Fed-batch fermentation strategy for efficient welan gum production by Sphingomonas sp. FM01.

BACKGROUND: As a novel type of extracellular polysaccharide produced by Sphingomonas sp., welan gum has been widely applied in various fields because of its excellent properties. The study has improved the fermentation process. RESULTS: The initial sucrose concentration, temperature and stirring speed were set to 20 g L-1, 33 °C and 400 rpm, respectively, and 13.3 g L-1 sucrose was added at 24, 40 and 56 h. The temperature and stirring speed were then set at 28 °C and 600 rpm from 24 to 48 h and 28 °C and 600 rpm from 48 to 72 h, respectively. As a result, welan gum production, dry cell weight, sucrose conversion rate and viscosity were correspondingly increased to 38.60 g L-1, 5.47 g L-1, 0.64 g g-1 and 3779 mPa·s, respectively. In addition, the mechanism by which fermentation strategy promotes welan gum synthesis was investigated by transcriptome analysis. CONCLUSION: Improving respiration and ATP supply, reducing unnecessary protein synthesis, and alleviating competition between cell growth and welan gum synthesis contribute to promoting the fermentation performance of Sphingomonas sp., thus providing a practical strategy for efficient welan gum production. © 2024 Society of Chemical Industry.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

Mosquito Feeding Habits in Coastal French Guiana: Mammals in the Crosshairs?

Pathogens transmitted by mosquitoes (Diptera, Culicidae) in sylvatic or urban cycles involve wild or domestic animals and humans, driven by various mosquito species with distinct host preferences. Understanding mosquito-host associations is crucial for ecological insights and pathogen surveillance. In this study, we analyzed mosquito blood meals from coastal French Guiana by amplifying and sequencing host DNA from blood-fed females. Using the 12S ribosomal RNA gene and Sanger sequencing, we identified blood meals from 26 mosquito species across six genera, with 59% belonging to the Culex genus. Nanopore sequencing of selected samples showed 12 mosquito species with one to three mixed blood-meal sources. Mammals were the primary hosts (88%), followed by birds (7%), squamates (3%), and amphibians (2%), indicating a strong preference for mammalian hosts. A total of 46 vertebrate host species were identified, demonstrating high host diversity. This research provides insights into mosquito host usage and highlights the complexities of monitoring arboviruses of public health concern.

Improvement of cellobiose 2-epimerase expression in Bacillus subtilis for efficient bioconversion of lactose to epilactose.

Epilactose, a lactose derivative known for its prebiotic properties and potential health benefits, has garnered significant interest. Cellulose 2-epimerase (CEase) is responsible for catalyzing the conversion of lactose to epilactose. In this study, the enhancement of food-grade CEase expression in Bacillus subtilis WB600 was systematically investigated. Among seven selected epilactose-producing CEases, Rhodothermus marinus CEase (RmCE) exhibited the highest epimerization activity when expressed in B. subtilis. Translational and transcriptional regulations were employed to enhance CEase expression by screening effective N-terminal coding sequences (NCSs) and promoters. The final strain demonstrated efficient production of CEase, with epimerization activity reaching 273.6 ±â€¯6.5 U/mL and 1255 ±â€¯26.4 U/mL in shake-flask and fed-batch cultivation, respectively. Utilizing only 0.25 % (V/V) of the fed-batch cultivation broth for lactose biotransformation, epilactose was efficiently produced from 300 g/L of lactose within 4 h, achieving a yield of 29.5 %. These findings provide significant support for the potential industrialization of enzymatic epilactose production.

CHO stable pool fed-batch process development of SARS-CoV-2 spike protein production: Impact of aeration conditions and feeding strategies.

Technology scale-up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and kLa must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale-up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS-CoV-2 spike protein in 1.8 L benchtop stirred-tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio-capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25-fold) and protein yield (2.52-fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio-capacitance signals.

Ni-Doped SFM Double-Perovskite Electrocatalyst for High-Performance Symmetrical Direct-Ammonia-Fed Solid Oxide Fuel Cells.

Ammonia has emerged as a promising fuel for solid oxide fuel cells (SOFCs) owing to its high energy density, high hydrogen content, and carbon-free nature. Herein, the electrocatalytic potential of a novel Ni-doped SFM double-perovskite (Sr1.9Fe0.4Ni0.1Mo0.5O6-δ) is studied, for the first time, as an alternative anode material for symmetrical direct-ammonia SOFCs. Scanning and transmission electron microscopy characterization has revealed the exsolution of Ni-Fe nanoparticles (NPs) from the parent Sr2Fe1.5Mo0.5O6 under anode conditions, and X-ray diffraction has identified the FeNi3 phase after exposure to ammonia at 800 °C. The active-exsolved NPs contribute to achieving a maximal ammonia conversion rate of 97.9% within the cell's operating temperatures (550-800 °C). Utilizing 3D-printed symmetrical cells with SFNM-GDC electrodes, the study demonstrates comparable polarization resistances and peak power densities of 430 and 416 mW cm-2 for H2 and NH3 fuels, respectively, with long-term stability and a negligible voltage loss of 0.48% per 100 h during ammonia-fed extended galvanostatic operation. Finally, the ammonia consumption mechanism is elucidated as a multistep process involving ammonia decomposition, followed by hydrogen oxidation. This study provides a promising avenue for improving the performance and stability of ammonia-based SOFCs for potential applications in clean energy conversion technologies.

Efficient production of phenyllactic acid in Escherichia coli via metabolic engineering and fermentation optimization strategies.

Phenyllactic acid (PhLA), an important natural organic acid, can be used as a biopreservative, monomer of the novel polymeric material poly (phenyllactic acid), and raw material for various medicines. Herein, we achieved a high-level production of PhLA in Escherichia coli through the application of metabolic engineering and fermentation optimization strategies. First, the PhLA biosynthetic pathway was established in E. coli CGSC4510, and the phenylalanine biosynthetic pathway was disrupted to improve the carbon flux toward PhLA biosynthesis. Then, we increased the copy number of the key genes involved in the synthesis of the PhLA precursor phenylpyruvic acid. Concurrently, we disrupted the tryptophan biosynthetic pathway and enhanced the availability of phosphoenolpyruvate and erythrose 4-phosphate, thereby constructing the genetically engineered strain MG-P10. This strain was capable of producing 1.42 ± 0.02 g/L PhLA through shake flask fermentation. Furthermore, after optimizing the dissolved oxygen feedback feeding process and other conditions, the PhLA yield reached 52.89 ± 0.25 g/L in a 6 L fermenter. This study successfully utilized metabolic engineering and fermentation optimization strategies to lay a foundation for efficient PhLA production in E. coli as an industrial application.

The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes.

BACKGROUND: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. RESULTS: Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. CONCLUSIONS: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.

Characterization of Runella zeae D-mannose 2-epimerase and its expression in Bacillus subtilis for D-mannose production from D-glucose.

D-Mannose 2-epimerase (MEase) catalyzes the bioconversion between D-glucose and D-mannose. It is an important potential biocatalyst for large-scale production of D-mannose, a functional monosaccharide used in pharmaceutical and food industries. In this study, a new microbial MEase was characterized from Runella zeae DSM 19591. The enzyme was purified by one-step nickel-affinity chromatography and determined to be a dimeric protein with two identical subunits of approximately 86.1 kDa by gel filtration. The enzyme showed the highest activity at pH 8.0 and 40 °C, with a specific activity of 2.99 U/mg on D-glucose and 3.71 U/mg on D-mannose. The melting temperature (Tm) was 49.4 °C and the half-life was 115.14 and 3.23 h at 35 and 40 °C, respectively. The purified enzyme (1 U/mL) produced 115.7 g/L of D-mannose from 500 g/L of D-glucose for 48 h, with a conversion ratio of 23.14 %. It was successfully expressed in Bacillus subtilis WB600 via pP43NMK as the vector. The highest fermentation activity was 10.58 U/mL after fed-batch cultivation for 28 h, and the whole cells of recombinant B. subtilis produced 114.0 g/L of D-mannose from 500 g/L of D-glucose, with a conversion ratio of 22.8 %.

Male Holstein calves fed a milk replacer and pelleted calf starter containing a botanical extract or a direct fed microbial (DFM) alone or in combination.

Botanical extracts (BE; Apex, Adisseo, North America, Atlanta, GA) are known to enhance DMI and gut health, while direct fed microbials (DFM), such as a lactobacillus acidophilus fermentation product (EX: Excel; Pacer Technology, Inc., Murtaugh, ID), has demonstrated improved gut health and growth performance when fed to growing neonatal Holstein calves. The hypothesis was this combination may be synergistic to neonatal calf growth performance and intestinal health. Eighty 2-5-d old Holstein bull calves were blocked by BW and randomly assigned to 1 of 4 treatments arranged using a randomized complete block design. Treatments were: 1) Control: no additives; 2) BE added at 496 mg/kg to the calf starter (CS); 3) EX added to the CS at 2.50 g/kg with EX added to the milk replacer (MR) at 5 g/d; and 4) BE&EX: BE and EX added to CS at same rates and added EX to MR. Calves received 0.283 kg MR in 1.9 L fed 2 x/d for the first 14 d, then increased to 0.42 kg in 2.84 L fed 2x/d through d 35, followed by 0.42 kg MR in 2.84 L fed 1x/d through d 42, followed by weaning. The CS was a 25% CP DM basis mini-pellet and the MR was a 22:20 (CP:fat) fed 2x/d at 0630 and 1800 h along with free choice water. Weaning occurred after d 49 of the 70-d experiment. Calves fed CS EX alone demonstrated greater BW gain compared with calves fed BE&EX with calves fed Control and BE being intermediate and similar (63.9, 63.5, 65.0, and 59.7 kg for Control, BE, EX, and BE&EX, respectively). Total DMI (MR+CS) was lower for calves fed BE&EX compared with calves fed the remaining treatments (116.5, 114.2, 116.4, and 104.9 kg). The feeding of a BE in combination with EX (DFM) to neonatal calves reduced BW during wk 8, 9, and 10 and reduced 0-70 d ADG (874.7, 870.0, 889.7, and 817.6 g/d) compared with calves fed the remaining treatments. Calves fed BE&EX demonstrated the lowest calf starter intake during wk 4 through 10 compared with calves fed the other treatments. The study average calf starter intake for calves fed Control and EX was greater compared with calves fed BE&EX with calves fed BE being similar and intermediate. Calves fed EX and BE&EX demonstrated greater total d of fecal scour score = 1 (5.4, 5.7, 8.0 and 8.3 d) compared with calves fed Control and BE. Gains in frame measurements of hip height, hip width, withers height, and body length were similar while calves fed BE&EX demonstrated lower heart girth gains (19.2, 18.3, 19.7 and 17.6 cm) during the milk feeding phase (0 - 7 wk) compared with calves fed Control and EX, with calves fed BE being similar and intermediate. It is not known why this study demonstrated an antagonism between BE and EX in growth performance and feed intake but further research is needed to identify the mechanism of action.

Immune modulation and cecal microbiome changes in broilers fed with fenugreek seeds and Bacillus-based probiotics.

Intensive broiler production systems face challenges like enteric diseases, impacting global food security. Strategies to enhance broiler immunity and gut health, particularly amidst antibiotic growth promoter restrictions, are crucial. The present study investigated the combined effects of fenugreek seeds (FS) and Bacillus-based direct-fed microbials (DFM) on immune-related gene expression in the ileum and alteration of microbial population in the cecum of broiler. The study involved 160 Ross 308 broiler chicks, which were divided into four groups consisting of 5 replicates, each containing eight birds. The chicks were grown for a period of 42 d, during which they had ad libitum access to feed and water. Dietary treatments were: Control (basal diet), FS5 (basal + 5g/kg fenugreek seeds), FS5DFM (basal + 5g/kg fenugreek seeds + 0.1g/kg Bacillus-based DFM), and DFM (basal + 0.1g/kg Bacillus-based DFM). Ileum tissue and cecal contents were collected on d 42 for gene expression and gut microbiome analysis. Ileal gene expression analysis revealed the downregulation of IL-6, IL-8L2, CASP6, PTGS2, and IRF7 in both FSs and DFMs groups compared to the control, suggesting individual immunomodulatory effects. However, avian ß-defensin genes exhibited complex regulation, highlighting the need for further investigation. Cecal microbiome diversity remained stable, with subtle shifts in specific taxa influenced by FSs and DFMs. Interestingly, the combination of the FSs and DFMs uniquely impacted specific taxa, including Clostridiales vadin BB60. These findings suggest that both FSs and DFMs demonstrated potential for improving broiler immunity through inflammation reduction. The combination of FSs and DFMs offers a synergistic effect in immune modulation and specific microbial modulation, warranting further investigation with pathogen challenge models for comprehensive understanding.

Enhancing Protein Content in Wild-Type Saccharomyces cerevisiae via Random Mutagenesis and Optimized Fermentation Conditions.

Single-cell protein (SCP) derived from microorganisms is widely recognized as a viable alternative protein source for the future. Nevertheless, the commercialization of yeast-based SCP is hampered by its relatively low protein content. Therefore, this study aimed to enhance the protein content of Saccharomyces cerevisiae via random mutagenesis. To achieve this, S. cerevisiae KCCM 51811, which exhibited the highest protein concentration among 20 edible S. cerevisiae strains, was selected as a chassis strain. Subsequently, a KCCM 51811 mutant library was constructed (through UV irradiation) and screened to isolate mutants exhibiting high protein content and/or concentration. Among the 174 mutant strains studied, the #126 mutant exhibited a remarkable 43% and 36% higher protein content and concentration, respectively, compared to the parental strain. Finally, the #126 mutant was cultured in a fed-batch system using molasses and corn-steep liquor, resulting in a protein concentration of 21.6 g/l in 100 h, which was 18% higher than that produced by the parental strain. These findings underscore the potential of our approach for the cost-effective production of food-grade SCP.

Enhancement of ε-poly-L-lysine production by Streptomyces albulus FQF-24 with feeding strategies using cassava starch as carbon source.

ε-Poly-L-lysine (ε-PL) is a natural and wide-spectrum antimicrobial additive. In this study, the production of ε-PL by Streptomyces albulus FQF-24 using cassava starch (CS) as carbon source and the effects of different feeding methods were investigated in a fermenter. The initial shake flask experiments demonstrated the efficient production of ε-PL with CS, achieving the ε-PL production of 1.18 g/L. Subsequent investigations in the fermenter identified that the ideal pH was 3.8 during the ε-PL synthesis phase. Under this condition, the production of ε-PL reached 1.35 g/L. When the pH was maintained at 3.8, the investigation of improvement of feeding composition was carried out in a 5 L fermenter. The intermittent feeding containing CS, inorganic and organic nitrogen sources resulted in the maximum ε-PL production and dry cell weight (DCW) reaching 17.17 g/L and 42.73 g/L. Additionally, continuous feeding with the composition of CS, organic and inorganic nitrogen sources, and inorganic salts further increased ε-PL production and DCW to 27.56 g/L and 38.5 g/L. Summarily, the above results indicate that the fermentation using low-cost CS and continuous feeding strategy with whole medium composition can provide a beneficial reference for the efficient production of ε-PL.

Stabilizing Pure Water-Fed Anion Exchange Membrane Water Electrolyzers through Membrane-Electrode Interface Engineering.

Nickel-iron (oxy)hydroxide (NiFeOxHy) stands as a cutting-edge nonprecious electrocatalyst for the oxygen evolution reaction (OER). However, the intrinsic thermodynamic instability of nickel and iron as anode materials in pure water-fed electrolyzers poses a significant durability challenge. In this study, an anion exchange ionomer coating was applied to NiFeOxHy to modify the local pH between a membrane and an electrode. This effectively extended the diffusion length of hydroxide anions toward the electrode, establishing an alkaline local pH environment. Stability tests with the ionomer coating showed reduced Ni dissolution. Moreover, locally resolved current density measurements were used to demonstrate a notably lower degradation rate during stability testing, revealing a 6-fold increase in stability with the ionomer on NiFeOxHy. In situ Raman spectroscopy in a neutral pH electrolyte confirmed inhibited Ni oxidation with the ionomer, mitigating Ni dissolution and enhancing stability of state-of-the-art NiFeOxHy catalysts in pure water-fed water electrolyzers.

Determination of phytic acid disappearance, ileal P digestibility at different dietary Ca levels, and relative P bioavailability in soybean meal, canola meal, distillers dried grains with solubles, corn fermented protein, and wheat middlings.

Three experiments were conducted to evaluate P utilization in soybean meal (SBM), canola meal (CM), distillers dried grains with solubles (DDGS), corn fermented protein (CFP), and wheat middlings (WM) using different assays. In Experiment 1, phytic acid disappearance (myo-inositol 1,2,3,4,5,6-hexakis; InsP6D) and inositol phosphate disappearance (InsP-PD) were determined using precision-fed cecectomized Leghorn roosters. Roosters were precision-fed 20 to 25 g of SBM, CM, DDGS, CFP, and WM. In Experiment 2, InsP6D, InsP-PD, and standardized ileal digestibility (SID) of P at different Ca levels were determined using ad libitum-fed broiler chickens. Semi-purified cornstarch-dextrose-based diets containing SBM, CM, DDGS, CFP, and WM as the sole source of P were fed. All diets contained 0.21% P and limestone was added at the expense of dextrose to provide 0.30, 0.45, 0.60, and 0.75% Ca. In Experiment 3, P bioavailability relative to KH2PO4 was determined based on tibia bone ash. Experiments contained 5 to 6 replicates per treatment. In Experiment 1 with precision-fed roosters, InsP6D and InsP-PD ranged from 8 to 71% among feedstuffs, with the lowest (P < 0.05) disappearance being observed in SBM. In Experiment 2 with ad libitum-fed chickens, there was a Ca × ingredient interaction (P < 0.05) whereby increasing Ca linearly decreased (P < 0.05) InsP6D, InsP-PD, and SID of P for all feedstuffs, excluding CFP. Estimated P digestibility calculated using InsP6D in Experiment 1 was in good agreement with SID in Experiment 2 determined at 0.75% Ca, except for SBM. In Experiment 3, regression of bone ash content (mg/tibia) on supplemental P intake yielded P bioavailability values ranging from 30 to 81% among feedstuffs relative to KH2PO4, with the highest (P < 0.05) bioavailability being observed for DDGS and CFP. In conclusion, 1) InsP6D in precision-fed roosters can provide preliminary indications of P digestibility in plant-based feedstuffs, 2) SID determined at 0.75% Ca was in good agreement with other bioassays, and 3) P in DDGS and CFP was highly available compared with other feedstuffs.

Pasture-finishing of cattle in Western U.S. rangelands improves markers of animal metabolic health and nutritional compounds in beef.

As environmental and health concerns of beef production and consumption mount, there is growing interest in agroecological production methods, including finishing beef cattle on pastures with phytochemically diverse grasses, forbs, and/or shrubs. The goal of this metabolomics, lipidomics, and fatty acid methyl ester profiling study was to compare meat (pectoralis profundus) of Black Angus cattle from two commercial US beef finishing systems (pasture-finished on Western U.S. rangeland; n = 18 and grain-finished in a Midwest U.S. feedlot; n = 18). A total of 907 out of 1575 compounds differed in abundance between pasture-finished and grain-finished beef samples (all, false discovery rate adjusted P < 0.05). Pasture-finished beef contained higher levels of phenolic antioxidants (2.6-fold), alpha-tocopherol (3.1-fold), nicotinate/vitamin B3 (9.4-fold), choline (1.2-fold), myo-inositol (1.8-fold), and omega-3 fatty acids (4.1-fold). Grain-finished beef contained higher levels of gamma-tocopherol (14.6-fold), nicotinamide/vitamin B3 (1.5-fold), pantothenate/vitamin B5 (1.3-fold), and pyridoxine/vitamin B6 (1.3-fold); indicating that feeding some grain (by-products) could be beneficial to increase levels of certain B-vitamins. Pasture-finished beef samples also displayed lower levels of oxidative stress (homocysteine, 0.6-fold; and 4-hydroxy-nonenal-glutathione, 0.4-fold) and improved mitochondrial function (1.3-fold) compared to grain-finished animals. Two potential metabolites of fluoroquinolone antibiotics, 2,8-quinolinediol and 2,8-quinolinediol sulfate, were only observed in grain-finished beef, though the source remains unknown. While pasture-finished cattle displayed improved markers of metabolic health and concentrated additional, potentially health-promoting compounds in their meat, our findings should not be interpreted as that grain-finished beef is unhealthy to consume. Randomized controlled trials in humans are required to further assess whether observed differences between pasture-finished and feedlot-finished beef have an appreciable effect on human health.

Enhanced Poly-γ-Glutamic Acid Production by a Newly Isolated Bacillus halotolerans F29.

Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications. In this study, we isolated a novel γ-PGA-producing strain, Bacillus halotolerans F29. The one-factor-at-a-time method was used to investigate the influence of carbon sources, nitrogen sources, and culture parameters on γ-PGA production. The optimal carbon and nitrogen sources were sucrose and (NH4)2SO4, respectively. The optimal culture conditions for γ-PGA production were determined to be 37 °C and a pH of 5.5. Response surface methodology was used to determine the optimum medium components: 77.6 g/L sucrose, 43.0 g/L monosodium glutamate, and 2.2 g/L K2HPO4. The γ-PGA titer increased significantly from 8.5 ± 0.3 g/L to 20.7 ± 0.7 g/L when strain F29 was cultivated in the optimized medium. Furthermore, the γ-PGA titer reached 50.9 ± 1.5 g/L with a productivity of 1.33 g/L/h and a yield of 2.23 g of γ-PGA/g of L-glutamic acid with the optimized medium in fed-batch fermentation. The maximum γ-PGA titer reached 45.3 ± 1.1 g/L, with a productivity of 1.06 g/L/h when molasses was used as a carbon source. It should be noted that the γ-PGA yield in this study was the highest of all reported studies, indicating great potential for the industrial production of γ-PGA.

Mating of unfed, engorged, and partially to fully gravid Aedes aegypti (Diptera: Culicidae) female mosquitoes in producing viable eggs.

BACKGROUND: Understanding the relationship between blood-feeding and mating is important in effectively managing the most well-adapted vector insect, Aedes aegypti (Linnaeus). Although extensive studies have investigated the behavioural aspects of Aedes such as blood-feeding, mating, and their relationship, several knowledge gaps still exist. Therefore, the present study was undertaken to determine the possibility of successful mating by unfed, engorged, and partially to fully gravid (up to 5 days after blood-feeding with fully developed eggs) female Ae. aegypti mosquitoes and production of viable eggs. METHODS: Mating of sexually mature adult Aedes aegypti was allowed in three different ways. In control 1, the females were allowed to mate before taking blood meal, and in control 2, the females were not at all allowed to mate. In the experiment, the females were separated into six categories, viz. D-0 to D-5. In D-0, the females were allowed to mate immediately after the bloodmeal and, in D-1, the females were allowed to mate on the first day of blood feeding, likewise, the females of D-2, D-3, D-4 and D-5 were allowed to mate on 2nd, 3rd, 4th and 5th day of blood feeding. Ovitrap was uniformly kept on the 4th day of blood feeding for the cages D-0 to D-3 for 1 h and then removed and for the cages D-4, and D-5, the ovitrap was kept on 4th and 5th day of blood feeding for 1h immediately after mating. The total number of eggs and the total number of hatching were counted. In the subsequent days, the entire experiment was replicated two times with different cohorts of mosquitoes, and the mean value of three experiments was used to draw Excel bars with 5% error bars and also for the statistical analysis. RESULTS: It was found that mating just before oviposition was sufficient to produce 1581 eggs (70% compared with control) and fertilize 1369 eggs (85% compared with total eggs laid), which is far higher than the 676 non-hatching (unfertilized) eggs (30%) laid by unmated females. Although mating is not essential for producing eggs, our study shows that even brief exposure to the semen and seminal fluids greatly enhances the oviposition and hatching efficiency, even if the mating occurs just before oviposition. However, those females mating before blood-feeding and those mating after blood-feeding produced 2266 and 2128 eggs, with hatching rates of 96.78% and 95.54%, respectively. Hence, the retention time of seminal fluid in the female seems to influence the number of eggs laid and the number of eggs hatched. CONCLUSIONS: In general, mating is possible in Ae. aegypti even minutes before oviposition and is sufficient to produce a greater number of viable eggs.

Application of a Novel Dissolution Medium with Lipids for In Vitro Simulation of the Postprandial Gastric Content.

Food can change various physiological parameters along the gastrointestinal tract, potentially impacting postprandial drug absorption. It is thus important to consider different in vivo conditions during in vitro studies. Therefore, a novel dissolution medium simulating variable postprandial pH values and lipid concentrations was developed and used in this study. Additionally, by establishing and validating a suitable analytical method, the effects of these parameters on the dissolution of a model drug, cinnarizine, and on its distribution between the lipid and aqueous phases of the medium were studied. Both parameters, pH value and lipid concentration, were shown to influence cinnarizine behavior in the in vitro dissolution studies. The amount of dissolved drug decreased with increasing pH due to cinnarizine's decreasing solubility. At pH values 5 and 7, the higher concentration of lipids in the medium increased drug dissolution, and most of the dissolved drug was distributed in the lipid phase. In all media with a lower pH of 3, dissolution was fast and complete, with a significant amount of drug distributed in the lipid phase. These results are in accordance with the in vivo observed positive food effect on cinnarizine bioavailability described in the literature. The developed medium, with its ability to easily adjust the pH level and lipid concentration, thus offers a promising tool for assessing the effect of co-ingested food on the dissolution kinetics of poorly soluble drugs.

Engineering regioselectivity of glycosyltransferase for efficient polydatin synthesis.

Resveratrol is a promising functional ingredient applied in food products. However, low bioavailability and poor water solubility, which can be improved by glycosylation, hinder its application. A uridine diphosphate-dependent glycosyltransferase (UGT) from Bacillus subtilis 168 (named UGTBS) presents potential application for resveratrol glycosylation; nonetheless, imprecise regioselectivity renders the synthesis of resveratrol-3-O-ß-D-glucoside (polydatin) difficult. Therefore, molecular evolution was applied to UGTBS. A triple mutant Y14I/I62G/M315W was developed for 3-OH glycosylation of resveratrol and polydatin accounted for 91% of the total product. Kinetic determination and molecular docking indicated that the enhancement of hydrogen bond interaction and altered conformation of the binding pocket increases the enzyme's affinity for the 3-OH group, stabilizing the enzyme-substrate intermediate and promoting polydatin formation. Furthermore, a fed-batch cascade reaction by periodic addition of resveratrol was conducted and nearly 20 mM polydatin was obtained. The mutant Y14I/I62G/M315W can be used for polydatin manufacture.