Novel method for screening probiotic candidates tolerant to human gastrointestinal stress.

Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress-gastrointestinal tolerance assay and culture (GTA-C) method-using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.

A forced aeration system for microbial culture of multiple shaken vessels suppresses volatilization.

Shake-flask culture, an aerobic submerged culture, has been used in various applications involving cell cultivation. However, it is not designed for forced aeration. Hence, this study aimed to develop a small-scale submerged shaking culture system enabling forced aeration into the medium. A forced aeration control system for multiple vessels allows shaking, suppresses volatilization, and is attachable externally to existing shaking tables. Using a specially developed plug, medium volatilization was reduced to less than 10%, even after 45 h of continuous aeration (~ 60 mL/min of dry air) in a 50 mL working volume. Escherichia coli IFO3301 cultivation with aeration was completed within a shorter period than that without aeration, with a 35% reduction in the time-to-reach maximum bacterial concentration (26.5 g-dry cell/L) and a 1.25-fold increase in maximum concentration. The maximum bacterial concentration achieved with aeration was identical to that obtained using the Erlenmeyer flask, with a 65% reduction in the time required to reach it.

Simultaneous removal of caesium and strontium using different removal mechanisms of probiotic bacteria.

When radioactive materials are released into the environment due to nuclear power plant accidents, they may enter into the body, and exposing it to internal radiation for long periods of time. Although several agents have been developed that help excrete radioactive elements from the digestive tract, only one type of radioactive element can be removed using a single agent. Therefore, we considered the simultaneous removal of caesium (Cs) and strontium (Sr) by utilising the multiple metal removal mechanisms of probiotic bacteria. In this study, the Cs and Sr removal capacities of lactobacilli and bifidobacteria were investigated. Observation using an electron probe micro analyser suggested that Cs was accumulated within the bacterial cells. Since Sr was removed non metabolically, it is likely that it was removed by a mechanism different from that of Cs. The amount of Cs and Sr that the cells could simultaneously retain decreased when compared to that for each element alone, but some strains showed only a slight reduction in removal. For example, Bifidobacterium adolescentis JCM1275 could simultaneously retain 55.7 mg-Cs/g-dry cell and 8.1 mg-Sr/g-dry cell. These results demonstrated the potentials of utilizing complex biological system in simultaneous removal of multiple metal species.

Understudied Hyperphosphatemia (Chronic Kidney Disease) Treatment Targets and New Biological Approaches.

Hyperphosphatemia is a secondary disorder of chronic kidney disease that causes vascular calcifications and bone-mineral disorders. As per the US Centers for Disease Control and Prevention, renal damage requires first-priority medical attention for patients with COVID-19; according to a Johns Hopkins Medicine report, SARS-CoV-2 can cause renal damage. Therefore, addressing the research inputs required to manage hyperphosphatemia is currently in great demand. This review highlights research inputs, such as defects in the diagnosis of hyperphosphatemia, flaws in understanding the mechanisms associated with understudied tertiary toxicities, less cited adverse effects of phosphate binders that question their use in the market, socioeconomic challenges of renal treatment and public awareness regarding the management of a phosphate-controlled diet, novel biological approaches (synbiotics) to prevent hyperphosphatemia as safer strategies with potential additional health benefits, and future functional food formulations to enhance the quality of life. We have not only introduced our contributions to emphasise the hidden aspects and research gaps in comprehending hyperphosphatemia but also suggested new research areas to strengthen approaches to prevent hyperphosphatemia in the near future.

Development of a simple cultured cell-anaerobic microbial co-culture system using liquid paraffin.

Conventional co-culture systems are complicated, lack versatility, and do not adequately replicate the intestinal lumen environment. This study aimed to devise a system that allows for (i) arbitrary sampling of the culture medium, (ii) monitoring the growth of co-cultured cells, (iii) aerobic-anaerobic co-culture, (iv) simple operation, and (v) evaluation of multiple samples. We built a simple cell-anaerobic microorganism co-culture system using liquid paraffin to separate growth spaces for aerobic cells and anaerobic bacteria. Mineral oil was added to the top of the anaerobic bacterial cultivation space to seal the space and reduce gas exchange. Co-culture of anaerobic, Bifidobacterium bifidum and aerobic, epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that the barrier function and viability of co-cultured MDCK cells were comparable to those of a pure MDCK culture after 24 h, and the growth curve of co-cultured B. bifidum was similar to that of pure B. bifidum. Furthermore, the growth of B. bifidum pure culture under sealed conditions was approximately 1.5 times greater than that under non-sealed conditions at 24 h. Glucose consumption at 24 h of co-culture under sealed conditions was 10%-15% higher than that under non-sealed conditions. This highly versatile culture method enabled the quantitative characterisation of B. bifidum and MDCK cells upon co-culture. The newly established co-culture system could be applied to various aerobic cell-anaerobic bacteria co-culture which will provide a strategy for basic and applied research on host-microbe interactions.

Development of an insertion device selectively operational as a helical/figure-8 undulator.

An insertion device capable of switching the operation mode between helical and figure-8 undulators, and thus referred to as a helical-8 undulator, has been developed. It has the advantage that the on-axis heat load can be kept low regardless of the polarization state, even when a high K value is required to lower the fundamental photon energy. This is in contrast to conventional undulators in which the on-axis heat load tends to be significantly high to generate linearly polarized radiation with a high K value, and optical elements can be seriously damaged. The principle of operation, specification and light source performance of the developed helical-8 undulator are presented together with further options to enhance its capability.

Screening and isolation of acid-tolerant bacteria using a novel pH shift culture method.

Acid-tolerant bacteria, which multiply under neutral pH and can survive under acidic pH conditions, have a potential role in various applications under acidic conditions. Despite higher biomass productivity, their isolation and utilisation are not sufficiently developed compared to those of acidophiles. It takes considerable effort to distinguish the acid-tolerant bacteria from the rest of the bacterial community using conventional screening methods. Thus, we developed a novel screening method for acid-tolerant bacteria, which involves shifting the pH between acidic and neutral conditions. With this method, the bacterium Enterobacter sp. AC06 was isolated. Based on comparisons with the results reported in previous studies, the strain can be classified as acid-tolerant bacteria. The decreases in the live cell concentrations were 3.87 and 6.16 log cycles after 3 h acid treatment under pH 3.0 and 2.5, respectively. These results suggest that it is possible to isolate acid-tolerant bacteria using the pH shift culture method. In summary, this is the first study on bacterial screening based on acid tolerance. Our novel method potentially contributes to the understanding and utilisation of acid-tolerant bacteria by enhancing screening efficiency. Furthermore, our novel concept shift culture is potentially valuable for screening previously uncultured bacteria tolerant to various selective stress conditions.

Effects of organic carbon sources on growth and oil accumulation by Desmodesmus subspicatus LC172266 under mixotrophic condition.

Energy crisis and environmental sustainability have attracted global attention to microalgal biofuels. The present study investigated the impact of organic carbon sources on growth and bio-oil accumulation by an oleaginous microalga Desmodesmus subspicatus LC172266 under mixotrophic culture condition. Glucose and glycerol supported higher growth rates and lipid productivities than sucrose, fructose, mannitol and acetate. Each of the organic carbon source tested supported significantly (P < 0.05) higher growth rates and lipid productivities than the photoautotrophic culture (without organic carbon source). The lipid productivity obtained with a mixture of optima concentrations of glucose and glycerol (5.0 gL-1 glycerol + 10.0 gL-1glucose) (0.14875 ± 0.002 g/L/day) was about 25% and 66% higher than the values obtained with only 10.0 gL-1glucose and 5.0 gL-1glycerol, respectively. When a batch culture with 5gL-1glycerol was fed with 0.5 gL-1glucose daily the cell growth and lipid productivity were lower than the values obtained in a batch culture with a mixture of glucose and glycerol. The lipid productivity obtained in a 4-L photobioreactor was 94% (0.217 gL-1 day-1), higher than the value obtained in a flask culture with 10.0 g/Lglucose (0.112 gL-1 day-1) and 46% higher than the value obtained in a flask culture with 5.0 gL-1glycerol (0.086 gL-1 day-1).

Light-Emitting-Diode-Assisted, Fungal-Pigment-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Activity.

Nanoparticle synthesis, such as green synthesis of silver nanoparticles (AgNPs) using biogenic extracts, is affected by light, which changes the characteristics of particles. However, the effect of light-emitting diodes (LEDs) on AgNP biosynthesis using fungal pigment has not been examined. In this study, LEDs of different wavelengths were used in conjunction with Talaromyces purpurogenus extracellular pigment for AgNP biosynthesis. AgNPs were synthesized by mixing 10 mL of fungal pigment with AgNO3, followed by 24 h exposure to LEDs of different wavelengths, such as blue, green, orange, red, and infrared. All treatments increased the yield of AgNPs. The solutions exposed to blue, green, and infrared LEDs exhibited a significant increase in AgNP synthesis. All AgNPs were then synthesized to determine the optimum precursor (AgNO3) concentration and reaction rate. The results indicated 5 mM AgNO3 as the optimum precursor concentration; furthermore, AgNPs-blue LED had the highest reaction rate. Dynamic light scattering analysis, zeta potential measurement, transmission electron microscopy, and Fourier transform infrared spectroscopy were used to characterize the AgNPs. All LED-synthesized AgNPs exhibited an antimicrobial potential against Escherichia coli and Staphylococcus aureus. The combination of LED-synthesized AgNPs and the antibiotic streptomycin demonstrated a synergistic antimicrobial activity against both bacterial species.

Effect of alcohol on productivity and quality of adeno-associated virus 2 in HEK293 cells.

Investigation of enhancers to improve recombinant adeno-associated virus 2 (rAAV2) productivity by human embryonic kidney 293 cells (HEK293) suspension culture showed that the addition of ethanol improved the productivity and packaged genome integrity of rAAV2. Further optimization showed that adding ethanol in the range of 0.09%-1.11% (v/v) during rAAV2 production effectively improved rAAV2 productivity and quality. In addition, ethanol addition improved cell viability. Furthermore, proteome and pathway analysis of the cells during rAAV2 production showed that the addition of ethanol resulted in the upregulation of pathways related to intercellular signaling, gene expression, cell morphology, intercellular maintenance, and others. In contrast, pathways related to cell death, immunity, and reactions to infection were downregulated. These changes in pathway regulation were responsible for the improvement in rAAV2 productivity, packaged genome integrity, and cell viability during rAAV2 production. The results of this study can be applied to the production of viral vectors for in vivo gene therapy in an inexpensive and safe manner.

Control of carbon dioxide concentration in headspace of multiple flasks using both non-electric bellows pump and shaking incubator.

Current methods of controlling gas in the headspace involve constant speed aeration and proportional-integral-differential (PID) controlled aeration using improved monitoring devices or gas cylinders. However, these approaches are restricted and inconvenient to use. In this study, we propose a method to control the CO2 concentration in the headspace while maintaining the convenience of shake-flask culture. A combination of a non-electric bellows pump for shake-flask (NeBP-sf) and a CO2 incubator was used to control the flask gas phase by shaking without additional external power. The CO2 half-life, as an indicator of the ventilation ability of the system, was measured using a circulation direct monitoring and sampling system, and the NeBP-sf was optimised. The ventilation capacity varied depending on the shaking speed, and under optimal conditions, was 10 min compared with 45 min when only a breathable culture plug was used. In conventional microbial shaking culture, the CO2 concentration in the flask gas phase remained higher than the 5% set-value with a maximum of 9%, resulting in a large concentration difference with the set point. Therefore, the ventilation capacity of the conventional shake-flask culture was insufficient for aerobic culture. Cultivation of Escherichia coli and Lactiplantibacillus plantarum using the system showed no significant difference between the set point and real point values. Thus, the system combined an NeBP-sf and a gas incubator built-in shaking table to achieve the reproducibility of gas control while maintaining a high level of convenience.

Use of Catharanthus roseus Cell Cultures for the Synthesis of Metal Nanoparticles.

Metal nanoparticles have found applications in many fields owing to their unique physicochemical characteristics and ease of surface functionalization. Crucial for these applications is the development of environmentally friendly strategies for nanoparticle preparation. Nanoparticles can be prepared using several physical and chemical methods; however, the use of nontoxic and eco-friendly approaches is receiving increasing attention. Plant cell cultures are sustainable sources of bioactive compounds that can act as reducing and stabilizing agents during nanoparticle synthesis. Here, we describe the procedures used to synthesize silver and gold nanoparticles with cultured cells of Catharanthus roseus. The bioreduction of silver ions to nanoparticles with extract of seed-derived callus of C. roseus is evident from UV-Vis spectroscopy results wherein an absorption maxima is observed at 425 nm, indicating the formation of elemental silver. Similarly, reaction mixtures containing cell-free suspension culture filtrate of C. roseus and gold III ions turn wine red after 24 h incubation because of gold nanoparticle formation. These methods can be easily adapted for use in the preparation of other metal nanoparticles.

Fungal Pigment-Assisted Silver Nanoparticle Synthesis and Their Antimicrobial and Cytotoxic Potential.

The biological production of nanoparticles has taken center stage among the various generation methods available owing to its environment-friendly characteristics and reduced energy requirements. A protocol for pigment-assisted silver nanoparticle (AgNP) synthesis was established, employing an extracellular composite pigment produced by the Ascomycota Talaromyces purpurogenus (presently Talaromyces purpureogenus). The extracellular pigment can reduce the precursor silver salt into nanoparticles in the presence of sodium hydroxide and light. Transmission electron microscopy may be used to characterize the bio-generated nanoparticles, which can also be tested for their biological properties using antimicrobial and anticancer assays.

Development of a device for cultivation and isolation of microbes using a specialized cellulose film.

A device was developed for the cultivation and isolation of microbes using a specialized cellulose film (SCF). This method can solve problems associated with the agar plate method. It can also be used for other culture-related operations, which is not possible with the traditional agar plate culture method.

Production and stability of pigments by Talaromyces purpurogenus LC128689 in an alternating air phase-liquid phase cultivation system.

Effects of carbon source, nitrogen source, and alternatingly submerging the cells and exposing to gaseous oxygen on pigment production by Talaromyces purpurogenus LC128689, as well as pH, temperature, and UV stability of the pigments were investigated. Although fructose supported higher cell growth, a mixture of glucose and glycerol resulted in higher pigment production. Out of the organic and inorganic nitrogen sources investigated, peptone gave the highest cell concentration (7.2 ± 1.1 g/L) and pigment production (p ≤ 0 .05). The cells were then immobilized in loofa sponge and cultivated under alternating liquid phase-air phase (ALAP) system whereby the cells were alternatingly submerged and exposed to gaseous oxygen. After 20 days of cultivation, the concentrations of the red, orange, and yellow pigments were 30.15 AU500 nm , 15 AU460 nm , and 6.25 AU400 nm , respectively. In comparison with submerged culture in flasks, the red and orange pigments were 100% and 50% higher (p ≤ 0.05) in ALAP system. On the other hand, the yellow pigment was 100% higher in flask cultures than in ALAP. The three pigments were stable within a pH range of 2-12, retained more than 80% of their color intensity after autoclaving at (121°C and 1.0 atm) for 15 min and exposure to UV (3 uW/cm2 ) for 24 h.

Tailored synbiotic powder (functional food) to prevent hyperphosphataemia (kidney disorder).

Hyperphosphataemia is treated with phosphate binders, which can cause adverse effects. Spray-dried synbiotic powder (SP) composed of Lactobacillus casei JCM1134 (a phosphate-accumulating organism; PAO) and Aloe vera is potentially a safer alternative for efficient phosphate removal. In this study, a novel strategy was developed; lysine-derivatized deacetylated A. vera (DAVK) was synthesised and fabricated on phosphate-deficient PAO (PDP) for efficient phosphate transfer and then spray-dried with the supernatant of DAV centrifugation to form a sacrificial layer on PDP for SP integrity during gastric passage. In vitro experiments revealed that PAO removed only 1.6% of the phosphate from synthetic media, whereas SP removed 89%, 87%, and 67% (w/v) of the phosphate from milk, soft drink, and synthetic media, respectively, confirming the protective role of A. vera and efficient phosphate transport. Compared with commercial binders, SP effectively removed phosphate from synthetic media, whereas SP and CaCO3 exhibited comparative results for milk and soft drink. Importantly, CaCO3 caused hypercalcaemia. Thus, the described SP presents a promising tool to prevent hyperphosphataemia. This study also revealed a novel factor: diets of patients with chronic kidney disease should be monitored to determine the optimal phosphate binders, as phosphate removal performance depends on the accessible phosphate forms.

Analysis of porous breathable stopper and development of PID control for gas phase during shake-flask culture with microorganisms.

We evaluated the ventilation ability of two types (plug-type and cap-type) of culture-stoppers having standard air permeability. The culture-stoppers were evaluated using the circulation direct monitoring and sampling system with CO2 concentration in the gas phase of a shake-flask culture as an index. The half-lives of CO2 in the headspace of the shake flask with the plug-type and cap-type stoppers were about 51.5 min and about 30.3 min, respectively. Based on these half-lives, we formulated a model equation to simulate the behaviour of CO2 with different culture-stoppers. After validating the model equation by shake-flask culture with Saccharomyces cerevisiae, we investigated the effect of different ventilation abilities of the culture-stoppers on the growth of Pelomonas saccharophila and Escherichia coli: the sensitivity of the culture-stopper to the ventilation ability was dependent on the microorganism species. In the case of P. saccharophila, when the plug-type culture-stopper was combined with controlled CO2 concentration (6%) in the flask, the maximum yield increased by twofold compared to that of the control. This study shows the importance of ventilation in headspace and conventional culture-stoppers during the shake-flask culture of microorganisms. The problems that may occur between the conventional shake-flask culture approach using a breathable culture-stopper and the next-generation shake-flask culture without a conventional culture-stopper were clarified from the evaluation of gas-permeable culture-stoppers. The importance of controlled gaseous phase in the headspace during shake-flask culture of the microorganisms was also elucidated. KEY POINTS: • Ventilation capacity of culture-stoppers was evaluated using the CO2 half-life concentration. • Behaviour of microorganisms varies with the type of culture-stopper. • Developed a PID system for control of CO2 in flask gas phase to enhance the shake-flask culture.

Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms.

Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations. The gaseous destination has not been deeply analyzed. Our aim was to investigate the effect of flame sterilization on the headspace of the flask and on the shake-flask culture. In this study, the headspace CO2 concentration was found to increase during flame sterilization ~0.5-2.0% over 5-20 s empirically using the Circulation Direct Monitoring and Sampling System. This CO2 accumulation was confirmed theoretically using Computational Fluid Dynamics; it was 9% topically. To evaluate the influence of CO2 accumulation without interference from other sampling factors, the flask gas phase formed by flame sterilization was reproduced by aseptically supplying 99.8% CO2 into the headspace, without sampling. We developed a unit that can be sampled in situ without interruption of shaking, movement to a clean bench, opening of the culture-plug, and flame sterilization. We observed that the growth behaviour of Escherichia coli, Pelomonas saccharophila, Acetobacter pasteurianus, and Saccharomyces cerevisiae was different depending on the CO2 aeration conditions. These results are expected to contribute to improving microbial cell culture systems.

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius.

Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius. The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol-1) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.