Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Acid adaptation increased the resistance of Escherichia coli O157:H7 in bok choy (Brassica rapa subsp. chinensis) juice to high-pressure processing.

IMPORTANCE: Based on the U.S. Food and Drug Administration regulations, E. coli O157:H7 is a pertinent pathogen in high acid juices that needs to be inactivated during the pasteurization process. The results of this study suggest that the effect of acid adaptation should be considered in the selection of HPP parameters for E. coli O157:H7 inactivation to ensure that pasteurization objectives are achieved.

Quantitative Proteomic Analysis on the Slightly Acidic Electrolyzed Water Triggered Viable but Non-Culturable Listeria monocytogenes.

This study undertakes a comprehensive exploration of the impact of slightly acidic electrolyzed water (SAEW) on Listeria monocytogenes, a common foodborne pathogen, with a particular focus on understanding the molecular mechanisms leading to the viable but nonculturable (VBNC) state. Given the widespread application of SAEW as an effective disinfectant in the food industry, uncovering these molecular pathways is crucial for improving food safety measures. We employed tandem mass tags (TMT), labeling proteomic techniques and LC-MS/MS to identify differentially expressed proteins under two doses of SAEW conditions. We indicated 203 differential expressed proteins (DEPs), including 78 up-regulated and 125 down-regulated DEPs. The functional enrichment analysis of these proteins indicated that ribosomes, biosynthesis of secondary metabolites, and aminoacyl-tRNA biosynthesis were enriched functions affected by SAEW. Further, we delved into the role of protein chlorination, a potential consequence of reactive chlorine species generated during the SAEW production process, by identifying 31 chlorinated peptides from 22 proteins, with a dominant sequence motif of Rxxxxx[cY] and functionally enriched in translation. Our findings suggest that SAEW might prompt alterations in the protein translation process and trigger compensatory ribosome biosynthesis. However, an imbalance in the levels of elongation factors and AARSs could hinder recovery, leading to the VBNC state. This research carries substantial implications for food safety and sanitation, as it adds to our understanding of the SAEW-induced VBNC state in L. monocytogenes and offers potential strategies for its control.

Can simultaneous immobilization of arsenic and cadmium in paddy soils be achieved by liming?

Liming acidic paddy soils to near-neutral pH is the most cost-effective strategy to minimize cadmium (Cd) accumulation by rice. However, the liming-induced effect on arsenic (As) (im)mobilization remains controversial and is called upon for further investigation, particularly for the safe utilization of paddy soils co-contaminated with As and Cd. Here, we explored As and Cd dissolution along pH gradients in flooded paddy soils and extracted key factors accounting for their release discrepancy with liming. The minimum As and Cd dissolution occurred concurrently at pH 6.5-7.0 in an acidic paddy soil (LY). In contrast, As release was minimized at pH < 6 in the other two acidic soils (CZ and XX), while the minimum Cd release still appeared at pH 6.5-7.0. Such a discrepancy was determined largely by the relative availability of Fe under overwhelming competition from dissolved organic carbon (DOC). A mole ratio of porewater Fe/DOC at pH 6.5-7.0 is suggested as a key indicator of whether co-immobilization of As and Cd can occur in flooded paddy soils with liming. In general, a high mole ratio of porewater Fe/DOC (≥ 0.23 in LY) at pH 6.5-7.0 can endow co-immobilization of As and Cd, regardless of Fe supplement, whereas such a case is not in the other two soils with lower Fe/DOC mole ratios (0.01-0.03 in CZ and XX). Taking the example of LY, the introduction of ferrihydrite promoted the transformation of metastable As and Cd fractions to more stable ones in the soil during 35 days of flooded incubation, thus meeting a class I soil for safe rice production. This study demonstrates that the porewater Fe/DOC mole ratio can indicate a liming-induced effect on co-(im)mobilization of As and Cd in typical acidic paddy soils, providing new insights into the applicability of liming practice for the paddy soils.

Inactivation of Listeria monocytogenes through the synergistic interaction between plasma-activated water and organic acid.

This study observed that when plasma-activated water (PAW) was combined with organic acid, it showed a synergistic inactivation effect on Listeria monocytogenes, which is highly resistant to PAW. When comparing various organic acids, lactic acid (LA) showed the greatest synergistic effect, followed by malic acid (MA), citric acid (CA), and acetic acid (AA), whereas propionic acid (PA) did not show a synergistic effect. Organic acid lowered the activity of ROS defense enzymes (catalase, superoxide dismutase) by reducing intracellular pH (pHi), which induced the increase in the accumulation of ROS of PAW within the cell. In the end, the synergistic inactivation effect appeared as the increased occurrence of oxidative damage when organic acid was combined as a series of preceding causes. In this case, LA with the greatest ability to lower the pH induced the greatest synergistic effect, suggesting that LA is the best candidate to be combined with PAW. As a result of observing changes in inactivation activity for L. monocytogenes of PAW combined with 1.0% LA while storing at - 80, -20, 4, 25, & 37 °C for 30 days, respectively, it was confirmed that the lower the temperature, the lower the activity loss during the storage period, and that it had an activity of 3.72 log reduction based on 10 min treatment when stored at - 80 °C for 30 days. Application of PAW combined with 1.0% LA stored at - 80 °C for 30 days to mackerel inoculated with L. monocytogenes in ice form resulted in a decrease of 4.53 log after 120 min treatment, without changing the quality of mackerel. These results suggest that combining LA with PAW can be an effective control strategy for L. monocytogenes with high resistance to PAW, and can be effectively utilized, even in ice form.

ZccE, a P-type ATPase contributing to biofilm formation and competitiveness in Streptococcus mutans.

Most living organisms require zinc for survival; however, excessive amounts of this trace element can be toxic. Therefore, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent introduction of exogenous zinc ions, present a serious challenge for bacteria colonizing the oral cavity. Streptococcus mutans is considered one of the main bacterial pathobiont in dental caries. Here, we verified the role of a P-type ATPase ZccE as the main zinc-exporting transporter in S. mutans and delineated the effects of zinc toxification caused by zccE deletion in the physiology of this bacterium. The deletion of the gene zccE severely impaired the ability of S. mutans to grow under high zinc stress conditions. Intracellular metal quantification using inductively coupled plasma optical emission spectrometer revealed that the zccE mutant exhibited approximately two times higher zinc accumulation than the wild type when grown in the presence of a subinhibitory zinc concentration. Biofilm formation analysis revealed less single-strain biofilm formation and competitive weakness in the dual-species biofilm formed with Streptococcus sanguinis for zccE mutant under high zinc stress. The quantitive reverse transcription polymerase chain reaction test revealed decreased expressions of gtfB, gtfC, and nlmC in the mutant strain under excessive zinc treatment. Collectively, these findings suggest that ZccE plays an important role in the zinc detoxification of S. mutans and that zinc is a growth-limiting factor for S. mutans within the dental biofilm.

Identification and characterization of probiotics isolated from indigenous chicken (Gallus domesticus) of Nepal.

BACKGROUND: Excessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). METHODS: Selective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37°C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. RESULTS: A total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16S rDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. CONCLUSION: Our study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

Deciphering the induction of Listeria monocytogenes into sublethal injury using fluorescence microscopy and RT-qPCR.

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA-/PI+ cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA-/PI-) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA+). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment.

Inactivation of Escherichia coli in apple cider using atmospheric cold plasma.

Atmospheric cold plasma (ACP) is a promising non-thermal technology that has the potential to inactivate microorganisms in foods. In this work, the inactivation of E. coli K12, acid-adapted E. coli K12, and E. coli O157:H7 in apple cider by ACP was investigated using feed gases as simulated air (SA) (80 % N2 + 20 % O2) and a mixture of 90 % N2 + 10 % O2 with various processing times (0 to 180 s). We obtained the reduced the populations of both acid-adapted and non-adapted E. coli K12 by 5 log CFU/mL within 120 s, and E. coli O157:H7 within 90 s. Additionally, no significant changes in the °Brix, pH, temperature, or titratable acidity (TA) of apple cider were observed after exposure to ACP. However, processing times longer than 120 s resulted in significant changes in the pH values. The highest concentration of ozone and hydrogen peroxide reached to 0.22 ± 0.1 mg/L for CG in 180 s and 0.07 ± 0.01 mg/L for SA in 150 s, respectively. Both acid-adapted and non-acid adapted E. coli K12 was found to be more resistant to ACP processing than E. coli O157:H7 after the 90 s, so it could serve as a surrogate for E. coli O157:H7. When we compared the effect of the gas type on inactivation, non-selective media, the results showed no significant differences between the gas types, while selective media demonstrated significant differences. In optical absorption spectroscopy measurements of plasma species, primarily ozone peaks were observed. Furthermore, the optical absorption spectroscopy also revealed that the inactivation of the bacteria could be attributed to some plasma species with wavelengths between 190 and 308 nm. The findings provided a perspective on the use of ACP as a method for decontaminating fruit juices as a non-thermal processing.

Efficacy of weak acid preservatives on spoilage fungi of bakery products.

Organic acids and their salts are usually the first choice in the bread industry to restrict fungal spoilage, but their efficacy is pH-dependent and spoilage by fungi remains as a common threat. Therefore, this study aimed to evaluate the susceptibility of spoilage fungi of bakery products to acetic, sorbic, and propionic acids at different pH. Penicillium roqueforti, Penicilium paneum, Aspergillus pseudoglaucus, Aspergillus montevidensis and Hyphopichia burtonii strains isolated from spoiled products had their minimum inhibitory concentration (MIC) defined by macrodilution. The concentrations tested were: (i) sorbic acid up to 32 mM; (ii) propionic acid up to 1024 mM and (iii) acetic acid up to 800 mM with pH adjusted in 4.5, 5.0, 5.0 and 6.0 after setting the agent concentration. The lowest MICs for all agents were obtained at pH 4.5, usually doubling with every 0.5 pH increase. P. roqueforti strains isolated from spoiled products were the most resistant to all tested preservatives; while strains of the related species P. paneum, showed similar tolerance to acetic and propionic acids but was double more susceptible to sorbic acid. Strains of A. pseudoglaucus and A. montevidensis were indistinctly susceptible to the preservatives and were the most susceptible species to propionic and acetic acids. H. burtonii strains demonstrated the most variable behaviour in comparison to the other strains being the most susceptible to sorbic acid, were like Aspergillus strains regarding propionic acid, but tolerate well acetic acid. Propionic acid concentrations usually allowed in baked goods are lower than the concentrations required to inhibit the most tolerant isolates tested in this study. The same is true for sorbic acid at higher pH levels. Spoilage species of bakery ware presents a distinct susceptibility profile to the preservatives commonly used in this sector, but the high tolerance observed is a cause of concern.

ubiF is involved in acid stress tolerance and symbiotic competitiveness in Rhizobium favelukesii LPU83.

The acidity of soils significantly reduces the productivity of legumes mainly because of the detrimental effects of hydrogen ions on the legume plants, leading to the establishment of an inefficient symbiosis and poor biological nitrogen fixation. We recently reported the analysis of the fully sequenced genome of Rhizobium favelukesii LPU83, an alfalfa-nodulating rhizobium with a remarkable ability to grow, nodulate and compete in acidic conditions. To gain more insight into the genetic mechanisms leading to acid tolerance in R. favelukesii LPU83, we constructed a transposon mutant library and screened for mutants displaying a more acid-sensitive phenotype than the parental strain. We identified mutant Tn833 carrying a single-transposon insertion within LPU83_2531, an uncharacterized short ORF located immediately upstream from ubiF homolog. This gene encodes a protein with an enzymatic activity involved in the biosynthesis of ubiquinone. As the transposon was inserted near the 3' end of LPU83_2531 and these genes are cotranscribed as a part of the same operon, we hypothesized that the phenotype in Tn833 is most likely due to a polar effect on ubiF transcription.We found that a mutant in ubiF was impaired to grow at low pH and other abiotic stresses including 5 mM ascorbate and 0.500 mM Zn2+. Although the ubiF mutant retained the ability to nodulate alfalfa and Phaseolus vulgaris, it was unable to compete with the R. favelukesii LPU83 wild-type strain for nodulation in Medicago sativa and P. vulgaris, suggesting that ubiF is important for competitiveness. Here, we report for the first time an ubiF homolog being essential for nodulation competitiveness and tolerance to specific stresses in rhizobia.

Didecyldimethylammonium bromide: Application to control biofilms of Staphylococcus aureus and Pseudomonas aeruginosa alone and in combination with slightly acidic electrolyzed water.

This project explored the antibacterial mechanism of didecyldimethylammonium bromide (DDAB) toward Staphylococcus aureus and Pseudomonas aeruginosa, and its removal effect on biofilms. Furthermore, we explored the effect of treatment by DDAB combined with slightly acidic electrolyzed water (SAEW) on biofilms of S. aureus or P. aeruginosa. First, DDAB has bacteriostatic and biofilm removal effects. Second, The effect of DDAB combined with SAEW on biofilm is more obvious than that of the two alone. DDAB at a concentration of 16 MIC combined with SAEW (ACC 30 mg/L, ORP 875 mV, pH 6.30) completely cleared the biofilm. In addition, the results of Confocal laser scanning microscopy (CLSM), Field emission scanning electron microscopy (FESEM) and Attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR) showed that DDAB combined with SAEW could disrupt the structure of biofilms, reduce polysaccharides, proteins and phospholipids in biofilms. This method has the potential to be used in food production chains to control S. aureus and P. aeruginosa and their biofilms, and it can be used in other industries.

Integrated metabolomics of "big six" Escherichia coli on pea sprouts to organic acid treatments.

Naturally occurring organic acids (OAs) have demonstrated satisfactory effects in inhibiting common pathogens on fresh produce; however, their effectiveness on "big six" Escherichia coli serotypes, comprised of E. coli O26:H11, O45:H2, O103:H11, O111, O121:H19 and O145, remained unaddressed. Regarding this, using nuclear magnetic resonance (NMR) spectroscopy and ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), the sanitising efficacy and the underlying antimicrobial mechanisms of 10-min treatments with 0.2 mol/L ascorbic acid (AA), citric acid (CA) and malic acid (MA) against the "big six" strains on pea sprouts were thoroughly investigated in this study. Despite the varying antimicrobial efficacy (AA: 0.12-0.99, CA: 0.36-1.72, MA: 0.75-3.28 log CFU/g reductions), the three OAs induced consistent metabolic changes in the E. coli strains, particularly in the metabolism of membrane lipids, nucleotide derivatives and amino acids. Comparing all strains, the most OA-resistant strain, O26 (0.36-1.12 log CFU/g reductions), had the largest total amino acids accumulated to resist osmotic stress; its ulteriorly suppressed cell activity further strengthened its endurance. In contrast, the lowest OA-resistance of O121 (0.99-3.28 log CFU/g reductions) might be explained by the depletion of putrescine, an oxidative stress regulator. Overall, the study sheds light on the effectiveness of a dual-platform metabolomics investigation in elucidating the metabolic responses of "big six" E. coli to OAs. The manifested antimicrobial effects of OAs, especially MA, together with the underlying metabolic perturbations detected in the "big six" strains, provided scientific basis for applying OA treatments to future fresh produce sanitisation.

Evaluating hop extract concentrations found in commercial beer to inhibit Streptococcus mutans biofilm formation.

AIMS: The purpose of this study was to compare the effect of hop extracts with diverse ß-acid concentrations on Streptococcus mutans biofilm formation. METHODS AND RESULTS: Ten different hop extracts, with α-acid concentrations similar to those found in commercial beer products and ß-acid concentrations ranging from 2.6 to 8.1%, were added to distilled water to make standardized concentrations. S. mutans isolates were treated with hop extract dilutions varying from 1:2 to 1:256. The minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were determined and the optical density was evaluated. Live/dead staining confirmed the bactericidal effects. Biofilm formation of several strains of S. mutans was significantly inhibited by hop extract dilutions of 1:2, 1:4, 1:8, 1:16 and 1:32. Strong negative correlations were observed between α- and ß-acid concentrations of the hop extracts and S. mutans total growth and biofilm formation. CONCLUSIONS: The use of hop extracts prepared similarly to commercial beer decreased S. mutans biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The inclusion of hops in the commercial beer products may provide beneficial health effects. Further studies are warranted to determine an effect in vivo on the development of dental caries.

In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in adult gills to investigate branchial H+-excreting functions in vivo. We removed the opercula from zebrafish and then performed long-term acid acclimation experiments. The results of Western blot and immunofluorescence showed that the protein expression of H+-ATPase (HA) and the number of H+-ATPase-rich ionocytes were increased under acidic situations. The SIET results proved that the H+ excretion capacity is indeed enhanced in the gills acclimated to acidic water. In addition, both HA and Na+/H+ exchanger (Nhe) inhibitors suppressed the branchial H+ excretion capacity, suggesting that H+ is excreted in association with HA and Nhe in zebrafish gills. These results demonstrate that SIET is effective for in vivo detection in fish gills, representing a breakthrough approach for studying the molecular physiology of fish ion regulation.

Synergistic effect and disinfection mechanism of combined treatment with ultrasound and slightly acidic electrolyzed water and associated preservation of mirror carp (Cyprinus carpio L.) during refrigeration storage.

This study evaluated the synergistic effects and disinfection mechanism of ultrasound (US, 200 W and 30 kHz) and slightly acidic electrolysed water (SAEW, 60 mg/L and pH of 6.2) treatment and the associated preservation of mirror carp during refrigeration storage (4 °C). US and SAEW alone and US combined with SAEW (US + SAEW) showed the lower water loss of fish. Fish treated with US + SAEW exhibited significantly lower degrees of lipid oxidation and protein degradation (P < 0.05). Microbiological results showed that US + SAEW inhibited the growth of Pseudomonas and activity of endogenous enzymes; also, US + SAEW decreased the relative activities of 2,3,5-triphenyl tetrazolium chloride-dehydrogenase (TTC-dehydrogenase) and ATP-ase in Pseudomonas by 65.89% and 10.26%, respectively. The combination of US + SAEW destroyed the cellular membrane and aggravated the leakage of nucleic acid and protein of Pseudomonas, and effectively inhibited the activity of antioxidant enzyme. This study reports a new industrial method for preserving refrigerated fish.

The acid tolerance responses of the Salmonella strains isolated from beef processing plants.

The development of the stationary-phase, low-pH-inducible acid tolerance response (ATR) in the Salmonella contaminant of beef during the processing arises food safety concerns, because it may evoke bacterial coping mechanisms against bactericidal insults and alter gene expression that contribute to pathogen virulence. However, information on the development of the ATR and the stability (defined as the capacity to maintain the acquired acid tolerance after induction) in the Salmonella during the production and distribution of beef is limited. After adaptation overnight, ATRs in the 79 strains of Salmonella isolated from beef processing plants were investigated by comparing the log reduction in the 2-h acid challenge trials at pH 3.0. Six representative strains were selected to further estimate the effect of three factors in the incubation period on the development of the ATR, including adapted pH values (5.0, 5.4, 6.0, and 7.0), temperatures (10 °C and 37 °C), and the adaptation media (meat extract and brain heart infusion media). The stability of acid tolerance during the long-time chilled storage (4 °C for 13 days) was also observed on two strains of serotypes S. Derby and S. Meleagridis. All the strains isolated from beef processing plants exhibited an enhanced acid tolerance indicating the widespread existence of ATR. The results also revealed that strain variability was present in the development of ATR. Significant tolerance to lethal acidic environments (pH 3.0) was found when the Salmonella strains had been acid-adapted in meat extract at pH 5.0, pH 5.4, or pH 6.0, which indicated the possible induction of ATR during beef production. After the acid adaptations, the population reduction after the acid challenge (BHI, pH = 3) in the strains was significantly lower than the non-induced at the 1d, 7 day and 13 day's storage in meat extract media at 4 °C, which revealed the persistence of ATR during beef distribution. Compared to 37 °C, adaptation in lower temperature (10 °C) significantly reduced the ATR and no ATR was developed when adapted in 4 °C. This emphasizes the importance of keeping a low temperature of beef throughout the supply chains of beef industry.

Cooperative Interaction between Acid and Copper Resistance in Escherichia coli.

The persistence of pathogenic Escherichia coli under acidic conditions poses a serious risk to food safety, especially in acidic foods such as kimchi. To identify the bacterial factors required for acid resistance, transcriptomic analysis was conducted on an acid-resistant enterotoxigenic E. coli strain and the genes with significant changes in their expression under acidic pH were selected as putative resistance factors against acid stress. These genes included those associated with a glutamatedependent acid resistance (GDAR) system and copper resistance. E. coli strains lacking GadA, GadB, or YbaST, the components of the GDAR system, exhibited significantly attenuated growth and survival under acidic stress conditions. Accordantly, the inhibition of the GDAR system by 3-mercaptopropionic acid and aminooxyacetic acid abolished bacterial adaptation and survival under acidic conditions, indicating the indispensable role of a GDAR system in acid resistance. Intriguingly, the lack of cueR encoding a transcriptional regulator for copper resistance genes markedly impaired bacterial resistance to acid stress as well as copper. Conversely, the absence of YbaST severely compromised bacterial resistance against copper, suggesting an interplay between acid and copper resistance. These results suggest that a GDAR system can be a promising target for developing control measures to prevent E. coli resistance to acid and copper treatments.

Acidic/Alkaline Stress Mediates Responses to Azole Drugs and Oxidative Stress in Aspergillus fumigatus.

A human host exploits stresses such as acidic/alkaline pH, antifungal drugs, and reactive oxygen species to kill microbial pathogens such as the fungus Aspergillus fumigatus. However, A. fumigatus is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of the human host? In this observation, we show that simultaneous exposure to acidic pH and oxidative stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills A. fumigatus synergistically in vitro. Interestingly, A. fumigatus is resistant to the combination of alkaline pH and oxidative stress. Quantitative real-time PCR analyses showed that acidic/alkaline pH stress can mediate oxidative stress responses in A. fumigatus by regulating the expression of catalase-encoding genes. We further show that A. fumigatus is sensitive to the combination of acidic/alkaline stress and azole drug stress. Transcriptome analysis revealed that the sensitivity of A. fumigatus to azole drugs under acidic/alkaline conditions may be related to changes in genetic stability, sphingolipid metabolism, lipid metabolism, and amino acid metabolism. Collectively, our findings suggest that combinatorial stress represents a powerful fungicidal mechanism employed by hosts against pathogens, which suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The human host combats fungal infections via phagocytic cells that recognize and kill fungal pathogens. Immune cells combat Aspergillus fumigatus infections with a potent mixture of chemicals, including reactive oxygen species, acidic/alkaline stress, and antifungal drugs. However, A. fumigatus is relatively resistant to these stresses in vitro. In this observation, we show that it is the combination of acidic/alkaline pH and oxidative or azole stress that kills A. fumigatus so effectively, and we define the molecular mechanisms that underlie this potency. Our findings suggest that combinatorial stress is a powerful fungicidal mechanism employed by hosts, which suggests novel approaches to potentiate antifungal therapy. This study provides a platform for future studies that will address the combinatorial impacts of various environmental stresses on A. fumigatus and other pathogenic microbes.