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.

Stability of S-HBsAg in long-term stored lyophilised plant tissue.

A potent plant-derived oral vaccine against Hepatitis B Virus (HBV) requires a durable and compact form for efficacious and convenient distribution and delivery. In the previous study we have devised a successful freeze-drying process of plant material containing the HBV small surface antigen (S-HBsAg) for the purpose of an oral vaccine against the virus, but product storage stability was limited to 4 °C. The aim of this study was to upgrade a freeze-dried product formula to facilitate successful long-term storage of S-HBsAg assembled into Virus-Like Particles (VLPs) at elevated temperatures. Series of additional excipients and storage conditions were tested. Atmosphere of nitrogen proved to preserve S-HBsAg VLPs most efficiently, with only minor degradation at the highest temperature of 37 °C. As a result, a semi-product for the oral plant-derived vaccine against HBV with good storage capabilities was obtained.

Antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion.

BACKGROUND: Broccoli is a common vegetable recognized as a rich source of antioxidants. To date, research on the antioxidant properties of broccoli, predominantly conducted on extracts, has not considered the lesions of composition and this activity after gastrointestinal digestion. Here the stability of antioxidants during gastrointestinal digestion was evaluated in conjunction with the protective effects of broccoli sprouts (BS) against oxidative stress in human colon cells. RESULTS: The obtained data suggest that, among the biocompounds identified in BS, glucosinolates were mainly degraded under gastrointestinal digestion, while phenolics, particularly hydroxycinnamic acid derivatives, were the most resistant constituents. The antioxidant capacity of BS extract subjected to gastrointestinal digestion was similar to or higher than that determined for non-digested BS. Gastrointestinal digested BS extract exhibited reactive oxygen species (ROS)-inhibitory capacity in NCM460 human colon cells, with 1 mg mL(-1) showing an ROS clearance of 76.59%. A 57.33% reduction in oxidative DNA damage in NCM460 cells due to treatment with digested BS extract was observed. CONCLUSION: The results lend support to the possible application of BS as a rich source of antioxidants to improve the defensive system against oxidative stress in the human colon mucosa.

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.

Current Trends in the Production of Probiotic Formulations.

Preparations containing probiotic strains of bacteria have a beneficial effect on human and animal health. The benefits of probiotics translate into an increased interest in techniques for the preservation of microorganisms. This review compares different drying methods and their improvements, with specific reference to processing conditions, microorganisms, and protective substances. It also highlights some factors that may influence the quality and stability of the final probiotic preparations, including thermal, osmotic, oxidative, and acidic stresses, as well as dehydration and shear forces. Processing and storage result in the loss of viability and stability in probiotic formulations. Herein, the addition of protective substances, the optimization of process parameters, and the adaptation of cells to stress factors before drying are described as countermeasures to these challenges. The latest trends and developments in the fields of drying technologies and probiotic production are also discussed. These developments include novel application methods, controlled release, the use of food matrices, and the use of analytical methods to determine the viability of probiotic bacteria.

ROS Modulating Effects of Lingonberry (Vaccinium vitis-idaea L.) Polyphenols on Obese Adipocyte Hypertrophy and Vascular Endothelial Dysfunction.

Oxidative stress and dysregulated adipocytokine secretion accompanying hypertrophied adipose tissue induce chronic inflammation, which leads to vascular endothelial dysfunction. The present study investigated the ability of anthocyanin (ACN) and non-anthocyanin polyphenol (PP) fractions from lingonberry fruit to mitigate adipose tissue hypertrophy and endothelial dysfunction using 3T3-L1 adipocytes and human umbilical vein endothelial cells (HUVECs). This study showed that the PP fraction decreased intracellular ROS generation in hypertrophied adipocytes by enhancing antioxidant enzyme expression (SOD2) and inhibiting oxidant enzyme expression (NOX4, iNOS). Moreover, PP and ACN fractions reduced triglyceride content in adipocytes accompanied by downregulation of the expression of lipogenic genes such as aP2, FAS, and DAGT1. Treatment with both fractions modulated the mRNA expression and protein secretion of key adipokines in hypertrophied adipocytes. Expression and secretion of leptin and adiponectin were, respectively, down- and upregulated. Furthermore, PP and ACN fractions alleviated the inflammatory response in TNF-α-induced HUVECs by inhibiting the expression of pro-inflammatory genes (IL-6, IL-1ß) and adhesion molecules (VCAM-1, ICAM-1, SELE). The obtained results suggest that consuming polyphenol-rich lingonberry fruit may help prevent and treat obesity and endothelial dysfunction due to their antioxidant and anti-inflammatory actions.

Elderberry (Sambucus nigra L.) Fruit Extract Alleviates Oxidative Stress, Insulin Resistance, and Inflammation in Hypertrophied 3T3-L1 Adipocytes and Activated RAW 264.7 Macrophages.

Oxidative stress and inflammation in hypertrophied adipose tissue with excessive fat accumulation play a crucial role in the development of obesity and accompanying metabolic dysfunctions. This study demonstrated the capacity of elderberry fruit (EDB) extract to decrease the elevated production of reactive oxygen species in hypertrophied 3T3-L1 adipocytes. Treatment with the EDB extract resulted in modulation of mRNA expression and protein secretion of key adipokines in hypertrophied adipocytes. Expression of leptin and adiponectin was, respectively, down- and up-regulated. Moreover, glucose uptake stimulation was noticed in mature adipocytes, both sensitive to insulin and insulin resistant. This may suggest a positive effect of EDB extract on insulin resistance status. The extract was also found to alleviate the inflammatory response in activated RAW 264.7 macrophages by down-regulating the expression of proinflammatory genes (TNF-α, IL-6, COX-2, iNOS) and suppressing the enhanced production of inflammatory mediators (TNF-α, IL-6, PGE2, NO). In vitro experiments showed that the EDB extract could inhibit digestive enzymes, including α-amylase, α-glucosidase, and pancreatic lipase, leading to reduced intestinal absorption of dietary lipids and carbohydrates. Further in vivo studies could be postulated to support EDB as a functional food component for the prevention and treatment of obesity and metabolic-immune comorbidities.

Defense responses of Thuja orientalis to infestation of anholocyclic species aphid Cinara tujafilina.

The aim of this study was to determine an interdependence between generation of semiquinone radicals, superoxide anion (O2-), manganese ions (Mn2+) and phenolic content in leaves of Thuja orientalis in response to infestation by varying populations of Cinara tujafilina, i.e. 40 or 80 aphids per plant. Also, superoxide dismutase (SOD) and ß-d-glucosidase activities in leaves of T. orientalis in a defense response to C. tujafilina was recorded. Analyses of electron paramagnetic resonance (EPR) showed generally a higher concentration of semiquinone radicals with g-values of 2.0051 ± 0.0005 and 20032 ± 0.0005 after C. tujafilina infestation in leaves in comparison to the control. Up to 48 h post-infestation in leaves infested by 80 aphids the level of semiquinone radicals was significantly higher than in the control, while in leaves infested by 40 aphids the highest concentrations of these radicals were recorded at later time points (i.e. at 72 and 96 hpi). In parallel, the highest total generation of O2- and low activity of SOD were recorded in 24-h leaves infested by 80 aphids. Additionally, analysis of confocal images showed that the strongest yellow fluorescence indicating O2- generation was detected in epidermal cells of leaves up to 48 hpi. Significant reduction of Mn2+ ions detected by EPR spectroscopy in relation to the control was observed in 4-w leaves infested by 80 and 40 aphids and in 48-h leaves infested by 40 aphids. Phenolic contents in leaves infested by 80 and 40 aphids at all time points were higher than in the control. The greatest ß-d-glucosidase activity and phenolic contents were recorded at 96 h of feeding. These results indicate that the perception of C. tujafilina infestation by T. orientalis leaves induces a specified sequence of defense mechanisms in the course of time.

Method of preservation and type of protective agent strongly influence probiotic properties of Lactococcus lactis: A complete process of probiotic preparation manufacture and use.

In order to assess the essential probiotic properties of a strain dedicated for administration in humans and animals, characteristics of finally formulated products, rather than the cells solely, seems to be of crucial importance. In this study, composition of protective blends for manufacture of L. lactis probiotic powders was optimized using a statistical experimental design. The powders, generated by either spray- or freeze-drying techniques, were subsequently subjected to storage testing, and in vitro digestion in simulated stomach and small intestine. Finally, maintenance of adherence capability to human enterocyte-like cell lines, was evaluated. Our data demonstrated that 10% trehalose ensures the highest viability of L. lactis bacteria upon both drying techniques (viability of 60-68%). Moreover, skimmed milk-protected spray-dried cells exhibit the highest resistance to harsh environmental conditions of stomach (53.9 ±â€¯7.6% survival rate) and higher adhesion ability to HT-29 cell line after digestion (528 ±â€¯29 cells per 100 epithelial cells).

A Gastrointestinally Digested Ribes nigrum L. Fruit Extract Inhibits Inflammatory Response in a Co-culture Model of Intestinal Caco-2 Cells and RAW264.7 Macrophages.

Blackcurrant fruits are a rich source of polyphenolic compounds with high antioxidant capacity and potent anti-inflammatory properties. In this study, blackcurrant extract digested in an artificial gastrointestinal tract and its intestinal permeable fraction were investigated for their ability to suppress inflammatory responses induced in a two-component cell culture system of intestinal epithelial cells and macrophages. The obtained results showed the capacity of the extract at a concentration of 1 mg of freeze-dried blackcurrant powder per mL to down-regulate the expression of inflammatory mediators, such as IL-8 (54 ± 7%) and COX-2 (17 ± 6%) in intestinal cells and IL-1α (76 ± 4%), IL-1ß (91 ± 2%), and IL-6 (61 ± 5%) in macrophages stimulated with lipopolysaccharides. Inhibited COX-2 (44 ± 6%) and iNOS (15 ± 7%) expression played a role in reducing the production of prostaglandin E2 (40 ± 20%) and NO (31 ± 9%), respectively. Decreased TNF-α secretion (24 ± 5%) by activated macrophages was also observed after treatment with blackcurrant extract. Moreover, the gastrointestinal-digested extract (0.01-1 mg/mL) dose dependently decreased the enhanced ROS generation (14-54%) and oxidative DNA damage (16-37%) induced in intestinal cells. The increased intestinal permeability caused by proinflammatory mediators, as assessed by transepithelial electrical resistance, was completely counteracted.

Purple carrot anthocyanins suppress lipopolysaccharide-induced inflammation in the co-culture of intestinal Caco-2 and macrophage RAW264.7 cells.

This study was designed to evaluate the anti-inflammatory effects of purple carrot anthocyanins (PCA) with respect to gut inflammation, simulated in a co-culture system consisting of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. The obtained results indicated that PCA extract down-regulates the mRNA expression of proinflammatory interleukins Il-1ß (↓91%) and Il-6 (↓69%) as well as inflammatory mediators, such as cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNos), in lipopolysaccharide-activated RAW264.7 cells. The decrease in the generation of prostaglandin E2 (↓48%) and nitric oxide (↓26%) was observed as a result of the inhibition of Cox-2 (↓25%) and iNos (↓12%) mRNA expressions, respectively. Moreover, the PCA reduced mRNA expression (↓40%) and production (↓17%) of IL-8 in intestinal cells. The anti-inflammatory effect of PCA was contributed to the protection of the intestinal barrier, which was disrupted upon the stimulation of macrophages. These findings may provide preliminary justification for the use of PCA in further studies focused on the prevention and therapy supporting the conventional treatment of inflammatory bowel diseases.

Gastrointestinal digested Sambucus nigra L. fruit extract protects in vitro cultured human colon cells against oxidative stress.

Elderberry (EDB) Sambucus nigra L. is one of the oldest medicinal plants which is useful for therapeutic and nutritional purposes due to a large amount of biologically active constituents, including compounds with a high antioxidant capacity. The present study focused on the antioxidant potential of the colon-available EDB fruit extract, derived from the artificial gastrointestinal tract, with regard to human colonic mucosa cells cultured in vitro. Despite the significant loss of EDB bioactive compounds due to the digestion process, the colon-digested extract was able to reduce the excessive intracellular ROS production (22%) and oxidative DNA damage (46%) in the colon cells at a dose of 1 mg of freeze-dried EDB powder/ml. Moreover, the colon-digested EDB extract inhibited oxidant-induced mutagenicity (26%) in the Salmonella typhimurium TA102 strain, as determined by the Ames test. In conclusion, the current in vitro study confirmed that the fruits of S. nigra are capable of protecting colonic cells against the detrimental effects of oxidative stress.

Antioxidant effects of gastrointestinal digested purple carrot extract on the human cells of colonic mucosa.

Purple carrot (PC) is a potential dietary constituent, which represents a valuable source of antioxidants and can modulate the reactive oxygen species (ROS) level in the gastrointestinal tract. Antioxidant capacity of a PC extract subjected to digestion process simulated in the artificial alimentary tract, including the stomach, small intestine and colon, was analyzed in normal human cells of colon mucosa. Results indicated that the extract obtained upon passage through the gastrointestinal tract, which could come into contact with the colonic cells in situ, was less potent than the extract, which was not subjected to digestion process. Digested PC extract exhibited intracellular ROS-inhibitory capacity, with 1mg/mL showing the ROS clearance of 18.4%. A 20.7% reduction in oxidative DNA damage due to colon mucosa cells' treatment with digested PC extract was observed. These findings indicate that PC extract is capable of colonic cells' protection against the adverse effects of oxidative stress.

Freeze-drying of plant tissue containing HBV surface antigen for the oral vaccine against hepatitis B.

The aim of this study was to develop a freeze-drying protocol facilitating successful processing of plant material containing the small surface antigen of hepatitis B virus (S-HBsAg) while preserving its VLP structure and immunogenicity. Freeze-drying of the antigen in lettuce leaf tissue, without any isolation or purification step, was investigated. Each process step was consecutively evaluated and the best parameters were applied. Several drying profiles and excipients were tested. The profile of 20°C for 20 h for primary and 22°C for 2 h for secondary drying as well as sucrose expressed efficient stabilisation of S-HBsAg during freeze-drying. Freezing rate and postprocess residual moisture were also analysed as important factors affecting S-HBsAg preservation. The process was reproducible and provided a product with VLP content up to 200 µg/g DW. Assays for VLPs and total antigen together with animal immunisation trials confirmed preservation of antigenicity and immunogenicity of S-HBsAg in freeze-dried powder. Long-term stability tests revealed that the stored freeze-dried product was stable at 4°C for one year, but degraded at elevated temperatures. As a result, a basis for an efficient freeze-drying process has been established and a suitable semiproduct for oral plant-derived vaccine against HBV was obtained.