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Functional insights into the interaction of Lactobacillus spp. and potentially protective agents
Natural resveratrol analogs differentially target endometriotic cells into apoptosis pathways
AbstractThe specific characteristics of endometriotic cells are their ability to evade the apoptotic machinery and abnormal response to apoptotic stimuli. Natural-originated compounds may constitute a beneficial strategy in apoptosis modulation in endometriosis. We investigated and compared the potency of natural resveratrol analogs, including piceatannol, polydatin, and pterostilbene, in targeting cell death pathways, including apoptosis-related morphologic and biochemical processes, alongside the modulation of the critical genes expression. Upon resveratrol and pterostilbene treatment, a significant reduction of endometriotic cell viability and an increased apoptotic proportion of cells were noted. The lower antiproliferative potential was found for piceatannol and polydatin. Endometrial stromal T HESC cells were significantly more resistant than endometriotic epithelial 12Z cells to the cytotoxic activity of all analyzed compounds. They differentially affected endometriotic cell viability, cell cycle, anti- and proapoptotic genes regulation, caspases expression and enzymatic activity, and DNA fragmentation. Pterostilbene-mediated endometriotic cell apoptosis modulation was confirmed to be most effective but without evident caspase 3 upregulation. Our study provides valuable insight into the apoptogenic activity of resveratrol and its natural analogs in endometriotic cells. Data obtained revealed the highest therapeutic potential of pterostilbene by effectively targeting cell death determinants in endometriosis, strengthening its optimization in further extensive research.
Resveratrol and Its Natural Analogs Mitigate Immune Dysregulation and Oxidative Imbalance in the Endometriosis Niche Simulated in a Co-Culture System of Endometriotic Cells and Macrophages
Background: Inflammation and immune cell dysfunction are critical facilitators of endometriosis pathophysiology. Macrophages are renowned for stimulating lesion growth, vascularization, innervation, and pain generation. By combining macrophages and endometriotic cells, we determined if resveratrol and its natural analogs can target the immune dysregulation and oxidative imbalance in endometriosis. Methods: After treatment with compounds (5, 10, 25 µM), we evaluated the expression of key inflammatory and oxidative stress markers, cytokines release, and ROS production by applying q-PCR, ELISA, Cytometric Beads Array, and multiplexed fluorogenic staining and flow cytometry analysis with bioimaging. Results: The results showed that endometriosis-related macrophages treated with stilbenes have impaired expression of pro-inflammatory markers (IL6, IL8, IL1B, TNF, CCL2, CXCL10, PTGS2). The effect of resveratrol, pterostilbene, and piceatannol was observed, especially in reducing IL1B, CCL2, and CXCL10 genes up to 3.5-, 5-, and 7.7-fold at 25 µM, respectively. Also, with piceatannol or polydatin exposure, the IL-6 decrease was noticeable. This study reported an antioxidant effect by reducing ROS-positive cells from 96% to 48% by pterostilbene. Results from flow cytometry correlated with the transcript activation of detoxification enzymes (SOD, GPX). Conclusions: Prospects for potential therapy based on regulating the immune microenvironment and reducing the accumulation of free radicals with stilbenes application were described in the article.
Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol
Fermentation strategies in mead production: A multitechnique volatilomic approach to aroma characterization
Anti-proliferative potential and oxidative reactivity of thermo-oxidative degradation products of stigmasterol and stigmasteryl esters for human intestinal cells
AbstractStigmasterol in free and esterified form is incorporated in LDL cholesterol-lowering food products, intended for direct consumption and cooking, baking, and frying. Under thermal treatment, stigmasterol compounds may constitute a source of thermo-oxidative degradation products and oxyderivatives with potentially adverse health effects. This study aimed to analyze the anti-proliferative potential and genotoxicity of thermo-oxidatively treated stigmasterol (ST), stigmasteryl linoleate (ST-LA), and oleate (ST-OA). The effects on cell viability and proliferation, cell cycle progression, intracellular reactive oxygen species (ROS) generation, and DNA damage were analyzed in normal human intestinal cells. The mutagenic potential was assessed in a bacterial reverse mutation test using Salmonella enterica serovar Typhimurium strains involving metabolic activation. Stigmasteryl esters showed a significantly lower potential to affect intestinal cell viability and proliferation than non-esterified ST, regardless of heating. Thermo-oxidatively treated ST suppressed intestinal cell proliferation by arresting the cell cycle in the G2/M phase and DNA synthesis inhibition. The enhanced intracellular ROS generation and caspase 3/7 activity suggest targeting intestinal cells to the apoptosis pathway. Also, heated ST-LA intensified ROS production and elicited pro-apoptotic effects. Thermo-oxidative derivatives of ST and ST-LA may evoke harmful gastrointestinal effects due to their high oxidative reactivity towards intestinal cells.
Economic Analysis of the Production Process of Probiotics Based on the Biological and Physiological Parameters of the Cells
Probiotic bacteria confer a range of health benefits and are a focus of a growing number of studies. One of the main issues is their stability during drying and storage, which is why techniques, such as fluid bed drying and coating or treatment with stress factors during culturing, are utilized. The methods of the evaluation of probiotic viability and quality are, however, lacking and we need a way of distinguishing between different subpopulations of probiotic bacteria. To address this issue, imaging flow cytometry (IFC) has been utilized to assess cells after simulated in vitro digestion of dried and coated preparations treated with pH stress and heat shock. Samples were analyzed fresh and after 12 months of storage using RedoxSensor green and propidium iodide dyes to assess metabolic activity and cell membrane integrity of the cells. The results were then used to design a drying process on an industrial scale and evaluate the economic factors in the SuperPro Designer v13 software. Based on the number of biologically active and beneficial cells obtained utilizing tested methods, the coating process and treatment with heat shock and pH stress have been the most effective and up to 10 times cheaper to produce than only by drying. Additionally, samples after 12 months of storage have shown an increase in the proportion of cells with intermediate metabolic activity and small amounts of cell membrane damage, which are still viable in probiotic products. This subpopulation of bacteria can still be considered live in probiotic products but is not necessarily effectively detected by pour plate counts.
The Effects of Cellular Membrane Damage on the Long-Term Storage and Adhesion of Probiotic Bacteria in Caco-2 Cell Line
Adhesion is one of the main factors responsible for the probiotic properties of bacteria in the human gut. Membrane proteins affected by cellular damage are one of the key aspects determining adhesion. Fluid-bed-dried preparations containing probiotic bacteria were analyzed in terms of their stability (temperature of glass transition) and shelf life in different conditions (modified atmosphere, refrigeration). Imaging flow cytometry was utilized to determine four subpopulations of cells based on their physiological and morphological properties. Lastly, adhesion was measured in bacteria cultured in optimal conditions and treated with heat shock. The results show that the subpopulations with no or low levels of cell membrane damage exhibit the ability to adhere to Caco-2 cells. The temperature of protein denaturation in bacteria was recorded as being between 65 °C and 70 °C. The highest glass transition temperature (Tg) value for hydroxypropyl methylcellulose (used as a coating substance) was measured at 152.6 °C. Drying and coating can be utilized as a sufficient treatment, allowing a long shelf-life (up to 12 months). It is, however, worth noting that technological processing, especially with high temperatures, may decrease the probiotic value of the preparation by damaging the bacterial cells.
Chlorogenic Acid Inhibits Rahnella aquatilis KM25 Growth and Proteolytic Activity in Fish-Based Products
This work verified the antiproliferative and antiproteolytic activities of chlorogenic acid against Rahnella aquatilis KM25, a spoilage organism of raw salmon stored at 4 °C. Chlorogenic acid limited the growth of R. aqatilis KM25 in vitro at a concentration of 2.0 mg/mL. The dead (46%), viable (25%), and injured (20%) cell subpopulations were identified by flow cytometry following treatment of R. aquatilis KM25 with the examined agent. The exposure of R. aquatilis KM25 to chlorogenic acid altered its morphology. Changes in cell dimensions, mostly in length parameters from 0.778 µm to 1.09 µm, were found. The length of untreated cells ranged from 0.958 µm to 1.53 µm. The RT–qPCR experiments revealed changes in the expression of genes responsible for the proliferation and proteolytic activity of cells. Chlorogenic acid caused a significant reduction in the mRNA levels of the ftsZ, ftsA, ftsN, tolB, and M4 genes (−2.5, −1.5, −2.0, −1.5, and −1.5, respectively). In situ experiments confirmed the potential of chlorogenic acid to limit bacterial growth. A similar effect was noted in samples treated with benzoic acid, where the growth inhibition of R. aquatilis KM25 was 85–95%. Reduction of microbial R. aquatilis KM25 proliferation significantly limited total volatile base nitrogen (TVB-N) and trimethylamine (TMA-N) formation during storage, extending the shelf life of model products. The TVB-N and TMA-N parameters did not exceed the upper levels of the maximum permissible limit of acceptability. In this work, the TVB-N and TMA-N parameters were 10–25 mg/100 g and 2.5–20.5 mg/100 g, respectively; for samples with benzoic acid-supplemented marinades, the parameters TVB-N and TMA-N were 7.5–25.0 mg/100 g and 2.0–20.0 mg/100 g, respectively. Based on the results of this work, it can be concluded that chlorogenic acid can increase the safety, shelf life, and quality of fishery products.
Optimization of the flow cytometry method of detection, quantification and qualification of microorganisms in carrot juice
Wykorzystanie cytometrii przepływowej w analizie żywotności i aktywności proteolitycznej komórek drożdży gorzelniczych Saccharomyces cerevisiae w celu monitorowania efektywności fermentacji etanolowej
Dynamics of active fluorescent units (AFU) and water activity (aw) changes in probiotic products—pilot study
The flow cytometry method (FCM) is a widely renowned practice increasingly used to assess the microbial viability of probiotic products. Additionally, the measurement of water activity (aw) can be used to confirm the presence of viable cells in probiotic products throughout their shelf lives. The aim of this study was to investigate the correlation between changes in aw and variations in active fluorescent units (AFU), a unit commonly used in flow cytometry method, during the aging of probiotic products containing freeze-dried bacteria. We controlled the stability of probiotic products for bacterial counts (using ISO 19344 method) and aw levels in commercially available capsules containing freeze-dried bacteria such as Lactobacillus sp. or combinations of Lactobacillus sp. and Bifidobacterium sp. in standard conditions (25 ± 2 °C and 60% relative humidity) over a period of 24 months. During this time, the bacterial contents decreased by 0.12 Log10 in the single-strain product, by 0.16 Log10 in the two-strain product and by 0.26 Log10 in the multi-strain product. With the increase in aw, the number of bacteria decreased but the aw at the end point of the stability study did not exceed 0.15 in each of the three tested products. FCM combined with aw is a prospective analysis that can be used to assess the stability of probiotic products, both for its ability to detect bacterial viability and for practical (analysis time) and economic reasons.
Imaging Flow Cytometry Demonstrates Physiological and Morphological Diversity within Treated Probiotic Bacteria Groups
Probiotic bacteria can be introduced to stresses during the culturing phase as an alternative to the use of protectants and coating substances during drying. Accurate enumeration of the bacterial count in a probiotic formulation can be provided using imaging flow cytometry (IFC). IFC overcomes the weak points of conventional, commonly used flow cytometry by combining its statistical power with the imaging content of microscopy in one system. Traditional flow cytometers only collect the fluorescence signal intensities, while IFC provides many more steps as it correlates the data on the measured parameters of fluorescence light with digitally processed images of the analyzed cells. As an alternative to standard methods (plate cell counts and traditional flow cytometry) IFC provides additional insight into the physiology and morphology of the cell. The use of complementary dyes (RedoxSensorTM Green and propidium iodide) allows for the designation of groups based on their metabolic activity and membrane damage. Additionally, cell sorting is incorporated to assess each group in terms of growth on different media (MRS-Agar and MRS broth). Results show that the groups with intermediate metabolic activity and some degree of cellular damage correspond with the description of viable but nonculturable cells.
Quantitative Analysis of Lactobionic Acid in Bioreactor Cultures and Selected Biological Activities
The aim of this study was to quantitatively analyse lactobionic acid obtained from bioreactor cultures using whey as a liquid medium with bacteria of the Pseudomonas taetrolens species. The most important culture parameters affecting the production of the acid are indicated and evaluated. The highest lactobionic acid yield was 37.42 g/L, selecting the appropriate strain (Pseudomonas taetrolens 4′) and environmental conditions (2% lactose concentration in the bioreactor). The amount of lactose and lactobionic acid was determined by high-performance liquid chromatography. Microorganism analysis was also carried out using a flow cytometer with imaging to study the metabolic activity of microorganisms during lactobionic acid production. In addition, during the study, Bifidobacteria were microencapsulated with lactobionic acid and their survival was evaluated in an in vitro model of the gastrointestinal tract, checking the prebiotic properties of the acid. The highest number of viable cells in the microcapsules after digestion was obtained using the Bifidobacterium bifidum strain DSM 20082. The antagonistic activity of lactobionic acid was also analysed.
Dose–Response Effect of Nitrogen on Microbial Community during Hydrocarbon Biodegradation in Simplified Model System
Knowledge about the influence of C:N ratio on the biodegradation process of hydrocarbon compounds is of significant importance in the development of biostimulation techniques. The purpose of this study was to assess the impact of nitrogen compounds on the environmental consortium during the process of biological decomposition of hydrocarbons. The experimental variants represented low, moderate, and excessive biostimulation with nitrogen compounds. The metabolic activity of the consortium was tested using the flow cytometry technique. The efficiency of the biodegradation of hydrocarbons of the consortium, based on the gas chromatography method, and metapopulation changes, based on the analysis of V4 16srRNA sequencing data, were assessed. The results of the research confirm the positive effect of properly optimized biostimulation with nitrogen compounds on the biological decomposition of polycyclic aromatic hydrocarbons. The negative impact of excessive biostimulation on the biodegradation efficiency and metabolic activity of microorganisms is also proven. Low resistance to changes in the supply of nitrogen compounds is demonstrated among the orders Xanthomonadales, Burkholderiales, Sphingomonadales, Flavobacteriales, and Sphingobacteriales. It is proven that quantitative analysis of the order of Rhizobiales, characterized by a high-predicted potential for the decomposition of polycyclic aromatic hydrocarbons, may be helpful during biostimulation optimization processes in areas with a high nitrogen deficiency.
Global transcriptome analysis of Pseudomonas aeruginosa NT06 response to potassium chloride, sodium lactate, sodium citrate, and microaerophilic conditions in a fish ecosystem
Abstract Pseudomonas aeruginosa is an opportunistic pathogen that recently has been increasingly isolated from foods, especially from minimally processed fish-based products. Those are preserved by the addition of sodium chloride (NaCl) and packaging in a modified atmosphere. However, the current trends of minimizing NaCl content may result in an increased occurrence of P. aeruginosa. NaCl can be replaced with potassium chloride (KCl) or sodium salts of organic acids. Herein, we examined the antimicrobial effects of KCl, sodium lactate (NaL), sodium citrate (NaC), and sodium acetate (NaA) against P. aeruginosa NT06 isolated from fish. Transcriptome response of cells grown in medium imitating a fish product supplemented with KCl and KCl/NaL/NaC and maintained under microaerophilic conditions was analysed. Flow cytometry analysis showed that treatment with KCl and KCl/NaL/NaC resulted in changed metabolic activity of cells. In response to KCl and KCl/NaL/NaC treatment, genes related to cell maintenance, stress response, quorum sensing, virulence, efflux pump, and metabolism were differentially expressed. Collectively, our results provide an improved understanding of the response of P. aeruginosa to NaCl alternative compounds that can be implemented in fish-based products and encourage further exploration of the development of effective methods to protect foods against the P. aeruginosa, underestimate foodborne bacteria.