Optimization of Conditions For Increasing of Saffron Cell Biomass And Crocin Production In Stirred Bioreactor

Bioreactors provide suitable conditions for the growth of cells and production of secondary metabolites by regulating physical and chemical factors. In this study, first, sucrose, 2-(N-morpholino) ethanesulfonic acid (MES) as a buffering agent and medium pH was optimized in the Erlenmeyer flask. This aim was then pursued in a stirred bioreactor through aeration and pH medium adjustment. Results of the first step showed that Schenk and Hildebrandt (SH) basal medium with naphthalene acetic acid (2 mg.l-1) and 6-benzylaminopurine (1 mg.l-1) supplemented with 2.5 mM of MES and gradually increment of sucrose from 3 to 6% caused to catch the highest cell biomass and crocin production. The spectrophotometry measurement showed that the highest crocin content of the cells was 0.8 mg/g after five weeks. The results of the second part revealed that in the stirred bioreactor, constant pH (5.8) during the growth period is a limited factor for the cell growth and crocin production. Although aeration initially found to be an inhibited factor for the production of crocin, results showed that, if the evaporated volume of water caused by aeration is constituted, it can be an effective factor to increase cell growth rate around 2 folds. In addition, total crocin content of the cells, based on the HPLC could be raised up to 2 mg/g. Based on this study, it can be concluded that MES and gradual increment of sucrose could increasing the cell growth and crocin production. Aeration in bioreactor can increase cell biomass, if the medium volume will be kept constant.

CoBG-11 preparation v1

CoBG-11 is a coculture medium used to coculture cyanobacteria and E. coli by Zhang et al (2020). It is optimized for E. coli growth. Here are its components: 1. 150 mM NaCl, 2. 4 mM NH4Cl 3. 3 g/L 2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl] amino] ethanesulfonic acid (TES) The pH value is adjusted with NaOH to 8.3. The protocol below describes the preparation of 100ml of coBG-11.

New Continuous Process for the Production of Lipopeptide Biosurfactants in Foam Overflowing Bioreactor

In this work, an original culture process in bioreactor named overflowing continuous culture (O-CC) was developed to produce and recover continuously mycosubtilin, a lipopeptide antifungal biosurfactant of major interest. The lipopeptide production was first investigated in shake conical flasks in different culture media [ammonium citrate sucrose (ACS), Difco sporulation medium (DSM), and Landy], followed by a pH condition optimization using 3-(N-morpholino)propanesulfonic acid (MOPS) and 2-(N-morpholino)ethanesulfonic acid (MES) buffered media. A simple theoretical modeling of the biomass evolution combined with an experimental setup was then proposed for O-CC processed in stirred tank reactor at laboratory scale. Seven O-CC experiments were done in modified Landy medium at the optimized pH 6.5 by applying dilution rates comprised between 0.05 and 0.1 h–1. The O-CC allowed the continuous recovery of the mycosubtilin contained in the foam overflowing out of the reactor, achieving a remarkable in situ product removal superior to 99%. The biomass concentration in the overflowing foam was found to be twofold lower than the biomass concentration in the reactor, relating advantageously this process to a continuous one with biomass feedback. To evaluate its performances regarding the type of lipopeptide produced, the O-CC process was tested with strain BBG116, a mycosubtilin constitutive overproducing strain that also produces surfactin, and strain BBG125, its derivative strain obtained by deleting surfactin synthetase operon. At a dilution rate of 0.1 h–1, specific productivity of 1.18 mg of mycosubtilin⋅g–1(DW)⋅h–1 was reached. Compared with other previously described bioprocesses using almost similar culture conditions and strains, the O-CC one allowed an increase of the mycosubtilin production rate by 2.06-fold.

Development of an ELAA kit to detect neomycin in milk

Antibiotics used in livestock production offer various benefits as an antimicrobial agent, growth promoter, and feed effective improvement. However, the abuse of antibiotics leads to antibiotic resistance which may seriously threaten human and animal welfare, and growing levels of antibiotics or antibiotic-resistant bacteria in the environment increase the numbers of drug-resistant infection outbreaks. Therefore, many detection methods have been being developed to quickly assess antibiotic content and its residues in foods. Among many analytical methods, the aptamer-based biosensor has considerable attention for its outstanding advantages such as high specificity, high sensitivity, and good selectivity. We use the ELAA (Enzyme-Linked Aptamer Assay) method - a variant of ELISA - which has a high affinity with neomycin. Firstly, we investigated different buffers to create the Neo-BSA complex. As result, 2-(N-morpholino) ethanesulfonic acid (MES) buffer pH 7 was found with the best results. Next, to help the Neo-BSA complex be fixed well on polystyrene wells, we surveyed various buffers and found the coating buffer (50mM Bicarbonate buffer, pH 9.6) rated as the most suitable for this process. In addition, the quality of the kit is also assessed through competitive ELAA reaction components. Therefore, we have investigated and optimized conditions such as aptamer concentration 25 nM in PBS buffer, and the biotinized aptamers did not need heat treatment prior to joining the reaction. From the results, we have successfully developed a calibration curve for antibiotic residue in milk using the ELAA technique, linear range 0,1 ng/mL and 100 ng/mL. Then, we initially surveyed 20 milk samples found that the ELAA method was consistent with the results from LC-MS/MS was obtained showing no difference between the two methods. We continued to test the samples to determine the kit’s sensitivity and specificity. The results showed that the kit has a specificity and sensitivity of 100%. Finally, LOD and LOQ value had xavg = 0.448; SD = 0.22, LOD = xavg + 3SD = 1.11 (ng / ml); LOQ = x tb + 10SD = 2.65 (ng / mL). We will continue to optimize the kit before being brought to the market.

The use of HEPES-buffer in the production of gallium-68 radiopharmaceuticals – time to reconsider strict pharmacopoeial limits?

HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) is a buffer that is used in the radiolabelling of gallium-68 compounds. The beneficial effects of HEPES on molar activity in bioconjugates have been well described. Current strict regulations on the HEPES content in radiopharmaceuticals limit its use when intended for parenteral administration.This short communication summarizes data from the literature on the toxicity of HEPES in dogs after intravenous infusion and the subsequent use in humans. We also highlight the use of HEPES in an FDA labelled intravenous drug formulation. Regulatory institutions may consider this data to review current strict limits.

Analysis of KATP Channels Opening Probability of Hippocampus Cells Treated with Kainic Acid

Background: Kainic acid (KA)-induced seizures may be a valuable tool in the assessment of anti-epileptic drug efficacy in complex partial seizures. This study investigated the effects of KA on ATP-sensitive K+ (KATP) channels opening probability (NPo), which plays a crucial role in neuronal activities. Methods: For the optimisation and validation protocol, β-cells were plated onto 35 mm plastic petri dishes and maintained in RPMI-1640 media supplemented with 10 mM glucose, 10% FCS and 25 mM of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES). The treatment effects of 10 mM glucose and 30 μM fluoxetine on KATP channels NPo of β–cells were assessed via cell-attached patch-clamp recordings. For hippocampus cell experiments, hippocampi were harvested from day 17 of maternal Lister-hooded rat foetus, and then transferred to a Ca2+ and Mg2+-free HEPES-buffered Hank’s salt solution (HHSS). The dissociated cells were cultured and plated onto a 25 mm round cover glasses coated with poly-d-lysine (0.1 mg/mL) in a petri dish. The KATP channels NPo of hippocampus cells when perfused with 1 mM and 10 mM of KA were determined. Results: NPo of β-cells showed significant decreasing patterns (P < 0.001) when treated with 10 mM glucose 0.048 (0.027) as well as 30 μM fluoxetine 0.190 (0.141) as compared to basal counterpart. In hippocampus cell experiment, a significant increase (P < 0.001) in mean NPo 2.148 (0.175) of neurons when applied with 1 mM of KA as compared to basal was observed. Conclusion: The two concentrations of KA used in the study exerted contrasting effects toward the mean of NPo. It is hypothesised that KA at lower concentration (1 mM) opens more KATP channels, leading to hyperpolarisation of the neurons, which may prevent neuronal hyper excitability. No effect was shown in 10 mM KA treatment, suggesting that only lower than 10 mM KA produced significant changes in KATP channels. This implies further validation of KA concentration to be used in the future.

Soman hydrolysis catalysed by hypochlorite ions

Sarin (GB) and soman (GD) are severely toxic nerve agents that react slowly in water, resulting in long-term poisoning of the water and a serious threat to personnel. Some ions can catalyse GB and GD hydrolysis in water; the relevant research for GB is detailed, whereas that for GD is relatively less so. In this paper, GD hydrolysis catalysed by hypochlorite (ClO−) ions was studied via kinetic experiments. A fluorite-ion-specific electrode was used to monitor F− ions produced, allowing the rate constant and half-life of the GD hydrolysis to be calculated. The results showed that ClO− ions promote GD hydrolysis well; the higher the concentration of ClO−, the faster the GD was hydrolysed. In NaClO solution at pH 8.0 with 3.22×10–3 M ClO− ions, the half-life of GD hydrolysis was 82.5 s, about 875 times shorter than that in water at pH 8.0. The rate constant for catalysis of GD hydrolysis by ClO- ions ++(kc1o−)++ was 2.6 M−1 s−1, about one quarter the value of ++koh− ++but over 1500 times greater than kB and ++kPO4,++ with B representing N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid present as a free base; this result indicated that ClO− ions catalyse GD hydrolysis well.

Mechanistic Insight of Sensing Hydrogen Phosphate in Aqueous Medium by Using Lanthanide(III)-Based Luminescent Probes

The development of synthetic lanthanide luminescent probes for selective sensing or binding anions in aqueous medium requires an understanding of how these anions interact with synthetic lanthanide probes. Synthetic lanthanide probes designed to differentiate anions in aqueous medium could underpin exciting new sensing tools for biomedical research and drug discovery. In this direction, we present three mononuclear lanthanide-based complexes, EuLCl3 (1), SmLCl3 (2), and TbLCl3 (3), incorporating a hexadentate aminomethylpiperidine-based nitrogen-rich heterocyclic ligand L for sensing anion and establishing mechanistic insight on their binding activities in aqueous medium. All these complexes are meticulously studied for their preferential selectivities towards different anions such as HPO42−, SO42−, CH3COO−, I−, Br−, Cl−, F−, NO3−, CO32−/HCO3−, and HSO4− at pH 7.4 in aqueous HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer. Among the anions scanned, HPO42− showed an excellent luminescence change with all three complexes. Job’s plot and ESI-MS support the 1:2 association between the receptors and HPO42−. Systematic spectrophotometric titrations of 1–3 against HPO42− demonstrates that the emission intensities of 1 and 2 were enhanced slightly upon the addition of HPO42− in the range 0.01–1 equiv and 0.01–2 equiv., respectively. Among the three complexes, complex 3 showed a steady quenching of luminescence throughout the titration of hydrogen phosphate. The lower and higher detection limits of HPO42− by complexes 1 and 2 were determined as 0.1–4 mM and 0.4–3.2 mM, respectively, while complex 3 covered 0.2–100 μM. This concludes that all complexes demonstrated a high degree of sensitivity and selectivity towards HPO42−.