Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Peptide supplementation relieves stress and enhances glycolytic flux in filamentous fungi during organic acid bioproduction.

Filamentous fungi occupy a uniquely favorable position in the bioproduction of organic acids. Intracellular stress is the main stimulator in filamentous fungi to produce and accumulate organic acids with high flux. However, stress can affect the physiological activities of filamentous fungi, thereby deteriorating their fermentation performance. Herein, we report that peptide supplementation during Rhizopus oryzae fermentation significantly improved fumaric acid production. Specifically, fumaric acid productivity was elevated by approximately 100%, fermentation duration was shortened from 72 to 36 h, while maintaining the final titer. Furthermore, transcriptome profile analysis and biochemical assays indicated that the overall capabilities of the stress defense systems (enzymatic and nonenzymatic) were significantly improved in R. oryzae. Consequently, glycolytic metabolism was distinctly enhanced, which eventually resulted in improved fumaric acid production and reduced fermentation duration. We expect our findings and efforts to provide essential insights into the optimization of the fermentation performance of filamentous fungi in industrial biotechnology and fermentation engineering.

The role of environmental calcium in the extreme acid tolerance of northern banjo frog (Limnodynastes terraereginae) larvae.

Many aquatically respiring animals acutely exposed to low pH waters suffer inhibition of ion uptake and loss of branchial (gill) epithelial integrity, culminating in a fatal loss of body Na+. Environmental calcium levels ([Ca2+]e) are pivotal in maintaining branchial junction integrity, with supplemental Ca2+ reversing the negative effects of low pH in some animals. Tolerance of some naturally acidic environments by aquatic animals is further complicated by low [Ca2+]e, yet many of these environments are surprisingly biodiverse. How animals overcome the damaging actions of low pH and low environmental Ca2+ remains unknown. We examined the effects of [Ca2+]e on the response to low pH in larvae of the highly acid-tolerant frog Limnodynastes terraereginae. Acute exposure to low pH water in the presence of low (5 µmol l-1) [Ca2+]e increased net Na+ efflux. Provision of additional [Ca2+]e reduced net Na+ efflux, but the effect was saturable. Acclimation to both low and high (250 µmol l-1) [Ca2+]e improved the resistance of larvae to Na+ efflux at low pH. Exposure to the Ca2+ channel inhibitor ruthenium red resulted in an abrupt loss of tolerance in low pH-acclimated larvae. Acclimation to acidic water increased branchial gene expression of the intracellular Ca2+ transport protein calbindin, consistent with a role for increased transcellular Ca2+ trafficking in the tolerance of acidic water. This study supports a role for [Ca2+]e in promoting branchial integrity and highlights a potential mechanism via the maintenance of transcellular Ca2+ uptake in the acid tolerance of L. terraereginae larvae.

Simultaneous Quantification of Organic Acids in Tamarillo (Solanum betaceum) and Untargeted Chemotyping Using Methyl Chloroformate Derivatisation and GC-MS.

Sixteen organic acids were quantified in peel and pulp of Amber, Laird's Large and Mulligan cultivars of tamarillo using GC-MS. Fourteen of these compounds had not previously been quantified in tamarillo. An untargeted metabolomics approach was used in parallel to identify and quantify 64 more metabolites relative to the internal standard, indicating abundances of glutamic acid, pro-line, aspartic acid and γ-aminobutyric acid as well as lower concentrations of several other essential fatty acids and amino acids. The main findings were that total organic acid concentration was significantly higher (p < 0.05) in pulp than in peel, with the highest concentration seen in Mulligan pulp (219.7 mg/g DW). Remarkably, after citric acid, the potent bactericide itaconic acid was the second most abundant organic acid. At least 95% of organic acids in tamarillo were one of these two acids, as well as cis-aconitic, malic and 4-toluic acids. Differences between cultivar chemotypes were as substantial as differences between tissues. These results suggest that the bitter flavour of the peel does not result from organic acids. The combination of targeted and untargeted metabolomics techniques for simultaneous qualitative and quantitative investigation of nutrients and flavours is efficient and informative.

Long-Term Biocide Efficacy and Its Effect on a Souring Microbial Community.

Reservoir souring, which is the production of H2S mainly by sulfate-reducing microorganisms (SRM) in oil reservoirs, has been a long-standing issue for the oil industry. While biocides have been frequently applied to control biogenic souring, the effects of biocide treatment are usually temporary, and biocides eventually fail. The reasons for biocide failure and the long-term response of the microbial community remain poorly understood. In this study, one-time biocide treatments with glutaraldehyde (GA) and an aldehyde-releasing biocide (ARB) at low (100 ppm) and high (750 ppm) doses were individually applied to a complex SRM community, followed by 1 year of monitoring of the chemical responses and the microbial community succession. The chemical results showed that souring control failed after 7 days at a dose of 100 ppm regardless of the biocide type and lasting souring control for the entire 1-year period was achieved only with ARB at 750 ppm. Microbial community analyses suggested that the high-dose biocide treatments resulted in 1 order of magnitude lower average total microbial abundance and average SRM abundance, compared to the low-dose treatments. The recurrence of souring was associated with reduction of alpha diversity and with long-term microbial community structure changes; therefore, monitoring changes in microbial community metrics may provide early warnings of the failure of a biocide-based souring control program in the field. Furthermore, spore-forming sulfate reducers (Desulfotomaculum and Desulfurispora) were enriched and became dominant in both GA-treated groups, which could cause challenges for the design of long-lasting remedial souring control strategies. IMPORTANCE Reservoir souring is a problem for the oil and gas industry, because H2S corrodes the steel infrastructure, downgrades oil quality, and poses substantial risks to field personnel and the environment. Biocides have been widely applied to remedy souring, but the long-term performance of biocide treatments is hard to predict or to optimize due to limited understanding of the microbial ecology affected by biocide treatment. This study investigates the long-term biocide performance and associated changes in the abundance, diversity, and structure of the souring microbial community, thus advancing the knowledge toward a deeper understanding of the microbial ecology of biocide-treated systems and contributing to the improvement of current biocide-based souring control practices. The study showcases the potential application of incorporating microbial community analyses to forecast souring, and it highlights the long-term consequences of biocide treatment in the microbial communities, with relevance to both operators and regulators.

From Laboratory towards Industrial Operation: Biomarkers for Acidophilic Metabolic Activity in Bioleaching Systems.

In the actual mining scenario, copper bioleaching, mainly raw mined material known as run-of-mine (ROM) copper bioleaching, is the best alternative for the treatment of marginal resources that are not currently considered part of the profitable reserves because of the cost associated with leading technologies in copper extraction. It is foreseen that bioleaching will play a complementary role in either concentration-as it does in Minera Escondida Ltd. (MEL)-or chloride main leaching plants. In that way, it will be possible to maximize mines with installed solvent-extraction and electrowinning capacities that have not been operative since the depletion of their oxide ores. One of the main obstacles for widening bioleaching technology applications is the lack of knowledge about the key events and the attributes of the technology's critical events at the industrial level and mainly in ROM copper bioleaching industrial operations. It is relevant to assess the bed environment where the bacteria-mineral interaction occurs to learn about the limiting factors determining the leaching rate. Thus, due to inability to accurately determine in-situ key variables, their indirect assessment was evaluated by quantifying microbial metabolic-associated responses. Several candidate marker genes were selected to represent the predominant components of the microbial community inhabiting the industrial heap and the metabolisms involved in microbial responses to changes in the heap environment that affect the process performance. The microbial community's predominant components were Acidithiobacillus ferrooxidans, At. thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus sp. Oxygen reduction, CO2 and N2 fixation/uptake, iron and sulfur oxidation, and response to osmotic stress were the metabolisms selected regarding research results previously reported in the system. After that, qPCR primers for each candidate gene were designed and validated. The expression profile of the selected genes vs. environmental key variables in pure cultures, column-leaching tests, and the industrial bioleaching heap was defined. We presented the results obtained from the industrial validation of the marker genes selected for assessing CO2 and N2 availability, osmotic stress response, as well as ferrous iron and sulfur oxidation activity in the bioleaching heap process of MEL. We demonstrated that molecular markers are useful for assessing limiting factors like nutrients and air supply, and the impact of the quality of recycled solutions. We also learned about the attributes of variables like CO2, ammonium, and sulfate levels that affect the industrial ROM-scale operation.

Role of aspartate ammonia-lyase in Pasteurella multocida.

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Effects of Replacing In-feed Antibiotics with Synergistic Organic Acids on Growth Performance, Health, Carcass, and Immune and Oxidative Statuses of Broiler Chickens Under Clostridium perfringens Type A Challenge.

This study was conducted to investigate the effects of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative statuses of broiler chickens under Clostridium perfringens (CP) type A challenge. Two organic acid products were tested: organic acid 1 (OA1), consisting of butyrate, medium-chain fatty acids, organic acids, and phenolics; and organic acid 2 (OA2), consisting of buffered short-chain fatty acids. Six hundred 1-day-old male Arbor Acres broiler chicks were randomly assigned to one of five treatments: Control 1, basal diet, nonchallenged birds; Control 2, basal diet, with CP challenge; antimicrobial growth promoters (AGP), basal diet supplemented with Aureomycin (chlortetracycline), with CP challenge; OA1, basal diet supplemented with OA1, with CP challenge; and OA1OA2, basal diet supplemented with OA1 and OA2, with CP challenge. Each treatment had eight replicate pens of 15 birds. The experiments lasted for 29 days. The disease challenge was performed on days 15-17, with an oral gavage of 0.5 mL of CP culture (2.0 × 108 colony-forming units [CFU]/mL) for each bird. Body weights (BWs), intestinal lesion scores, immune organ indices, and serum malondialdehyde (MDA) concentrations were measured on days 19, 22, and 29, respectively, in three birds per pen. Carcass characteristics were determined on day 29. No treatment-related differences in mortality were noted before (P = 0.28) or after (P = 0.64) challenge or over the whole study period (days 0-28; P = 0.66). On day 19, the BW of Control 2 was lower than other treatments (P < 0.0001). On day 22, AGP, OA1, and OA1OA2 had higher BW than Control 2 (P = 0.001). The breast muscle yield of OA1 and OA1OA2 was higher than AGP (P < 0.05). The abdominal fat yield of OA1OA2 was lower than AGP and Control 2 (P < 0.05). On day 22, the birds fed OA1OA2 showed lower intestinal lesion scores than OA1 (P < 0.05). No treatment-related differences in immune organ (spleen, thymus, and bursa) indices were noted (P > 0.05). On day 29, the MDA concentration of OA1 and OA1OA2 was lower than those of Control 1 and AGP (P < 0.05). In conclusion, the addition of organic acids may protect broiler chickens from severe intestinal lesions and oxidative stress and may help reduce abdominal fat mass deposition. There is potential for organic acid-based products as alternatives for AGP in preventing necrotic enteritis in broilers.

Lactobacillus commensals autochthonous to human milk have the hallmarks of potent probiotics.

Maternal milk is an important source of essential nutrients for the optimal growth of infants. Breastfeeding provides a continuous supply of beneficial bacteria to colonize the infant gastrointestinal tract (GIT) and offers health benefits for disease prevention and immunity. The purpose of this study was to isolate novel probiotic strains from the breast milk of native Pakistani mothers and to evaluate their probiotic potential. We isolated 21 strains of bacteria from the colostrum and mature milk of 20 healthy mothers, who had vaginal deliveries and were not taking antibiotics. After phenotypic and genotypic characterization, these isolates were tested for survival in the GIT using in vitro acid and bile tests. Nine strains showing good acid tolerance were assessed for their growth rate, bile resistance and ability to hydrolyze bile salts. Out of the four Lactobacillus isolates adjudged to be most promising as probiotics, three were Lactobacillus fermentum strains and one was a strain of Lactobacillus oris. This study demonstrates that human milk is a viable source of commensal bacteria beneficial to both adults and babies.

Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose.

BACKGROUND: Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources. RESULTS: Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC-MS/MS-TripleQ and GC-MS, while untargeted metabolite profiling was performed using LC-MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures. CONCLUSIONS: Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.

Negative dietary cation-anion difference and amount of calcium in prepartum diets: Effects on milk production, blood calcium, and health.

Acidogenic prepartum diets with negative dietary cation-anion difference (DCAD) induce compensated metabolic acidosis, which stimulates calcium (Ca) mobilization before calving and decreases clinical and subclinical hypocalcemia postpartum. This strategy is often combined with limiting dietary Ca concentrations, which historically has been used to mobilize Ca prepartum to prepare cows for lactation. Supplemental dietary Ca in combination with a negative DCAD formulation that does not reverse the effect of compensated metabolic acidosis may be beneficial. Our objective was to determine the effects of prepartum dietary strategies on dry matter intake (DMI), milk production, peripartal Ca status, and health during the transition period in multiparous Holstein cows (n = 81). Treatments during the last 28 d before calving were: (1) positive DCAD diet, +6 mEq/100 g of DM, target urine pH >7.5, low dietary Ca (0.40% DM; CON); (2) negative DCAD diet, -24 mEq/100 g of DM, target urine pH 5.5 to 6.0, low dietary Ca (0.40% DM; ND); or (3) negative DCAD diet, -24 mEq/100 g of DM, target urine pH 5.5 to 6.0, , high dietary Ca (2.0% DM; NDCA). Preplanned treatment contrasts were: (1) CON versus (ND and NDCA), and (2) ND versus NDCA. Individual DMI were recorded daily. Cows were milked 3 times daily, with individual DMI and milk yield summarized by week. Whole blood sampled at calving and 24 h, 48 h, and 4 d after calving was analyzed for ionized Ca concentration, and serum was analyzed for total Ca. Prepartum urine pH for cows fed ND or NDCA averaged 5.7, whereas cows fed CON remained >7.5. During the 3 wk before calving, cows fed CON had greater DMI than cows fed ND or NDCA, with NDCA greater than ND. Postpartum DMI (% of body weight) tended to be less for cows fed CON than for those fed ND or NDCA prepartum. Thresholds for subclinical hypocalcemia were ionized Ca <1.0 mM at 24 h, and total Ca ≤2.125 mM at 48 h after calving. On average, blood Ca for cows fed CON indicated subclinical hypocalcemia, whereas blood Ca for cows fed ND or NDCA was greater than subclinical hypocalcemia thresholds for both ionized Ca and total Ca. No milk production differences were detected. Cows fed CON had an elevated adverse health score (calculated by assigning numerical values to recorded health events) and tended to have an elevated somatic cell count during the fresh period compared with cows fed ND or NDCA. Overall, an acidogenic diet prepartum without or with high Ca improved postpartum Ca status and health. Supplementation of additional Ca to the acidogenic diet had little effect.

Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface.

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

Amino acids other than glutamate affect the expression of the GAD system in Listeria monocytogenes enhancing acid resistance.

The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety.

Inoculation of pqqE gene inhabiting Pantoea and Pseudomonas strains improves the growth and grain yield of wheat with a reduced amount of chemical fertilizer.

OBJECTIVE: The present study was performed to examine the role of pqqE inhabiting rhizobacteria in organic acid production and relationship of the organic acids with phosphate solubilization by the bacteria in vitro as well as in vivo. METHODS AND RESULTS: The pqqE gene was PCR amplified and sequenced in genomic DNA of Pantoea sp. WP-5 and Pseudomonas sp. NN-4. Nucleotide sequence obtained from WP-5 and NN-4 showed maximum sequence similarity (88 and 89%, respectively) with the pqqE gene of Pseudomonas fluorescens strain CMR12a (KM251420). Deduced amino acid sequence from pqqE gene of Pseudomonas sp. NN-4 and Pantoea sp. WP-5 showed 75 and 93% similarity, respectively, with protein pyrroloquinoline quinone. Phosphate solubilization and acid production assay were quantified on spectrophotometer and high-profile liquid chromatograph, respectively, by each bacterial strain. Both strains produced organic acids such as acetic, citric, gluconic, succinic and malic acid and lowered the pH of Pikovskaya broth medium under laboratory conditions. Phosphate solubilization by Pantoea sp. WP-5 was 311 ± 4 and 204 ± 3 µg ml-1 in the culture medium supplemented with glucose and sucrose as carbon source, respectively. Pseudomonas sp. NN-4 solubilized 176 ± 3 and 298 ± 5 µg ml-1 phosphate in Pikovskaya broth medium under similar conditions. In field experiments conducted during two consecutive years, the concentration of acetic acid and gluconic acid was higher in root exudates of plants treated with Pantoea sp. WP-5 at 30% reduced doses of nitrogen (N)- and phosphorus (P)-based chemical fertilizers as compared to non-inoculated plants. Values of chlorophyll contents, crop growth rate, leaf area index, straw yield and P contents were recorded higher in plants inoculated with Pantoea sp. WP-5 and Pseudomonas sp. NN-4 as compared to non-inoculated control. Grain yield was increased by 10-12% due to inoculation with Pantoea sp. WP-5 and Pseudomonas sp. NN-4 over non-inoculated control in the field experiments. CONCLUSIONS: These results lead to the conclusions that the rhizobacteria inhabiting pqqE gene produced organic acids and solubilized the phosphate in vitro. On inoculation to wheat plants in field experiments, these strains produced the organic acids, solubilized the phosphate, and improved the P uptake and productivity of wheat. SIGNIFICANCE AND IMPACT OF THE STUDY: The Pantoea sp. WP-5 and Pseudomonas sp. NN-4 are the potential candidates for inoculation to wheat as phosphate solubilizer even with reduced chemical fertilizer dose. The inoculation of the strains may enhance grain yield and net income of the farmer even with less chemical fertilizer application. This practice will be helpfull inminimizing environmental pollution.

Biotransformation of green tea (Camellia sinensis) by wine yeast Saccharomyces cerevisiae.

Wine yeast Saccharomyces cerevisiae 71B was used in fermentation of green tea to modulate the volatiles and nonvolatiles. After fermentation, higher alcohols, esters, and acids, such as isoamyl alcohol, isobutanol, ethyl octanoate, ethyl decanoate, octanoic, and decanoic acids were generated. Some key aroma compounds of tea including linalool, hotrienol, dihydroactinidiolide, and 2-phenylethanol increased significantly. Among these compounds, linalool and 2-phenylethanol increased by 1.3- and 10-fold, respectively, which impart floral and fruity notes to fermented green tea. Alkaloids including caffeine, theobromine, and theophylline were reduced significantly after fermentation, while the most important free amino acid in tea, theanine, was not metabolized by S. cerevisiae. Tea catechins decreased whereas gallic and caffeic acids increased significantly, resulting in the unchanged antioxidant capacity of the fermented green tea. Hence, this work highlighted the potential of using S. cerevisiae to modulate green tea aroma and nonvolatiles. PRACTICAL APPLICATION: A novel fermented tea is produced by yeast fermentation. Saccharomyces cerevisiae led to significant changes in tea volatiles and nonvolatiles. Antioxidant capacity remained stable after fermentation.

Effects of Hop Bitter Acids, Bitter Components in Beer, on Cognition in Healthy Adults: A Randomized Controlled Trial.

The present study aimed to investigate the effects of matured hop bitter acids (MHBAs) on human cognition, mental fatigue, and mood state. In this randomized double-blind placebo-controlled study, 60 healthy adults (age 45-64 years) with self-awareness of cognitive decline were randomly divided into 2 groups and received either orally administered MHBAs (35 mg/day) or placebo for 12 weeks. Cognitive functions and mental states were assessed using neuropsychological tests or questionnaires at baseline and weeks 6 and 12 of the intervention. The change in verbal fluency score at week 6 compared with that at baseline was significantly higher in the MHBAs-treated group compared with that in the placebo group (P = 0.034), and Stroop test score at week 12 was significantly lower in the MHBAs-treated group compared with the placebo group (P = 0.019). Furthermore, subjective fatigue and anxiety at week 12 were significantly improved in the MHBAs-treated group (P = 0.008 and 0.043, respectively) compared with the placebo group. This is the first study to evaluate the effects of bitter ingredients in beer on cognition, subjective mood, and mental fatigue in a clinical trial. Our findings suggest that hop-derived bitter acids might be beneficial for cognition and mood state.

Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo.

Lysosomal acidification is a key feature of healthy cells. Inability to maintain lysosomal acidic pH is associated with aging and neurodegenerative diseases. However, the mechanisms elicited by impaired lysosomal acidification remain poorly understood. We show here that inhibition of lysosomal acidification triggers cellular iron deficiency, which results in impaired mitochondrial function and non-apoptotic cell death. These effects are recovered by supplying iron via a lysosome-independent pathway. Notably, iron deficiency is sufficient to trigger inflammatory signaling in cultured primary neurons. Using a mouse model of impaired lysosomal acidification, we observed a robust iron deficiency response in the brain, verified by in vivo magnetic resonance imaging. Furthermore, the brains of these mice present a pervasive inflammatory signature associated with instability of mitochondrial DNA (mtDNA), both corrected by supplementation of the mice diet with iron. Our results highlight a novel mechanism linking impaired lysosomal acidification, mitochondrial malfunction and inflammation in vivo.

Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms.

There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. Label="OBJECTIVE">To evaluate at the hard tissue level the effect of C. albicans on the cariogenic potential of S. mutans biofilms focusing on the mineral profile of induced carious lesions. This study also aimed to evaluate the effect of C. albicans on the acidogenic potential of S. mutans biofilms. METHODOLOGY Dual-species (CA+SM) and single-species biofilms (CA or SM) were grown on the surface of enamel slabs in the presence of glucose/sucrose supplemented culture medium for 24, 48 and 72 hours. Demineralization was evaluated through percentage of surface microhardness change (%SMC) and transversal microradiography analysis (ILM and LD) and pH of the spent medium was recorded daily. Data were analyzed by two-way ANOVA followed by Bonferroni correction. RESULTS%SMC was statistically different among the biofilms at each time point being the highest for SM biofilms and the lowest for CA biofilms which also differed from CA+SM biofilms [SM (24 h: 47.0±7.3; 48 h: 66.3±8.3; 72 h: 75.4±3.9); CA (24 h: 7.3±3.3; 48 h: 7.1±6.4; 72 h: 6.6±3.6); CA+SM (24 h: 35.9±7.39.1; 48 h: 47.2±9.5; 72 h: 47.6±9.5)]. pH of spent medium was statistically lower for SM biofilms compared to the other biofilms at each time point and remained constant over time while pH values increased from 24 to 72 h for both CA and CA+SM biofilms [SM (24 h: 4.4±0.1; 48 h: 4.4±0.1; 72 h: 4.5±0.1); CA (24 h: 6.9±0.3; 48 h: 7.2±0.2; 72 h: 7.5±0.2); CA+MS (24 h: 4.7±0.2; 48 h: 5.1±0.1; 72 h: 6.1±0.6)]. IML and LD for SM biofilms increased over time while no difference was observed from 24 to 72 h for the other biofilms. CONCLUSIONS The present data suggest that C. albicans has low enamel demineralization potential and the presence of C. albicans can reduce both the cariogenic and acidogenic potentials of S. mutans biofilms.

Effects of encapsulated essential oils and organic acids on laying performance, egg quality, intestinal morphology, barrier function, and microflora count of hens during the early laying period.

The objective of this study was to investigate the effect of encapsulated essential oils and organic acids (EOA) on the growth performance, egg quality, intestinal morphology and functions, and microbial count of laying hens from week 21 to 30. A total of five hundred and four 21-wk-old layers were randomly allotted into 4 groups consisting of 7 replicates with 18 birds per replicate. The birds were fed a basic diet (CON) or diets with EOA at 150 mg/kg, 300 mg/kg, and 450 mg/kg in the other 3 groups, respectively. Compared to the CON group, the addition of 150 mg/kg EOA significantly increased laying rate (P < 0.05) of hens from week 21 to 25. A linear increasing (linear, P < 0.01) in ileal villus height of laying hens fed EOA from 150 to 300 mg/kg was observed at week 30. At week 25, the supplementation of 300 mg/kg EOA significantly increased (P < 0.05) mRNA relative expression of aminopeptidase, sodium-glucose cotransporter 1, and Na+-independent neutral amino acid transporter in duodenum and glucose transporter 2 in jejunum of laying hens compared to the CON groups. Meanwhile, the relative expression of glucose transporter 2 mRNA in the jejunum was upregulated with increasing concentration of EOA in diets (linear, P < 0.05). Hens in EOA 300 group had higher mRNA relative expression of mucin-2 in ileum (P < 0.05) than hens in CON group. Additionally, the secretory immunoglobulin in ileum A were linear decreased (linear, P < 0.01) with the increasing supplement of EOA. Dietary supplementation with EOA tended to increase (P = 0.083) the counts of Bifidobacterium in cecal digesta at week 25 and 30. In conclusion, dietary with EOA may maintain intestinal tract morphology and promote digestive and absorptive capacities and barrier function, especially at 300 mg/kg. This study provided evidence of using EOA as a potential feed additive for laying hens.

Changes in chemical profile of Cascade hop cones according to the growing area.

BACKGROUND: The growing area has a substantial effect on plants, affecting secondary metabolism. For hops, different authors have studied the effect of growing area on the chemical composition of cones with the aim of verifying and understanding the changes in hop characters. Despite the scant literature the subject receives increasing attention by brewers and hop growers. The present study aimed to characterize, using gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography with ultraviolet detection (HPLC-UV), cones of hop (Humulus lupulus L.) cultivar Cascade. Plant material was obtained from nine different areas of Italy and compared with Cascade samples grown in the United States, Germany and Slovenia. RESULTS: Differences in bitter acids and xanthohumol content were observed. Nevertheless, no correlation between bitter acids and xanthohumol production, on the one hand, and rainfall, temperatures and latitude, on the other hand, were observed in our samples. The Slovenia samples were richer in molecules that confer hoppy, woody and flower notes; USA2 samples were more characterized by woody, earthy, grassy and floral aroma, quite different characters if compared to USA1, which had the lowest presence of grassy aromatic compounds. In the Italian samples, TRENTINO was the genotype most characterized by limonene presence. CONCLUSION: The results of this study are indicative of the importance for hop users to know and characterize hops coming from different growing regions. The study pays special attention to the characterization of the differences in chemical characters of Cascade hop in Italy, where hop cultivation has developed only recently, but is in continuous expansion. © 2019 Society of Chemical Industry.