Comparison of bacteria disintegration methods and their influence on data analysis in metabolomics

Metabolomic experiments usually contain many different steps, each of which can strongly influence the obtained results. In this work, metabolic analyses of six bacterial strains were performed in light of three different bacterial cell disintegration methods. Three strains were gram-negative (Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae), and three were gram-positive (Corynebacterium glutamicum, Bacillus cereus, and Enterococcus faecalis). For extraction, the methanol–water extraction method (1:1) was chosen. To compare the efficiency of different cell disintegration methods, sonication, sand mill, and tissue lyser were used. For bacterial extract metabolite analysis, 1H NMR together with univariate and multivariate analyses were applied. The obtained results showed that metabolite concentrations are strongly dependent on the cell lysing methodology used and are different for various bacterial strains. The results clearly show that one of the disruption methods gives the highest concentration for most identified compounds (e. g. sand mill for E. faecalis and tissue lyser for B. cereus). This study indicated that the comparison of samples prepared by different procedures can lead to false or imprecise results, leaving an imprint of the disintegration method. Furthermore, the presented results showed that NMR might be a useful bacterial strain identification and differentiation method. In addition to disintegration method comparison, the metabolic profiles of each elaborated strain were analyzed, and each exhibited its metabolic profile. Some metabolites were identified by the 1H NMR method in only one strain. The results of multivariate data analyses (PCA) show that regardless of the disintegration method used, the strain group can be identified. Presented results can be significant for all types of microbial studies containing the metabolomic targeted and non-targeted analysis.

Application of Emerging Cell Disintegration Techniques for the Accelerated Recovery of Curcuminoids from Curcuma longa

Curcuminoids, the bioactive compounds with many beneficial effects on human health, exist in Curcuma longa (turmeric). In the present study, the impact of different cell disintegration techniques to enhance total curcuminoid recovery (TC) from fresh and dried turmeric was investigated. The impact of thermal pretreatment (TP), ultrasound pretreatment (UP), enzyme pretreatment (EP), and pulsed electric field pretreatment (PEF) on the recovery of curcumin (CUR), demethoxycurcumin (DMC), and bis-demethoxycurcumin (BDMC) from fresh and dried turmeric were studied. The cell disintegration index (Zp) and high-performance liquid chromatography (HPLC) analysis of curcuminoids were performed to evaluate the efficiency of the applied techniques. With fresh turmeric, the highest curcuminoid recovery was 83.6 mg/g dry basis with EP. The highest structural tissue damage was obtained with UP achieving a cell disintegration level of 92.5%. The technology with the highest time-saving and low specific energy input was PEF with a total curcuminoid recovery of 80.9 mg/g dry basis. Working with dried turmeric, the drying required high specific energy input for 72 h at 50 °C; however, the untreated dried sample reached 125.3 mg/g dry basis of TC without further pretreatment after drying.

Pulse Electric Field Technology for Wastewater and Biomass Residues’ Improved Valorization

Development and adoption of more efficient and robust technologies for reuse of wastewater embedded resources, in particular materials and energy, is becoming an unavoidable necessity. Among many emerging technologies in the sector of wastewater treatment residuals valorization, Pulsed Electric Field (PEF) processes have shown interesting potential, although they have not yet entered the sector’s mainstream as a consolidated commercial technology, as in other industrial applications, such as the food, medical, and bio-based industries. PEF is a non-thermal technology suitable to biological applications, involving gentle cell disintegration and enhanced cell membrane permeability and as such applicable to disinfection, sterilization, and to those processes that benefit from an enhanced extraction of organic compounds from biological matter, such as anaerobic digestion, biological processes for recovery of nutrients, and biorefinery of cell-embedded compounds. PEF technology applications in wastewater/biomass residues management are reported and advantages, drawbacks, and barriers of the technology are discussed in this paper.

Selective Release of Recombinant Periplasmic Protein From E. coli Using Continuous Pulsed Electric Field Treatment

To date, high-pressure homogenization is the standard method for cell disintegration before the extraction of cytosolic and periplasmic protein from E. coli. Its main drawback, however, is low selectivity and a resulting high load of host cell impurities. Pulsed electric field (PEF) treatment may be used for selective permeabilization of the outer membrane. PEF is a process which is able to generate pores within cell membranes, the so-called electroporation. It can be readily applied to the culture broth in continuous mode, no additional chemicals are needed, heat generation is relatively low, and it is already implemented at industrial scale in the food sector. Yet, studies about PEF-assisted extraction of recombinant protein from bacteria are scarce. In the present study, continuous electroporation was employed to selectively extract recombinant Protein A from the periplasm of E. coli. For this purpose, a specifically designed flow-through PEF treatment chamber was deployed, operated at 1.5 kg/h, using rectangular pulses of 3 μs at specific energy input levels between 10.3 and 241.9 kJ/kg. Energy input was controlled by variation of the electric field strength (28.4–44.8 kV/cm) and pulse repetition frequency (50–1,000 Hz). The effects of the process parameters on cell viability, product release, and host cell protein (HCP), DNA, as well as endotoxin (ET) loads were investigated. It was found that a maximum product release of 89% was achieved with increasing energy input levels. Cell death also gradually increased, with a maximum inactivation of -0.9 log at 241.9 kJ/kg. The conditions resulting in high release efficiencies while keeping impurities low were electric field strengths ≤ 30 kV/cm and frequencies ≥ 825 Hz. In comparison with high-pressure homogenization, PEF treatment resulted in 40% less HCP load, 96% less DNA load, and 43% less ET load. Therefore, PEF treatment can be an efficient alternative to the cell disintegration processes commonly used in downstream processing.

Bacterial nanotubes as a manifestation of cell death

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells’ genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.

Effect of fuzzy-controlled slow freezing on pumpkin (Cucurbita Moschata Duch) cell disintegration and phenolics

Freezing has been widely used to preserve vegetables including seasonal pumpkins. This work aimed to investigate the effects of freezing on pumpkin cell disintegration and phenolics. A fuzzy logic control (FLC) system was built to obtain better temperature control of the freezing system. Changes in cellular disintegration, electrical conductivity and phenolics content were evaluated. The angle measure technique and principal component analysis were used to delineate the surface texture changes of the frozen pumpkin cells. The results showed that FLC offered reliable temperature control performance. Freezing at -18 °C for 7 h caused the highest cell degradation of 0.467 on the disintegration scale. Decomposition was also indicated by an almost double increase in electrical conductivity. The changes in texture were accurately reflected in the mean angle spectra and 81.3% and 7.4% of the variability due to treatments could be explained by two principal components respectively. Freezing pumpkin at -18 °C for 6 h correlated to the maximum increase in total phenolics of 70.44%. The increased phenolics were dominated by caffeic acid, chlorogenic acid and p-coumaric acid. In conclusion, as the freezing system exhibits positive effects on the phenolics content of pumpkin, it may be employed to process seasonal pumpkin to obtain higher value from the produce.

Myricetin Abrogates Cisplatin-Induced Oxidative Stress, Inflammatory Response, and Goblet Cell Disintegration in Colon of Wistar Rats

Cisplatin [cis-diamminedichloroplatinum II] is an extensively prescribed drug in cancer chemotherapy; it is also useful for the treatment of diverse types of malignancies. Conversely, cisplatin is associated with a range of side effects such as nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, and so on. Myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4chromenone) is a very common natural flavonoid found in fruits, tea, and plants. It has been found to have high-value pharmacological properties and strong health benefits. To examine the role of myricetin in colon toxicity induced by cisplatin, we conducted a concurrent prophylactic study in experimental animals that were treated orally with myricetin for 14 days at two doses—25 and 50 mg/kg of body weight. On the 14th day, a single intraperitoneal injection of cisplatin (7.5 mg/kg body weight) was administered in all groups except control. The effects of myricetin in cisplatin-induced toxicity in the colon were assessed in terms of antioxidant status, phase-II detoxification enzymes, the level of inflammatory markers, and goblet cell disintegration. Myricetin was found to restore the level of all the antioxidant enzymes analyzed in the study. In addition, the compound ameliorated cisplatin-induced lipid peroxidation, increase in xanthine oxidase activity, and phase-II detoxifying enzyme activity. Myricetin also attenuated deteriorative effects induced by cisplatin by regulating the level of molecular markers of inflammation (NF-κB, Nrf-2, IL-6, and TNF-α), restoring Nrf-2 levels, and controlling goblet cell disintegration. The current study reinforces the conclusion that myricetin exerts protection in colon toxicity via up-regulation of inflammatory markers, improving anti-oxidant status, and protecting tissue damage.