagulation process by using aluminum (Al) electrodes, the experimental design methodology was applied. Indeed, all factors considered have an important effect to treat leachates which likely contaminate groundwater, rivers and grounds. The investigated variables were initial pH (U1), reaction time (U2) and applied current density “J” (U3). The response surface methodology was applied by using the Doehlert Matrix. The statistical analysis was performed by using NemrodW software (LPRAI, version 2000). Suitability of the model and the success of Doehlert Matrix design for the optimization of the electrocoagulation process indicates that the predicted and experimental values were in fair agreement. In addition, the postulated model is valid and predictive. According to the response surface methodology, the optimal conditions for 98% of COD and 97% of color intensity removal responses were found at the current density 14.4 mA cm−2; the reaction time 72.5 min and the initial pH 6.2.

Optimization of fermentation conditions for the production of recombinant feruloyl esterase from Burkholderia pyrrocinia B1213.

Statistical experimental designs were used to optimize conditions for recombinant Burkholderia pyrrocinia feruloyl esterase (BpFae) production in bacteria under lactose induction. After optimization by single factor design, Plackett-Burman design, steepest ascent design and the response surface method, the optimal conditions for BpFae production were: 6g/L lactose, pH 5.5, pre-induced period 5h, 23°C, shaker rotational speed of 240rpm, medium volume of 50mL/250mL, inoculum size 0.2% (v/v), and a post-induced period of 32h in a Luria-Bertani culture. The produced BpFae activity was 7.43U/mL, which is 2.92 times higher than that obtained under optimal conditions using IPTG as the inducer. BpFae activity was 4.82U/mL in a 5L fermenter under the abovementioned optimal conditions. BpFae produced a small amount of ethyl acetate but had no effect on the synthesis of other important esters in Baijiu. The results underpin further investigations into BpFae characterization and potential applications.

Analysis of sensitivity and specificity: precise recognition of neutrophils during regeneration of contused skeletal muscle in rats

In this report, we applied the TissueFAXS 200 digital pathological analysis system to rapidly and accurately identify neutrophils during regeneration of contused skeletal muscle, and to provide information for follow-up studies on neutrophils to estimate wound age. Rat injury model was established, and skeletal muscle samples were obtained from the control group and contusion groups at 1, 1.5, 2, 3, 4, and 6 h, as well as at 1, 3, 5, and 15 d post-injury (n = 5 per group). The expression of nuclei and neutrophils was detected by hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. A total of 20 injury site areas of 0.25 mm2 (0.5 mm × 0.5 mm) were then randomly selected at all time points. A TissueFAXS 200 digital pathological analysis system was used to identify the positive and negative numbers. Knowledge of five professional medical workers were considered the gold standard to measure the false positive rate (FPR), false negative rate (FNR), sensitivity, specificity, and area under the curve (AUC) of receiver operating characteristic (ROC) curves. As a result, with a staining area of neutrophils from 8 µm2 to 15 µm2, the FPR was 4.28%–12.14%, the FNR was 12.42%–64.08%, the sensitivity was 35.92%–87.58%, the specificity was 87.86%–95.72%, the Youden index was 0.316–0.754, the accuracy was 82.80%–88.30%, and the AUC was 0.771–0.826. The AUC was largest when the cut-off value of the staining area was 12 µm2. Our results show that this software-based method is more accurate than the human eye in evaluating neutrophil infiltration. Based on the sensitivity and specificity, neutrophils can be accurately identified during regeneration of contused skeletal muscle. The TissueFAXS 200 digital pathological analysis system can also be used to optimize conditions for different cell types under various injury conditions to determine the optimal cut-off value of the staining area and provide optimal conditions for further study. Furthermore, it will provide evidence for forensic pathology cases.

Production of Extracellular Rennin-Like Enzyme by a Newly Isolate Mucor circinelloides (von Tieghem) and its Application in Camembert Cheese Making

Introduction: Aspartic proteases produced by different non-pathogenic fungi belonging to mucorales, are commonly used as milk-coagulants. The present study aims to optimize conditions for milk-clotting protease production by a new strain of Mucor genus and to assess its ability in soft cheese making. Materials and Methods: About 20 fungal strains isolated from soil were investigated for their potential to produce milk-clotting proteases for further applications in cheese making. The hyper producer strain Mucor circinelloides 2095-2047 was selected for optimization of rennin-like enzyme production under solid-state fermentation (SSF) using the stepwise modifications of the selected parameters. The enzyme produced under the optimal conditions was partially purified and then applied in Camembert cheese making trials compared to the crude extract and commercial rennet. Results: The maximum milk-curdling activity achieved after optimization (571.43 SU/mL) was obtained using wheat bran (10 g) as the mainly source of carbon containing 1% galactose; moistened with the M-9 solution (pH 6.0) and incubated at 30°C for 96 hours. The enzyme of M. circinelloides was partially purified with a high recovery of 105% and 6.23-fold purity after (NH4)2SO4 fractionation and dialysis. The physicochemical properties of the three produced cheeses were very close. A low sensory quality of cheese was obtained with the crude extract which was getting better using the pre-purified enzyme. This extract was able to develop a very close or even a similar sensory quality to that obtained by the commercial rennet. Conclusions: According to findings, it is possible to propose the purified enzyme of M. circinelloides as a new alternative for rennet, but further optimization, purification and cheese production tests are required.

Factorial Experimental Design for Optimization of Zinc Oxide Nanoparticles Production

Background: Biosynthetic nanomaterials have recently received increasing attention because they are non-toxic, clean, environmentally acceptable, safe, and biocompatible. Objective: In the present study, cell-free culture filtrate of Aspergillus sp. was used for extracellular synthesis of zinc oxide (ZnO) nanoparticles. Method: Plackett-Burman and Taguchi designs were implemented to optimize conditions for maximum ZnO nanoparticle production. In the Plackett-Burman design, 15 factors, representing different carbon and nitrogen sources, were studied. For the Taguchi design, an L-27 (313) standard orthogonal array was constructed to examine nine factors. Results: The maximum yield of ZnO nanoparticles of 21.73 g/L was achieved with 1.0 mM ZnSO4 under optimal conditions of peptone extract (20 g/L), yeast extract (10 g/L), meat extract (10 g/L), K2HPO4 (0.25 g/L), FeSO4⋅7H2O (0.002 g/L), NaCl (2.5 g/L), pH 6, 32°C, and a 200-mL volume. The ZnO nanoparticles’ production was confirmed by the formation of white aggregates. The UV absorption spectrum showed one peak at 376 nm, which also confirmed the formation of nanoparticles. Transmission electron microscopy revealed that the nanoparticles were large rods of 11.6-43.97 nm diameter, and 355.91 nm length. Importantly, the ZnO nanoparticles exhibited broad antimicrobial activity against gram-positive and gram-negative bacteria and a unicellular fungus. Conclusion: The concentrations of ZnSO4 ions, ferrous ions, and peptone and meat extracts, and the interactions between them, were observed to be the main parameters influencing ZnO nanoparticles’ yield.