Steam quality monitoring as a strategy to reduce wet packs and sterilization failure.

BACKGROUND: Hospital articles processed by steam are widely used in the Central Sterile Supply Department (CSSD), responsible for due sterilization. Steam sterilization is discussed worldwide, aiming to protect patients. If steam is outside the specified requirements, the sterilization process may fail, resulting in the wet packs at the end of the sterilization cycle. METHODS: The present study evaluated the steam quality at Santa Catarina Hospital (São Paulo, SP, Brazil) from 2016 to 2022. Saturated steam containing noncondensable gases, excess condensate, or even superheat was characterized using the methodology indicated in the European Standard EN 285:2015. RESULTS: From 2016 to 2020, qualification tests showed that the saturated steam quality does not achieve standard limit parameters. Infrastructural maintenance actions were taken to adjust the saturated steam quality. In 2021, the steam quality followed technical standards, and its adequacy was confirmed in 2022. CONCLUSIONS: The points developed by the hospital's maintenance department, the adoption of appropriate devices for this purpose, and the correct preventive maintenance in the autoclaves, together with the correct qualification of the equipment and proof of the steam quality, contributed to improve the safety of the hospital sterilization process and reduce the incidence of wet packages.

A Classical Phenotype of Fabry Disease with Novel Mutation Found by Kidney Biopsy, A Case Report.

Fabry disease (FD) is a multi-organ disorder caused by a deficiency of alpha-galactosidase (α-GLA) or reduced activity of the enzyme due to mutations in the GLA gene on the X chromosome, making it an X-linked hereditary disease. A 37-year-old man previously diagnosed with sudden deafness and cardiac hypertrophy was referred to our department after an abnormal urine finding during a public health checkup. A renal biopsy revealed characteristic findings, and he was diagnosed with FD with a novel GLA abnormality (c.714dupT (p.I239Yfs*11)). We are currently administering enzyme replacement therapy (ERT) with agalsidase α. This case shows that a novel genetic abnormality in FD can be overlooked for 37 years, even in the presence of typical symptoms. The significance of a renal biopsy in diagnosing FD is emphasized, highlighting the crucial role of nephrologists.  DOI: 10.52547/ijkd.7595.

Directional and amplitude characteristics of pulsed call sequences in captive free-swimming Pacific white-sided dolphins (Lagenorhynchus obliquidens).

We investigated the directional properties and gain control of a pulsed call sequence that functions as a contact call in Pacific white-sided dolphins (Lagenorhynchus obliquidens). The pulsed call sequences were stereotyped patterns composed of three or more pulsed call elements and were collected from two dolphins, separated into adjacent pools, and allowed to swim freely. Eight hydrophones and an overhead camera were used to determine the positions and directions of the participants. The mean peak frequency and source levels were 8.4 ± 4.4 (standard deviation)-18.7 ± 12.7 kHz and 160.8 ± 3.8 to 176.4 ± 7.9 dB re 1 µPa (peak-to-peak), respectively, depending on the element types. The elements were omnidirectional, with mean directivity index of 0.9 ± 3.4 dB. The dolphins produced sequences, regardless of their relative position and direction to the lattice, leading to the adjacent pool where the conspecific was housed. They increased the amplitude by 6.5 ± 4.6 dB as the distance from the caller to an arbitrary point in the adjacent pool doubled. These results suggest that callers broadcast pulsed call sequences in a wide direction to reach dispersed conspecifics. However, they can adjust the acoustic active space by controlling the source levels.

Obtaining Bioproducts from the Studies of Signals and Interactions between Microalgae and Bacteria.

The applications of microalgae biomass have been widely studied worldwide. The classical processes used in outdoor cultivations of microalgae, in closed or open photobioreactors, occur in the presence of bacteria. Understanding how communication between cells occurs through quorum sensing and evaluating co-cultures allows the production of microalgae and cyanobacteria to be positively impacted by bacteria, in order to guarantee safety and profitability in the production process. In addition, the definition of the effects that occur during an interaction, promotes insights to improve the production of biomolecules, and to develop innovative products. This review presents the interactions between microalgae and bacteria, including compounds exchanges and communication, and addresses the development of new pharmaceutical, cosmetic and food bioproducts from microalgae based on these evaluations, such as prebiotics, vegan skincare products, antimicrobial compounds, and culture media with animal free protein for producing vaccines and other biopharmaceutical products. The use of microalgae as raw biomass or in biotechnological platforms is in line with the fulfillment of the 2030 Agenda related to the Sustainable Development Goals (SDGs).

Analysis of water quality over non-condensable gases concentration on steam used for sterilization.

BACKGROUND: Non-condensable gases (NCGs) are all gases that do not undergo liquefaction during the saturated steam sterilization process. During a sterilization cycle, the NCGs presence inside the chamber is one of the biggest threats to the sterilization process compromising process validation and product quality. METHODS: In this work, 170 testing of NCGs concentrations performed between September 2016 and August 2021 were carried out by Orionce Serviços de Metrologia Ltda (Barueri, SP, Brazil), according to EN285:2015 procedure. For steam generation, the types of water used were softened water (SW), one-step reverse osmosis (RO), purified water (PW), and water for injection (WFI). The data obtained were analyzed using Minitab® software, version 18.1, to identify a relationship between the water quality used for steam generation and the concentration of non-condensable gases determined into equipment qualification. FINDINGS: From total tests performed, 109 tests passed, and 61 tests failed, representing 64.2% and 35.8%, respectively. A higher failure rate was observed in terms of concentration of non-condensable gases in systems that used purified water for steam generation (64.7%), followed by softened water (55.6%), one-step reverse osmosis (42.9%), and water for injection (7.6%). CONCLUSION: System processes using WFI for steam generation showed better results for steam quality approvement, in terms of NCGs concentration, compared to softened, purified, or reverse osmosis water treatments in the concentration of non-condensable gases in steam used for sterilization processes of industrial utilities.

Evaluation of the effectiveness of manual and automated dialyzers reprocessing after multiple reuses.

A cross-sectional study was conducted to evaluate the effectiveness of manual and automated dialyzer reprocessing. Dialyzers were filled with fluid thioglycollate medium from blood and dialysate chambers after being reprocessed and chemically sterilized with 0.2% peracetic acid. They were incubated for 14 days at 35°C ± 2°C, and microbiologic analysis was performed. Microorganisms were identified in 3 of the 11 samples (27.3%) from the blood chambers: Sphingomonas paucimobilis (2/3) and Penicillium spp (1/3) and in 11 of the 11 samples (100%) from the dialysate chambers: S paucimobilis (7/11), Stenotrophomonas maltophilia (4/11), Pseudomonas aeruginosa (3/11), Candida spp (1/11), and Acinetobacter baumannii (1/11). Of the 4 manually reprocessed dialyzers, gram-positive bacillus were identified in 1 sample (25%) from the blood chamber, and Bacillus spp and Burkholderia spp were identified in 1 sample (25%) from the dialysate chamber. The dialyzers reprocessing can pose risks safety because of exposure patient to microorganisms.

Ligand-based design, synthesis, and experimental evaluation of novel benzofuroxan derivatives as anti-Trypanosoma cruzi agents.

A set of substituted-[N'-(benzofuroxan-5-yl)methylene]benzohydrazides (4a-t), previously designed and synthesized, was experimentally assayed against Trypanosoma cruzi, the etiological agent of Chagas' disease, one of the most neglected tropical diseases. Exploratory data analysis, Hansch approach and VolSurf formalism were applied to aid the ligand-based design of novel anti-T. cruzi agents. The best 2D-QSAR model showed suitable statistical measures [n = 18; s = 0.11; F = 42.19; R(2) = 0.90 and Q(2) = 0.77 (SDEP = 0.15)], and according to the optimum 3D-QSAR model [R(2) = 0.98, Q(2) = 0.93 (SDEP = 0.08)], three latent variables explained 62% of the total variance from original data. Steric and hydrophobic properties were pointed out as the key for biological activity. Based upon the findings, six novel benzofuroxan derivatives (4u-z) were designed, synthesized, and in vitro assayed to perform the QSAR external prediction. Then, the predictability for the both models, 2D-QSAR (Rpred(2) = 0.91) and 3D-QSAR (Rpred(2) = 0.77), was experimentally validated, and compound 4u was identified as the most active anti-T. cruzi hit (IC50 = 3.04 µM).

Synthesis, molecular modeling and preliminary biological evaluation of a set of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole as potential antibacterial, anti-Trypanosoma cruzi and antifungal agents.

A series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives was synthesized and their activity screened in vitro against Staphylococcus aureus, Trypanosoma cruzi, and Candida albicans. The bioactivity was expressed as minimum inhibitory concentration (MIC) for S. aureus strains, and as fifty-percent inhibitory concentration (IC(50)) of parasite population growth for T. cruzi. A molecular modeling approach was performed to establish qualitative relationships regarding the biological data and the compounds' physicochemical properties. The 5-(4-OC(4)H(9)Ph, 5l), and 5-(4-CO(2)CH(3)Ph, 5o) derivatives were the most active compounds for S. aureus ATCC 25923 (MIC=1.95-1.25 µg/mL) and T. cruzi (IC(50)=7.91 µM), respectively. Also, a preliminary evaluation against C. albicans involving some compounds was performed and the 5-(4-CH(3)Ph, 5e) derivative was the most active compound (MIC=3.28-2.95 µg/mL). In this preliminary study, all synthesized 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives were active against all microorganisms tested.

Novel benzofuroxan derivatives against multidrug-resistant Staphylococcus aureus strains: design using Topliss' decision tree, synthesis and biological assay.

The aim of this study was the design of a set of benzofuroxan derivatives as antimicrobial agents exploring the physicochemical properties of the related substituents. Topliss' decision tree approach was applied to select the substituent groups. Hierarchical cluster analysis was also performed to emphasize natural clusters and patterns. The compounds were obtained using two synthetic approaches for reducing the synthetic steps as well as improving the yield. The minimal inhibitory concentration method was employed to evaluate the activity against multidrug-resistant Staphylococcus aureus strains. The most active compound was 4-nitro-3-(trifluoromethyl)[N'-(benzofuroxan-5-yl)methylene]benzhydrazide (MIC range 12.7-11.4 µg/mL), pointing out that the antimicrobial activity was indeed influenced by the hydrophobic and electron-withdrawing property of the substituent groups 3-CF(3) and 4-NO(2), respectively.

Sterilization of medical devices by ethylene oxide, determination of the dissipation of residues, and use of Green Fluorescent Protein as an indicator of process control.

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (BI) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/-3.2)% to 7.5 (+/-2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/-3.0)% to 21.5 (+/-6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/-3.2)% to 23.9 (+/-3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor.

Study on the thermal stability of green fluorescent protein (GFP) in glucose parenteral formulations.

Large volume parenteral solutions (LVPS) that are widely used in the healthcare system must be processed by moist-heat treatment to an assured sterility level in which the efficacy is measured by a bioindicator (BI) that provides fast, accurate and reliable results. This study evaluated the thermal stability of green fluorescent protein (GFP) into glucose-based LVPS (1.5-50%) solutions to determine its utility as a BI for thermal processes. GFP, expressed by Escherichia coli, isolated/purified by TPP/HIC, was diluted in buffered (each 10mM: Tris-EDTA, pH 8; phosphate, pH 6 and 7; acetate, pH 5) and in water for injection (WFI; pH 6.70+/-0.40) glucose solutions (1.5-50%) and exposed to constant temperatures from 80 degrees C to 95 degrees C. The thermal stability was expressed in decimal reduction time (D-value, time required to reduce 90% of the GFP fluorescence intensity). At 95 degrees C, the D-values for GFP in 1.5-50% glucose were: (i) 1.63+/-0.23 min (pH 5); (ii) 2.64+/-0.26 min (WFI); (iii) 2.50+/-0.18 min (pH 6); (iv) 3.24+/-0.28 min (pH 7); (v) 2.89+/-0.44 min (pH 8). By the convenient measure of fluorescence intensity and its thermal stability, GFP has the potential as a BI to assay the efficacy of moist-heat processing of LVPS at temperatures < or =100 degrees C.

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride.

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH.

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.

Aplicação da Proteína Verde Fluorescente, GFPuv, como indicador biológico na validação da autoclavação de soluções parenterais e da esterilização por óxido de etileno de itens termolábeis. Comparação com esporos de Bacillus subtilis / Use of the Green Fluorescent Protein as a biological indicator for the validation of parenteral solutions sterilization and for the ethylene oxide sterilization process of thermal labile items

A Proteína Verde Fluorescente recombinante, GFPuv, é um sistema marcador atrativo pois, sua presença pode ser visualizada através da intensidade de fluorescência emitida, sem o uso de substratos ou meios complexos. Sendo uma molécula estável à presença de substâncias orgânicas, temperaturas acima de 70’graus’C e ampla faixa de pH, é um potencial Indicador Biológico (IB) para diversas aplicações. A estabilidade térmica da GFPuv, foi avaliada pela medida da perda de intensidade de fluorescência, expressa em valores D (min), tempo de exposição necessário para redução de 90% da intensidade de fluorescência inicial da GFPuv. GFPuv (3,5-9,0 ‘mü’g/mL), expressa por E. coli e isolada por extração de Partição em Três Fases (TPP) e purificação por Cromatografia de Interação Hidrofóbica (HIC), foi diluída nas soluções parenterais preparadas em tampão (10 mM cada: Tris-EDTA, pH8; Fosfato, pH 6 e 7, e Acetato, pH 5) e em água para injeção, WFI; pH = 6,70’+ou-‘0,40), e expostas a temperaturas de 25’graus’C e ao intervalo entre 80’graus’C e 100’graus’C. A 95’graus’C, os valores D para GFPuv em soluções de 1,5% a 50% de glicose variaram de: (i) 1,63 (‘+ou-‘0,23) min em acetato pH 5; (ii) 2,64 ‘+ou’ 0,26 min em WFI; (iii) 2,50 `mais ou menos' 0,18 min em fosfato pH 6; (iv) 3,24 `mais ou menos' 0,28 min em fosfato pH 7 e, (v) 2,89 `mais ou menos' 0,44 min em Tris-EDTA ph 8...

Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values.

The stability at room temperature (25 degrees C) of recombinant green fluorescent protein (GFPuv), expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction column, was studied. The GFPuv was diluted in buffered (each 10 mM: Tris-HCl, pH 8.0; phosphate, pH 6.0 and 7.0 and acetate, pH 5.0) and in unbuffered (water for injection [WFI]; pH 6.70 +/- 0.40) glucose solutions (from 1.5 to 50%). By assaying the loss of fluorescence intensity as a measure of denaturation, the stability of GFPuv in these solutions was evaluated relative to glucose concentration, pH, osmolarity, density, conductivity, and viscosity. The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time required to reduce 90% of the initial fluorescence intensity of GFPuv. The D-value between 56 and 83 h of GFPuv at 1.5-15% glucose in WFI was equivalent to 20-30% glucose in a phosphate. The stability of GFPuv in 50% glucose was similar for all buffers studied and four times higher than in WFI. By the convenient measure of fluorescence intensity, GFPuv can be used as an indicator to report the extent of denaturation rates of other proteins in glucose solutions.

Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins.

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4 degrees C prior to incubation, agitation speed, isopropyl-beta-D-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfate polyacrylamide gel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37 degrees C, 24-h incubation).

Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values.

The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5-9.0 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees C) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85 degrees C, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate). The stability of GFPuv in Tris-HCl (pH >8.0) was constant at 90 and 95 degrees C, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100 degrees C.

Effect of media on spore yield and thermal resistance of Bacillus stearothermophilus.

The interference of eight components in the yield of sporulation and thermal resistance to moist heat (121 degrees C) of Bacillus stearothermophilus spores suspended in 0.02 M calcium acetate solution and inoculated on paper strips previously treated with calcium acetate/calcium hydroxide was studied. The spore yield of 1.0 x 10(8)mL was developed at 62 degrees C in 17 media containing different concentrations of D-glucose, sodium chloride, L-glutamic acid, yeast extract, peptone, manganese sulfate, potassium phosphate, and ammonium phosphate. The combined effects of yeast extract, peptone, and glucose contributed positively to the spore yield and to the stability of the thermal resistance of both spores in suspension and on strips.

The effect of bioindicator preparation and storage on thermal resistance of Bacillus stearothermophilus spores.

Paper strips inoculated with spores of Bacillus stearothermophilus ATCC 7953 were conventionally dried (lot 1) and lyophilized (lot 2); stored in defined environments of 32 and 86% relative humidity at 10, 25 and 33 degrees C for 210 d; and submitted to moist heat treatments at 121 degrees C. A significant decrease in thermal resistance from initial starting levels was found for lyophilized bioindicators stored at 86% relative humidity. The respective average D121 degrees C values were 1.55+/-0.05 and 1.37+/-0.10 min for lyophilized bioindicators stored at 32 and 86% relative humidity; and 1.65+/-0.15 min and 1.57+/-0.11 min for dried bioindicators stored in the same environments.

The effect of composition of parenteral solution on the thermal resistance of Bacillus stearothermophilus and Bacillus subtilis spores.

Large-volume parenteral solutions were submitted to heat treatments after being inoculated with Bacillus stearothermophilus ATCC 7953 (Tr = 121 degrees C) and Bacillus subtilis ATCC 9372 (Tr = 104.5 degrees C) spores. The average decimal reduction time for B. stearothermophilus ranged from a D121 degrees C value of 1.31 to 3.14 min, in glucophysiologic and Ringer's solutions respectively. For B. subtilis, D104.5 degrees C value increased from 0.69 to 1.37 min, in Ringer's (pH=5.91) and 50% glucose (pH 3.05) solutions respectively. The z value ranged from 7.95 degrees C (20% mannitol solution) to 13.14 degrees C (50% glucose solution), corresponding to an activation energy (Ea) of 81.48 and 49.30 kcal/mol, respectively.