In vitro selection and characterization of new probiotic candidates from table olive microbiota.

To date, only a few studies have investigated the complex microbiota of table olives in order to identify new probiotic microorganisms, even though this food matrix has been shown to be a suitable source of beneficial lactic acid bacteria (LAB). Two hundred and thirty eight LAB, belonging to Lactobacillus plantarum, Lactobacillus pentosus and Leuconostoc mesenteroides species, and isolated from Nocellara Etnea table olives, have been screened in this survey through an in vitro approach. A simulation of transit tolerance in the upper human gastrointestinal tract, together with autoaggregation and hydrophobicity, have been decisive in reducing the number of LAB to 17 promising probiotics. None of the selected strains showed intrinsic resistances towards a broad spectrum of antibiotics and were therefore accurately characterized on an undifferentiated and 3D functional model of the human intestinal tract made up of H4-1 epithelial cells. As far as the potential colonization of the intestinal tract is concerned, a high adhesion ratio was observed for Lb. plantarum O2T60C (over 9%) when tested in the 3D functional model, which closely mimics real intestinal conditions. The stimulation properties towards the epithelial barrier integrity and the in vitro inhibition of L. monocytogenes adhesion and invasion have also been assessed. Lb. plantarum S1T10A and S11T3E enhanced trans-epithelial electrical resistance (TEER) and therefore the integrity of the polarized epithelium in the 3D model. Moreover, S11T3E showed the ability to inhibit L. monocytogenes invasion in the undifferentiated epithelial model. The reduction in L. monocytogenes infection, together with the potential enhancement of barrier integrity and an adhesion ratio that was above the average in the 3D functional model (6.9%) would seem to suggest the Lb. plantarum S11T3E strain as the most interesting candidate for possible in vivo animal and human trials.

Improvement of lipid profile by probiotic/protective cultures: study in a non-carcinogenic small intestinal cell model.

Plasma lipid levels are important risk factors for the development of atherosclerosis and coronary heart disease. Previous findings have shown that probiotic bacteria exert positive effects on hypercholesterolemia by lowering serum cholesterol and improving lipid profile that, in turn, leads to a reduced risk of coronary heart disease and atherosclerosis. Most of these studies were carried out with tumoral cell lines that have a metabolism quite different from that of normal cells and may thus respond differently to various stimuli. Here, we demonstrate the beneficial effects of some probiotics on cholesterol levels and pathways in normal small intestinal foetal epithelial tissue cells. The results show that Lactobacillus plantarum strain PCS 26 efficiently removes cholesterol from media, exhibits bile salt hydrolase activity, and up-regulates several genes involved in cholesterol metabolism. This study suggests that Lactobacillus plantarum PCS 26 might act as a liver X receptor agonist and help to improve lipid profiles in hypercholesterolemic patients or even dislipidemias in complex diseases such as the metabolic syndrome.

ELISA kit for peanut protein determination: collaborative study.

A collaborative study in 10 laboratories was performed to validate an ELISA method developed for the quantitative determination of peanut protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit does not produce any false-positive results or cross-reactivity with a broad range of peanut-free food matrixes. All participants obtained the peanut ELISA kit with standard operational procedures, a list of samples, the samples, and a protocol for recording test results. The study included 15 food samples. Three food matrix samples of zero peanut content showed peanut protein content lower than the first standard (0.10 mg/kg). Three samples with peanut declared as an ingredient revealed peanut protein content outside the calibration curve (absorbance was above the highest standard) in all laboratories, and three samples had the peanut content reported either above the highest standard or within the calibration curve, depending on the laboratory. Six samples with peanut declared as an ingredient gave the peanut protein content within the calibration curve. Only these six samples, together with a positive control sample (CS2), were used for statistical evaluation. The statistical tests (Cochran, Grubbs, and Mandel) and analysis of variance were used for the evaluation of the collaborative study results. Repeatability and reproducibility limits, as well as an LOQ (LOQcollaborative 0.22 mg peanut proteins/kg) and an LOD (LODcollaborative 0.07 mg peanut proteinslkg) for the kit were calculated.

Skeletal muscle-derived cell cultures as potent models in regenerative medicine research.

Cell cultures have been used extensively by many scientists in recent decades to study various cell and tissue mechanisms. The use of cell cultures has many advantages over use of in vivo experimental models, but there are also limitations. As skeletal muscle-derived cell cultures become more commonly utilized in studies of muscle regeneration processes the question of their relevance in experimentation is highlighted with regard to in vivo experimental models. This article reviews studies that have been performed simultaneously in in vivo and in vitro experiments on skeletal muscle and assesses the correlation of results. Although they seem to correlate, no such studies on humans have been performed so far.

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.

Application of Gut Cell Models for Toxicological and Bioactivity Studies of Functional and Novel Foods.

The concept of functional and novel foods undoubtedly bears great potential as an asset to human health. However, this very same quest for ever new bioactive ingredients calls for reliable and distinct risk assessment as they may be potentially hazardous to human health. Most of today's methodologies still rely on decades old routines of animal trials and use of tumor-derived cell lines. Since such methodologies are not in line with the actual processes in the human body and with the 3R (replacement, reduction, refinement) concept, the results are often unreliable and misleading. Therefore, in this paper we propose the utilization of available untransformed small intestinal cell lines derived from human and pig tissue of non-tumor origin and describe several available cell models of the gut that offer a functional, close resemblance with the in vivo environment.

Nitrate content in dandelion (Taraxacum officinale) and lettuce (Lactuca sativa) from organic and conventional origin: intake assessment.

To estimate the actual intake of nitrate by consumption of different lettuce varieties, 52 samples of lettuce of different origins and dandelion from 15 different areas of northeast Slovenia were analysed. For determination of actual nitrate content, a continuous flow method was used. The lowest nitrate content was detected in dandelion, with a mean value of 195 mg kg(-1) (ranging 47-487 mg kg(-1)). Nitrate content in lettuce of different origins ranged 85-3237 mg kg(-1), with a mean value of 1196 mg kg(-1). The mean nitrate content in organically cultivated lettuce was 890 mg kg(-1), which was considerably lower than the nitrate level in conventionally cultivated lettuce (1298 mg kg(-1)). Consumption of 100 g of dandelion would result in a maximal nitrate intake corresponding to 22% of the acceptable daily intake (ADI), with values up to seven times higher for lettuce.

The use of a porcine intestinal cell model system for evaluating the food safety risk of Bacillus cereus probiotics and the implications for assessing enterotoxigenicity.

The use of porcine intestinal cell lines in assessing toxicity of Bacillus cereus probiotics in conjunction with animal challenge trials with toxigenic B. cereus was investigated. Toxigenic and toxin deletion mutants of B. cereus and two probiotic strains (Paciflor and Toyocerin) were examined for bacterial attachment, cytotoxicity and ability to induce nitric oxide as markers of toxicity. Both cytotoxicity and production of nitric oxide were detected in wild-type toxigenic strains and the Paciflor probiotic strain but not Toyocerin. Attachment of B. cereus was low (less than 1%) in all strains. Discrimination between toxigenic B. cereus and the probiotic strains was possible semi-quantitatively via dilution. Despite cytotoxicity in vitro, challenge experiments using 10(8)-10(9) spores of the toxigenic B. cereus NVH75/95 in weaned piglets did not induce diarrhoea or intestinal lesions. Thus, the pig small intestinal epithelial intestinal cell line PSI is appropriate for identification of potential toxicity in B. cereus strains and sets a low threshold for risk of enterotoxicity to humans.

Tylosema esculentum extractives and their bioactivity.

The investigation of Tylosema esculentum (Morama) husks, cotyledons, and tuber yielded griffonilide 2, compound 1, griffonin 3, gallic acid 4, protocatechuic acid 5, ß-sitosterol 6, behenic acid 7, oleic acid 8, sucrose 9, 2-O-ethyl-α-D-glucopyranoside 10, kaempferol 11 and kaempferol-3-O-ß-D-glucopyranoside 12. The structures of the isolates were determined by NMR, HR-TOF EIMS, IR and UV-vis spectroscopy, and by comparison with literature data. The husk EtOAc and n-butanol extracts demonstrated >90% DPPH radical scavenging activity at concentrations of 25, 50 and 250 µg/mL. Furthermore the husk extracts showed higher total phenolic content (233 mg GAE/g). The extractives exhibited minimum inhibitory quantities of 50-100 µg or no activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans. The tuber extracts were inactive against Caco-2 and Hela cell lines, while the husk extracts showed low activity against Caco-2 and Vero cell line with IC(50) values >400 µg/mL. The GC-MS analysis showed the beans and tuber non-polar (n-hexane) extracts major constituents as fatty acids.

Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection.

Tylosema esculentum (marama) beans and tubers are used as food, and traditional medicine against diarrhoea in Southern Africa. Rotaviruses (RVs) are a major cause of diarrhoea among infants, young children, immunocompromised people, and domesticated animals. Our work is first to determine anti-RV activity of marama bean and tuber ethanol and water extracts; in this case on intestinal enterocyte cells of human infant (H4), adult pig (CLAB) and adult bovine (CIEB) origin. Marama cotyledon ethanolic extract (MCE) and cotyledon water extract (MCW) without RV were not cytotoxic to all cells tested, while seed coat and tuber extracts showed variable levels of cytotoxicity. Marama cotyledon ethanolic and water extracts (MCE and MCW, resp.) (≥0.1 mg/mL), seed coat extract (MSCE) and seed coat water extract (MSCW) (0.01 to 0.001 mg/mL), especially ethanolic, significantly increased cell survival and enhanced survival to cytopathic effects of RV by at least 100% after in vitro co- and pre-incubation treatments. All marama extracts used significantly enhanced nitric oxide release from H4 cells and enhanced TER (Ω/cm(2)) of enterocyte barriers after coincubation with RV. Marama cotyledon and seed coat extracts inhibited virion infectivity possibly through interference with replication due to accumulation of nitric oxide. Marama extracts are therefore promising microbicides against RV.

Novel and established intestinal cell line models - An indispensable tool in food science and nutrition.

This review presents the applications of intestinal cell models of human and pig origin in food and nutritional sciences and highlights their potential as in vitro platforms for preclinical research. Intestinal cell models are used in studies of bioavailability, adsorption and transport in nutritional or toxicological settings, allergic effects of food components, as well as probiotics and/or host-pathogen gut interactions. In addition, this review discusses the advantages of using specialized and functional cell models over generic cancer-derived cell lines.

The morama bean (Tylosema esculentum): a potential crop for southern Africa.

The morama bean is an underutilized leguminous oilseed native to the Kalahari Desert and neighboring sandy regions of Botswana, Namibia, and South Africa (Limpopo, North-West, Gauteng, and Northern Cape provinces), and forms part of the diet of the indigenous population in these countries. It is also known as gemsbok bean, moramaboontjie, elandboontjie, braaiboonjie, marama, marumana, tsi, tsin, gami, and ombanui. It is reported as an excellent source of good quality protein (29-39%); its oil (24-48%) is rich in mono- and di-unsaturated fatty acids and contains no cholesterol. Morama is a good source of micronutrients such as calcium, iron, zinc, phosphate, magnesium, and B vitamins including folate. It is also reported to be a potential source of phytonutrients including phenolic compounds (e.g., tannins), trypsin inhibitors, phytates, and oligosaccharides, components which have been shown in other foods to contribute to health in particular, prevention of noncommunicable diseases such as cardiovascular diseases, diabetes, and some cancers. From a nutritional and health perspective, the morama bean has potential commercial value as a cash crop and value-added products, particularly in the communities where it is found.

Pancreatic beta cell lines and their applications in diabetes mellitus research.

During the past 30 years great effort has been put into establishing an insulin-secreting beta cell line that retains normal regulation of insulin secretion, but only few of these attempts have been successful. To overcome the limited availability of primary beta cells and to include the principles of the 3Rs into the field of diabetes mellitus research, numerous investigators used X-rays or viruses to induce insulinomas, in vitro transformation, derivation of cells from transgenic mice or even non-islet cells to produce immortalised beta cell lines. The most widely used insulin-secreting cell lines are RIN, HIT, MIN, INS-1 and TC cells. These cells produce insulin and small amounts of glucagon and somatostatin. Some of them are only poorly responsive to glucose, others respond to glucose well, but their concentration-dependence curve is markedly shifted to higher sensitivity. Despite problems associated with beta cell cultures, these cell lines have provided some valuable information about physiological processes. However, an urgent need to establish a "normal" beta cell line of human or pig origin remains.

Functional cell models of the gut and their applications in food microbiology--a review.

Animal experimentation has a long tradition for risk assessment of new drugs before they reach the clinic. To reduce expensive animal experimentation, attempts have been made to build inexpensive and convenient intestinal functional cell models to study toxicity and bioavailability of new substances along with providing relevant models to study interactions between the host, pathogens and intestinal microflora. We review the available cell lines and models of the intestine and their potential uses. Tumor derived cell lines such as Caco-2, T84 and HT-29 are widely used despite many drawbacks, which are discussed with respect to complexity of the gut, where various cell types interact with commensal microbiota and gut-associated lymphoid tissue. To address this complexity, 3D models of human and animal gut represent a promising in vitro system to mimic in vivo situation without the use of transformed cell lines.

Circulating nucleic acids as possible damage-associated molecular patterns in different stages of renal failure.

Chronic renal failure (CRF) is a condition associated with the risk of cardiovascular complications. Systemic inflammatory response, initiated by the pathogen-associated molecular-pattern (PAMP) molecules, exerts many similarities with the damage-associated molecular-pattern (DAMP) molecule-induced systemic response. Up to now, a number of DAMP molecules were identified. We hypothesized that the available circulating nucleic acids, acting as DAMPs, may modulate immunoinflammatory reaction in CRF. Patients with the different stages of chronic kidney disease, kidney transplantation, and patients on dialysis were included in the study. Obtained results about higher concentration of circulating ribonucleic acid (RNA), according to the stages of kidney diseases, may contribute to the hypothesis that damaged kidney tissue releases nucleic acids. Circulating RNAs expressed maximal absorbance peak at 270 nm in spectrophotometric scan analysis, which corresponded to polyC, compared to different standard samples. During in vitro conditions, by using the culture of human residential macrophages, circulating RNA isolated from patients with IV-V-stage renal diseases, patients on hemodialysis, and patients who underwent renal transplantation were able to significantly change signal transduction proteins related to inflammation and antiviral response. They significantly increased the intracellular concentration of active nuclear transcription factor nuclear factor kappa B (NF-kappaB), interferon regulatory factors (IRF)-3, and IRF-7 and significantly decreased melanoma differentiation-associated protein-5 (MDA-5) and p38. In this way, it seems that circulating RNA, acting as DAMP, may contribute to the mechanisms of additional inflammatory reaction, possible immune destruction, and decreased antiviral response, related to complications in kidney diseases.

Lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection.

This study aimed to examine the potential antiviral activity of lactic acid bacteria (LAB) using animal and human intestinal and macrophage cell line models of non tumor origin. To this end, LAB strains selected on the basis of previous in vitro trials were co-incubated with cell line monolayers, which were subsequently challenged with rotavirus (RV) and transmissible gastroenteritis virus (TGEV). In order to elucidate the possible mechanism responsible for the antiviral activity, the induction of reactive oxygen species (ROS) release as well as the attachment ability of LAB on the cell lines was investigated. Various strains were found to exhibit moderate to complete monolayer protection against viral RV or TGEV disruption. Highest protection effects were recorded with the known probiotics Lactobacillus rhamnosus GG and Lactobacillus casei Shirota against both RV and TGEV, while notable antiviral activity was also attributed to Enterococcus faecium PCK38, Lactobacillus fermentum ACA-DC179, Lactobacillus pentosus PCA227 and Lactobacillus plantarum PCA236 and PCS22, depending on the cell line and virus combination used. A variable increase (of up to 50%) on the release of NO(-) and H(2)O(2) (ROS) was obtained when LAB strains were co-incubated with the cell lines, but the results were found to be LAB strain and cell line specific, apart from a small number of strains which were able to induce strong ROS release in more than one cell line. In contrast, the ability of the examined LAB strains to attach to the cell line monolayers was LAB strain but not cell line specific. Highest attachment ability was observed with L. plantarum ACA-DC 146, L. paracasei subsp. tolerans ACA-DC 4037 and E. faecium PCD71. Clear indications on the nature of the antiviral effect were evident only in the case of the L. casei Shirota against TGEV and with L. plantarum PCA236 against both RV and TGEV. In the rest of the cases, each interaction was LAB-cell line-virus specific, barring general conclusions. However, it is probable that more than one mechanism is involved in the antiviral effect described here. Further investigations are required to elucidate the underlying mode of action and to develop a cell line model as a system for selection of probiotic strains suited for farm animal applications.

The role of functional foods, nutraceuticals, and food supplements in intestinal health.

New eating habits, actual trends in production and consumption have a health, environmental and social impact. The European Union is fighting diseases characteristic of a modern age, such as obesity, osteoporosis, cancer, diabetes, allergies and dental problems. Developed countries are also faced with problems relating to aging populations, high energy foods, and unbalanced diets. The potential of nutraceuticals/functional foods/food supplements in mitigating health problems, especially in the gastrointestinal (GI) tract, is discussed. Certain members of gut microflora (e.g., probiotic/protective strains) play a role in the host health due to its involvement in nutritional, immunologic and physiological functions. The potential mechanisms by which nutraceuticals/functional foods/food supplements may alter a host's health are also highlighted in this paper. The establishment of novel functional cell models of the GI and analytical tools that allow tests in controlled experiments are highly desired for gut research.

Attachment, invasion, and translocation of Campylobacter jejuni in pig small-intestinal epithelial cells.

Campylobacters are susceptible to environmental conditions such as starvation, temperature, and oxidative stress. Species such as Campylobacter jejuni have developed a number of mechanisms for responding to these conditions. We conducted a study to investigate whether survival of C. jejuni and pathogen-host cell interactions such as adherence, invasiveness, and intraepithelial survival in pig small-intestinal (PSI) epithelial cells are altered in response to starvation, changes in temperature, and atmospheric oxygen concentration. We assessed the ability of C. jejuni to translocate across polarized intestinal epithelial cell monolayers by measuring transepithelial electrical resistance (TER). Following heat stress, we observed loss of C. jejuni culturability but not viability. Heat-stressed C. jejuni adhered efficiently to pig intestinal epithelial cells, but their invasiveness was significantly impaired when compared with unstressed C. jejuni. Prolonged exposure to atmospheric oxygen reduced the ability of C. jejuni to adhere to intestinal epithelial cells, whereas brief exposure increased invasiveness and intraepithelial survival. By comparison, nutrient limitation reduced adherence, invasiveness, and intracellular survival of C. jejuni. Adherence of C. jejuni strongly affected the pig intestinal epithelium, as reflected by a significant decrease in TER of polarized intestinal epithelial cells. No correlation between TER and the translocation capacity of C. jejuni was observed. Additionally, campylobacters were detected in the basal chamber of a functional small-intestinal epithelial cell model at 3 hours post infection, without a significant reduction in the TER value, suggesting transcellular transport of C. jejuni into the body.

Gut health promoting activity of new putative probiotic/protective Lactobacillus spp. strains: a functional study in the small intestinal cell model.

In interaction studies with the host intestine, the use of the appropriate gut functional cell model is essential. Therefore, we examined the protective properties of selected lactobacilli in a newly established intestinal cell model. Bacteria were cocultured with the pig small intestinal epithelial cells (PSIc1) and pig blood monocytes (PoM2) in a functional intestinal cell model. Intercellular intestinal integrity was measured by transepithelial electrical resistance (TER), before and after coculture with selected bacterial strains. All selected bacterial strains showed important gut health promoting activity by: enhancing the intestinal integrity and increasing metabolic activity of intestinal cells. Stimulation of immune response was strain specific. The best stimulants were unidentified lactobacillus strains obtained from fermented food in Africa (PCK87 and 66), followed by Lactobacillus plantarum (PCS26). Their activity was significantly higher (p<0.05) than that of the commercial Lactobacillus casei Shirota strain.

Dideoxy fluoro-ketopyranosyl nucleosides as potent antiviral agents: synthesis and biological evaluation of 2,3- and 3,4-dideoxy-3-fluoro-4- and -2-keto-beta-d-glucopyranosyl derivatives of N(4)-benzoyl cytosine.

The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-4-ulose)-N(4)-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-2-ulose)-N(4)-benzoyl cytosine (13a) and their detritylated analogues 8a and 14a, respectively, is described. Condensation of peracetylated 3-deoxy-3-fluoro-D-glucopyranose (1) with silylated N(4)-benzoyl cytosine, followed by selective deprotection and isopropylidenation afforded compound 2. Routine deoxygenation at position 2', followed by a deprotection-selective reprotection sequence afforded the partially tritylated dideoxy nucleoside of cytosine 6, which upon oxidation of the free hydroxyl group at the 4'-position, furnished the desired tritylated 2,3-dideoxy-3-fluoro ketonucleoside 7a in equilibrium with its hydrated form 7b. Compound 2 was the starting material for the synthesis of the dideoxy fluoro ketopyranonucleoside 13a. Similarly, several subsequent protection and deprotection steps as well as routine deoxygenation at position 4', followed by oxidation of the free hydroxyl group at the 2'-position of the partially tritylated dideoxy nucleoside 12, yielded the desired carbonyl compound 13a in equilibrium with its hydrated form 13b. Finally, trityl removal from 7a/b and 13a/b provided the unprotected 2,3-dideoxy-3-fluoro-4-keto and 3,4-dideoxy-3-fluoro-2-ketopyranonucleoside analogues 8a and 14a, in equilibrium with their gem-diol forms 8b and 14b. None of the compounds showed inhibitory activity against a wide variety of DNA and RNA viruses at subtoxic concentrations, except 7a/b that was highly efficient against rotavirus infection. Nucleoside 7a/b also exhibited cytostatic activity against cells of various cancers. BrdU-cell cycle analysis revealed that the mechanism of cytostatic activity may be related to a delay in G1/S phase and initiation of programmed cell death.