In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

Synergistic effects of mixing cocksfoot and sainfoin on in vitro rumen fermentation: role of condensed tannins

Some bioactive secondary metabolites in forage legumes can cause digestive interactions, so that the rumen fermentation pattern of a mixture of forages can differ from the average values of its components. The objective of this study was to investigate the potential role of condensed tannins (CT) on the synergistic effects between one grass species, cocksfoot, and one CT-containing legume species, sainfoin, on in vitro rumen fermentation characteristics. Cocksfoot and sainfoin in different proportions (in g/kg, 1000:0, 750:250, 500:500, 250:750 and 0:1000) were incubated under anaerobic conditions in culture bottles containing buffered rumen fluid from sheep. Incubations were carried out using artificial saliva with and without polyethylene glycol (PEG), which binds and thus inactivates CT. Rumen fermentation parameters describing the degradation and the fate of the energetic and nitrogenous substrates were measured at 3.5 and 24 h. At the early fermentation stage, when the sainfoin level increased from 0 to 1000 g/kg, the ammonia concentration in the medium quadratically decreased from 3.20 to 0.53 mmol/l in absence of PEG (P<0.01) but not in its presence. This result demonstrates that sainfoin CT decreased the rumen degradation of the proteins in the whole mixture, including the proteins in cocksfoot, rather than just the proteins in sainfoin. Interestingly, the total gas and methane productions were lower in mixtures incubated in absence of PEG than in presence of PEG (P<0.001) while no significant PEG effect was observed on digestibility. At the late fermentation stage, a positive quadratic effect on dry matter digestibility was detected without PEG (P<0.05), indicating a synergistic action of cocksfoot plus sainfoin on plant substrate degradation due to CT. The presence of PEG increased gas production (P<0.001) and NH3-N concentration in the medium (P<0.001). Our results suggest that CT could allow a better utilization of plant substrates in mixtures by the rumen ecosystem by improving the partitioning of degraded substrates toward lower gas losses, and decreasing the protein degradation.

In vitro fermentation of potential prebiotic flours from natural sources: impact on the human colonic microbiota and metabolome

Scope: Fibers and prebiotics represent a useful dietary approach for modulating the human gut microbiome. Therefore, aim of the present study was to investigate the impact of four flours (wholegrain rye, wholegrain wheat, chickpeas and lentils 50:50, and barley milled grains), characterized by a naturally high content in dietary fibers, on the intestinal microbiota composition and metabolomic output. Methods and results: A validated three-stage continuous fermentative system simulating the human colon was used to resemble the complexity and diversity of the intestinal microbiota. Fluorescence in situ hybridization was used to evaluate the impact of the flours on the composition of the microbiota, while small-molecule metabolome was assessed by NMR analysis followed by multivariate pattern recognition techniques. HT29 cell-growth curve assay was used to evaluate the modulatory properties of the bacterial metabolites on the growth of intestinal epithelial cells. All the four flours showed positive modulations of the microbiota composition and metabolic activity. Furthermore, none of the flours influenced the growth-modulatory potential of the metabolites toward HT29 cells. Conclusion: Our findings support the utilization of the tested ingredients in the development of a variety of potentially prebiotic food products aimed at improving gastrointestinal health.

In vitro assessment of the bioaccessibility of brominated flame retardants in indoor dust using a colon extended model of the human gastrointestinal tract

An in vitro colon extended physiologically based extraction test (CEPBET) which incorporates human gastrointestinal tract (GIT) parameters (including pH and chemistry, solid-to-fluid ratio, mixing and emptying rates) was applied for the first time to study the bioaccessibility of brominated flame retardants (BFRs) from the 3 main GIT compartments (stomach, small intestine and colon) following ingestion of indoor dust. Results revealed the bioaccessibility of γ-HBCD (72%) was less than that for α- and β-isomers (92% and 80% respectively) which may be attributed to the lower aqueous solubility of the γ-isomer (2 μg L−1) compared to the α- and β-isomers (45 and 15 μg L−1 respectively). No significant change in the enantiomeric fractions of HBCDs was observed in any of the studied samples. However, this does not completely exclude the possibility of in vivo enantioselective absorption of HBCDs, as the GIT cell lining and bacterial flora – which may act enantioselectively – are not included in the current CE-PBET model. While TBBP-A was almost completely (94%) bioaccessible, BDE-209 was the least (14%) bioaccessible of the studied BFRs. Bioaccessibility of tri-hepta BDEs ranged from 32–58%. No decrease in the bioaccessibility with increasing level of bromination was observed in the studied PBDEs.

A comparison of the anticancer properties of isoxanthohumol and 8-prenylnaringenin using in vitro models of colon cancer

The hops plant (Humulus lupulus L.) is an essential ingredient in beer and contains a number of potentially bioactive prenylflavonoids, the predominant being the weakly estrogenic isoxanthohumol (Ix), which can be converted to the more strongly estrogenic 8-PN by the colonic microbiota. The aim of this study was to investigate the biological activity of 8-PN and Ix using in vitro models representing key stages of colorectal carcinogenesis, namely cell growth and viability (MTT assay), cell-cycle progression (DNA content assay), DNA damage (Comet assay), and invasion (Matrigel assay). A significant decrease in Caco-2 cell viability was noted after both 8-PN and Ix treatments at the higher doses (40 and 50 μM, respectively) although the impact on cell cycle differed between the two compounds. The decreased cell viability observed after Ix treatment was associated with a concentration-dependent increase in G2/M and an increased sub-G1 cell-cycle fraction, whereas treatment with 8-PN was associated with an elevated G0/G1 and an increased sub-G1 cell-cycle fraction. Significant antigenotoxic activity was noted at all 8-PN concentrations tested (5-40 μM). Although significant antigenotoxic activity was also noted with Ix treatment at ≤25 μM, at a higher dose, Ix itself exerted genotoxic activity. In a dose-dependent manner, both compounds inhibited HT115 cell invasion with reductions up to 52 and 46% for Ix and 8-PN, respectively, in comparison to untreated cells. This study demonstrated that both Ix and its gut microbial metabolite 8-PN exert anticancer effects on models of key stages of colon tumourigenesis.

Design, synthesis and in vitro degradation of a novel co-drug for the treatment of psoriasis

Psoriasis is a common, chronic and relapsing inflammatory skin disease. It affects approximately 2% of the western population and has no cure. Combination therapy for psoriasis often proves more efficacious and better tolerated than monotherapy with a single drug. Combination therapy could be administered in the form of a co-drug, where two or more therapeutic compounds active against the same condition are linked by a cleavable covalent bond. Similar to the pro-drug approach, the liberation of parent moieties post-administration, by enzymatic and/or chemical mechanisms, is a pre-requisite for effective treatment. In this study, a series of co-drugs incorporating dithranol in combination with one of several non-steroidal anti-inflammatory drugs, both useful for the treatment of psoriasis, were designed, synthesized and evaluated. An ester co-drug comprising dithranol and naproxen in a 1:1 stoichiometric ratio was determined to possess the optimal physicochemical properties for topical delivery. The co-drug was fully hydrolyzed in vitro by porcine liver esterase within four hours. When incubated with homogenized porcine skin, 9.5% of the parent compounds were liberated after 24 h, suggesting in situ esterase-mediated cleavage of the co-drug would occur within the skin. The kinetics of the reaction revealed first order kinetics, Vmax = 10.3 μM/min and Km = 65.1 μM. The co-drug contains a modified dithranol chromophore that was just 37% of the absorbance of dithranol at 375 nm and suggests reduced skin/clothes staining. Overall, these findings suggest that the dithranol-naproxen co-drug offers an attractive, novel approach for the treatment of psoriasis.

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40 μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10 μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin

The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions

Purpose: Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions. Methods: Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays. Results: Keratocytes cultured with RA up to 10×10-6 M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media. Conclusions: RA was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behaviour and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Diversity of condensed tannin structures affects rumen in vitro methane production in sainfoin (Onobrychis viciifolia) accessions

Sainfoin is a non-bloating temperate forage legume with a moderate-to-high condensed tannin (CT) content. This study investigated whether the diversity of sainfoin accessions in terms of CT structures and contents could be related to rumen in vitro gas and methane (CH4) production and fermentation characteristics. The aim was to identify promising accessions for future investigations. Accessions differed (P < 0·0001) in terms of total gas and CH4 productions. Fermentation kinetics (i.e. parameters describing the shape of the gas production curve and half-time gas production) for CH4 production were influenced by accession (P ≤ 0·038), but not by PEG. Accession, PEG and time affected (P < 0·001) CH4 production, but accession and PEG interaction showed only a tendency (P = 0·08). Increase in CH4 due to PEG addition was not related to CT content. Further analysis of the relationships among multiple traits (nutritional composition, CT structure and CH4 production) using principal component analysis (PCA) based on optimally weighted variables revealed differences among accessions. The first two principal component axes, PC1 (57·6%) and PC2 (18·4%), explained 76·0% of the total variation among accessions. Loading of biplots derived from both PCAs made it possible to establish a relationship between the ratio of prodelphinidin:procyanidin (PD:PC) tannins and CH4 production in some accessions. The PD:PC ratio seems to be an important source of variation that is negatively related to CH4 production. These results suggested that sainfoin accessions collected from across the world exhibited substantial variation in terms of their effects on rumen in vitro CH4 production, revealing some promising accessions for future investigations.

Virgin olive oil phenolics extract inhibit invasion of HT115 human colon cancer cells in vitro and in vivo

The decreased cancer risk associated with consumption of olive oil may be due to the presence of phenolics which can modulate pathways including apoptosis and invasion that are relevant to carcinogenesis. We have previously shown that a virgin olive oil phenolics extract (OVP) inhibited invasion of HT115 colon cancer cells in vitro. In the current study we assessed the in vitro effects of OVP (25 μg mL(-1)) on HT115 cell migration, spreading and integrin expression. Furthermore, the anti-metastatic activity of OVP - at a dose equivalent to 25 mg per kg per day for 2, 8 or 10 weeks - was assessed in a Severe Combined ImmunoDeficiency (SCID) Balb-c mouse model. After 24 h OVP did not inhibit cell migration but significantly reduced cell spreading on fibronectin (65% of control; p < 0.05) and expression of a range of α and β integrins was modulated. In vivo, OVP by gavage significantly (p < 0.05) decreased not only tumour volume but also the number of metastases in SCID Balb-c mice. Collectively, the data suggest that - possibly through modulation of integrin expression - OVP decreases invasion in vitro and also inhibits metastasis in vivo.

Impacts of plant-based foods in ancestral hominin diets on the metabolism and function of gut microbiota in vitro

Ancestral human populations had diets containing more indigestible plant material than present-day diets in industrialized countries. One hypothesis for the rise in prevalence of obesity is that physiological mechanisms for controlling appetite evolved to match a diet with plant fiber content higher than that of present-day diets. We investigated how diet affects gut microbiota and colon cells by comparing human microbial communities with those from a primate that has an extreme plant-based diet, namely, the gelada baboon, which is a grazer. The effects of potato (high starch) versus grass (high lignin and cellulose) diets on human-derived versus gelada-derived fecal communities were compared in vitro. We especially focused on the production of short-chain fatty acids, which are hypothesized to be key metabolites influencing appetite regulation pathways. The results confirmed that diet has a major effect on bacterial numbers, short-chain fatty acid production, and the release of hormones involved in appetite suppression. The potato diet yielded greater production of short-chain fatty acids and hormone release than the grass diet, even in the gelada cultures, which we had expected should be better adapted to the grass diet. The strong effects of diet on hormone release could not be explained, however, solely by short-chain fatty acid concentrations. Nuclear magnetic resonance spectroscopy found changes in additional metabolites, including betaine and isoleucine, that might play key roles in inhibiting and stimulating appetite suppression pathways. Our study results indicate that a broader array of metabolites might be involved in triggering gut hormone release in humans than previously thought. IMPORTANCE: One theory for rising levels of obesity in western populations is that the body's mechanisms for controlling appetite evolved to match ancestral diets with more low-energy plant foods. We investigated this idea by comparing the effects of diet on appetite suppression pathways via the use of gut bacterial communities from humans and gelada baboons, which are modern-day primates with an extreme diet of low-energy plant food, namely, grass. We found that diet does play a major role in affecting gut bacteria and the production of a hormone that suppresses appetite but not in the direction predicted by the ancestral diet hypothesis. Also, bacterial products were correlated with hormone release that were different from those normally thought to play this role. By comparing microbiota and diets outside the natural range for modern humans, we found a relationship between diet and appetite pathways that was more complex than previously hypothesized on the basis of more-controlled studies of the effects of single compounds.

O6-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat

Scope Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). Methods and Results In this study, the NOC-derived DNA adduct O6-carboxymethylguanine (O6-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O6-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose–response association with O6-CMG (p = 0.004) and MDA (p = 0.008) formation. Conclusion The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.

Addition of different fats to a carbohydrate food: impact on gastric emptying, glycaemic and satiety responses and comparison with in vitro digestion

In vitro, the addition of lipids to a carbohydrate food has been found to increase the digestibility of starch. In contrast, in vivo studies have shown that the addition of fat to a food can reduce the glycaemic response (GR). The aim of this study was to assess if delayed gastric emptying (GE) causes reduced GR with the addition of lipids to a carbohydrate food and if a relationship between GR and in vitro digestion of starch exists for high fat foods. Ten healthy volunteers were tested on five occasions after consuming pancakes containing 50 g of available carbohydrate and 202 kcal of sunflower oil, olive oil, butter, medium chain triglyceride (MCT) oil or a control containing no oil. GR was measured using fingerpick blood samples, satiety using visual analogue scales and GE using the 13C octanoic acid breath test. There was a significant difference in GR between the different pancake breakfasts (p = 0.05). The highest GR was observed following the control pancakes and the lowest following the olive oil pancakes. There were significant differences in GE half time, lag phase and ascension time (p < 0.05) between the different pancakes with the control pancakes having the shortest GE time and the MCT pancakes the longest. There was a significant difference in satiety parameters fullness (p = 0.003) and prospective consumption (p = 0.050), with satiety being lowest following the control pancakes. There was a significant inverse correlation between the GR and all satiety parameters. A significant inverse correlation (p = 0.009) was also observed between the digestibility of starch in vitro and GR in vivo. The paper indicates that the digestibility of starch in vitro does not predict the GR for high fat containing foods