The high temperature induces conformational changes in β-glucosidase, making it inactive and limiting its application field. In this paper, the effect of trehalose on the thermostability of β-glucosidase from low-moisture Hevea brasiliensis seeds was investigated. The results showed that the residual enzyme activities of β-glucosidase supplemented with trehalose after high-temperature treatment were significantly higher than that of the control group. The improvement of thermostability could be explained by low-field nuclear magnetic resonance (LF-NMR) and molecular dynamics (MD) simulations at the molecular level. Moreover, adding trehalose increased the water activity and water content of β-glucosidase, leading to a more stable conformation. Trehalose replaced some water and formed a stable network of hydrogen bonds with protein and surrounding water. The glass formed by trehalose also reduced molecular movement, thus providing good protection for enzymes.

Proton batteries have attracted increasing interests because of their potential for grid-scale energy storage with high safety and great low-temperature performances. However, their development is significantly retarded by electrolyte design due to free water corrosion. Herein, we report a layer intercalatable electrolyte (LIE) by introducing trimethyl phosphate (TMP) into traditional acidic electrolyte. Different from conventional role in batteries, the presence of TMP intriguingly achieves co-intercalation of solvent molecules into the interlayer of anode materials, enabling a new working mechanism for proton reactions. The electrode corrosion was also strongly retarded with expanded electrochemical stability window. The half-cell therefore showed an outstanding long-term cycling stability with 91.0% capacity retention at 5 A g-1 after 5000 cycles. Furthermore, the assembled full batteries can even deliver an ultra-long lifetime with a capacity retention of 74.9% for 2 months running at -20 °C. This work provides new opportunities for electrolyte design of aqueous batteries.

Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0-10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na+, K+, and NH4 +. It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD+. Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD+-binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD+ under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L-tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent.

This study aimed to investigate the behavior of Salmonella enterica and Listeria monocytogenes in processed date paste and syrup at different temperatures. Commercial products were inoculated with approximately 6 log CFU/mL of S. enterica or L. monocytogenes and stored at 4, 10, and 24°C for 90 days. S. enterica was able to survive in date products until the end of storage at 4°C. At this temperature, numbers decreased by 2.1 log CFU/g in date paste and by 3.4 log CFU/g in date syrup; however, at 10°C, cells were reduced >4.2 log CFU/g and were undetectable by direct plating in date paste or by enrichment (complete elimination) in syrup. Further, at 24°C, complete elimination of S. enterica was achieved in date paste and syrup by 30 and 7 days, respectively. L. monocytogenes numbers decreased by 1.4, 4.4, and >4.6 log CFU/g in date paste stored at 4, 10, and 24°C for 90 days, respectively. In date syrup, numbers of L. monocytogenes decreased to undetectable levels by 50, 14, and 4 days at 4, 10, and 24°C, respectively, by direct plating and complete elimination was observed at 10 and 24°C by 50 and 30 days of storage, respectively. The initial pH values of date paste and syrup were 4.7 and 4.8, respectively, and remained stable until the end of storage except for L. monocytogenes-inoculated syrup. PRACTICAL APPLICATION: Salmonella enterica and Listeria monocytogenes can easily survive in date paste and syrup particularly at refrigerator temperature, which explains the necessity of preventing the contamination of date products with foodborne pathogens.

Microbial challenge studies using nonpathogenic surrogates provide a practical means for validating thermally based pathogen controls for low-moisture foods. Because the relative thermal resistance, or kill ratio, of Enterococcus faecium NRRL B-2354 (a nonpathogenic surrogate) to Salmonella is greatly influenced by food composition, this study assessed relative thermal resistance of a five-strain Salmonella cocktail and E. faecium in skim milk powder (SMP), lactose-free skim milk powder (LSMP), 90% milk protein isolate (MPI), and lactose powder (LP). The impact of sugar composition (lactose versus glucose-galactose) on resuscitation of bacterial survivors, by using SMP and LSMP, was also determined. Dairy powders were inoculated with agar-grown cultures, mixed, preequilibrated at 0.25 water activity (aw), ground to achieve homogeneity, reequilibrated, and subjected to isothermal treatment. After enumeration on nonselective differential media, log-linear and Bigelow models were fit to the survivor data via one-step global regression. The aw changes and glass transition temperature were assessed at elevated temperatures by using uninoculated, equilibrated powder samples. Estimated D90°C-values were approximately two times higher for E. faecium (P < 0.05) than for Salmonella in SMP, LP, and MPI, but statistically similar (P > 0.05) in LSMP. Addition of sugars to recovery media did not influence survivor resuscitation from heat-treated SMP and LSMP, confirming that microbial inactivation was impacted primarily by the thermal treatment, not the recovery step. Thermally induced changes in aw were seen only for LP and MPI, with the glass transition temperature observed only for SMP and MPI. In conclusion, rather than always requiring greater lethality of E. faecium than Salmonella, these findings suggest that sufficient pathogen controls for low-moisture foods can also be validated by thoroughly documenting the appropriate kill ratios of E. faecium to Salmonella.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of β-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Injecting supercritical CO2 (scCO2) into basalt formations for long-term storage is a promising strategy for mitigating CO2 emissions. Mineral carbonation can result in permanent entrapment of CO2; however, carbonation kinetics in thin H2O films in humidified scCO2 is not well understood. We investigated forsterite (Mg2SiO4) carbonation to magnesite (MgCO3) via amorphous magnesium carbonate (AMC; MgCO3·xH2O, 0.5 < x < 1), with the goal to establish the fundamental controls on magnesite growth rates at low H2O activity and temperature. Experiments were conducted at 25, 40, and 50 °C in 90 bar CO2 with a H2O film thickness on forsterite that averaged 1.78 ± 0.05 monolayers. In situ infrared spectroscopy was used to monitor forsterite dissolution and the growth of AMC, magnesite, and amorphous SiO2 as a function of time. Geochemical kinetic modeling showed that magnesite was supersaturated by 2 to 3 orders of magnitude and grew according to a zero-order rate law. The results indicate that the main drivers for magnesite growth are sustained high supersaturation coupled with low H2O activity, a combination of thermodynamic conditions not attainable in bulk aqueous solution. This improved understanding of reaction kinetics can inform subsurface reactive transport models for better predictions of CO2 fate and transport.

The use of baking ovens as a microbial kill step should be validated based on results of thermal inactivation models. Although traditional isothermal models may not be appropriate for these dynamic processes, they are being used by the food industry. Previous research indicates that the impact of additional process conditions, such as humidity, should be considered when validating thermal processes for the control of microbial hazards in low-moisture foods. In this study, the predictive performance of traditional and modified thermal inactivation kinetic models accounting for process humidity were assessed for predicting inactivation of Enterococcus faecium NRRL B-2354 in a multi-ingredient composite food during baking. Ingredients (milk powder, protein powder, peanut butter, and whole wheat flour) were individually inoculated to achieve ∼6 log CFU/g, equilibrated to a water activity of 0.25, and then mixed to form a cookie dough. An isothermal inactivation study was conducted for the dough to obtain traditional D- and z-values (n = 63). In a separate experiment, cookies were baked under four dynamic heating conditions: 135°C, high humidity; 135°C, low humidity; 150°C, high humidity; and 150°C, low humidity. Process humidity measurements; time-temperature profiles for the product core, surface, and bulk air; and microbial survivor ratios were collected for the four conditions at six residence times (n = 144). The traditional isothermal model had a high root mean square error (RMSE) of 856.51 log CFU/g, significantly overpredicting bacterial inactivation during the process. The modified model accounting for the dynamic time-temperature profile and process humidity data was a better predictor with an RMSE of 0.55 log CFU/g. These results indicate the importance of accounting for additional process parameters in baking inactivation models and that model performance can be improved by utilizing model parameters obtained directly from industrial-scale experimental data.