Biocomposites of nanofibrillated cellulose, polypyrrole, and silver nanoparticles with electroconductive and antimicrobial properties.

In this work, flexible and free-standing composite films of nanofibrillated cellulose/polypyrrole (NFC/PPy) and NFC/PPy-silver nanoparticles (NFC/PPy-Ag) have been synthesized for the first time via in situ one-step chemical polymerization and applied in potential biomedical applications. Incorporation of NFC into PPy significantly improved its film formation ability resulting in composite materials with good mechanical and electrical properties. It is shown that the NFC/PPy-Ag composite films have strong inhibition effect against the growth of Gram-positive bacteria, e.g., Staphylococcus aureus. The electrical conductivity and strong antimicrobial activity makes it possible to use the silver composites in various applications aimed at biomedical treatments and diagnostics. Additionally, we report here the structural and morphological characterization of the composite materials with Fourier-transform infrared spectroscopy, atomic force microscopy, and scanning and transmission electron microscopy techniques.

Pinoresinol stimulates keratinocyte proliferation and downregulates TNF-α secretion in peripheral blood mononuclear cells: An experimental in vitro study.

Background and Aims: Natural coniferous resins are used in traditional medicine for the treatment of skin wounds. Coniferous wood resins ("callus" resin) are a mixture of abietic (resin) acids, lignans such as pinoresinol, and p-coumaric acid. The wound-healing properties of resins are thought to be related to their antimicrobial properties, but also to their effects on cell proliferation and inflammation. The purpose of this study was to identify and investigate the effects of novel aqueous dispersions of resin and its main components in the proliferation of human primary keratinocytes in vitro and in the expression of proinflammatory cytokines in human peripheral blood mononuclear cells. Methods: The proliferation studies were performed under low and high calcium conditions with or without added growth stimulators at the time points of 2 and 6 days using AlamarBlue Cell Viability Reagent. The cytokine release assay was carried out by incubating the cells with the test articles for 18 h, after which the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and IL-8 were measured in the supernatant by enzyme-linked immunosorbent assay. Results: Resin and the purified lignan PINO, but not p-coumaric acid or abietic acid (industrial tall oil rosin), enhanced the proliferation of human keratinocytes in vitro and inhibited the expression of TNF-α, and to a lesser extent the expression of IL-1ß in peripheral blood mononuclear cells. Conclusions: In this study, novel aqueous dispersions of spruce resin were used to investigate the effects of main resin components on keratinocyte proliferation and on the expression of key proinflammatory cytokines known to be associated with chronic wounds. The observations suggest that lignans, such as PINO, but not resin acids, are the components of resins that mediate the proliferative and TNF-α-suppressing effects. Lignans including PINO were identified as novel potential compounds in the treatment of chronic skin ulcers.

An efficient Pulsed-Field Gel Electrophoresis (PFGE) method for typing autolytic Lacticaseibacillus rhamnosus strains.

Species of lactic acid bacteria, due to their versatile metabolism, are commonly used in food and feed products, both as technological starters and as health- and welfare-promoting agents. Correct strain identification in microbe-containing products is vital, and the Pulsed-Field Gel Electrophoresis (PFGE) typing method is considered the 'gold standard' for this purpose. This typing technique is widely used in molecular epidemiology, especially for the early detection of emerging isolates with food-safety implications, for outbreak surveillance, and for infection control. The autolytic behavior that we encountered when typing Lacticaseibacillus rhamnosus strains using the PFGE technique led us to modify the current method used for typing lactic acid bacteria. This study describes a PFGE method for the molecular typing of autolytic members of the lactic acid bacteria.•An efficient method for overcoming DNA degradation during PFGE analysis for typing Lacticaseibacillus rhamnosus strains is described.•The method described herein could be considered for typing autolytic lactic acid bacteria.

Antimicrobial activity of weak acids in liquid feed fermentations, and its effects on yeasts and lactic acid bacteria.

BACKGROUND: The efficacy of weak organic acids in the control of yeasts in pig liquid feed was studied taking into account the effects on lactic acid bacteria (LAB) responsible for beneficial fermentation. RESULTS: The yeast population in pig liquid feed was taxonomically identified. Kazachstania exigua, Debaryomyces hansenii and Pichia deserticola dominated the fermentation in liquid feed and whey. Pichia deserticola was found in whey and foaming liquid feed and dominated the fermentation after incubation. The sensitivity of the isolates against weak acids was measured in culture medium as well as in fermented and non-fermented liquid feed. Formic acid and potassium sorbate successfully reduced the growth of yeasts in all media without interfering with LAB development. Both of these organic acids showed an increased antifungal effect when used in liquid feed fermented by a Lactobacillus plantarum strain. CONCLUSION: The loss of energy, reduced palatability and other practical problems due to the high growth of yeasts in fermented liquid diets can be reduced by organic acids without affecting lactic acid fermentation.

Effect of bovine lactoferrin on the internalization of coagulase-negative staphylococci into bovine mammary epithelial cells under in-vitro conditions.

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were of Staphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. cohnii urealyticus; two strains of Staph. aureus were used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that of Staph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

Comparative study of sugar fermentation and protein expression patterns of two Lactobacillus plantarum strains grown in three different media.

A comparative study of two strains of Lactobacillus plantarum (REB1 and MLBPL1) grown in commercial medium (MRS broth), cucumber juice, and liquid pig feed was performed to explore changes to the metabolic pathways of these bacteria, using a proteomics approach (two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry) combined with analyses of fermentable sugars and fermentation end products. The protein expression showed that even with an excess of glucose in all media, both strains could metabolize different carbohydrates simultaneously and that hexoses could also be used via a phosphoketolase pathway with preferential expression in liquid feed. Sugar analyses showed that the fermentation of sugars was homolactic for all media, with some heterolactic activity in liquid feed, as shown by the production of acetate. Cucumber juice (the medium with the highest glucose content) showed the lowest hexose consumption (10%), followed by liquid feed (33%) and MRS broth (50%). However, bacterial growth was significantly higher in cucumber juice and liquid feed than in MRS broth. This discrepancy was due to the growth benefit obtained from the utilization of the malate present in cucumber juice and liquid feed. Despite different growth conditions, the synthesis of essential cellular components and the stress response of the bacteria were unaffected. This study has improved our understanding of the mechanisms involved in the growth performance of an appropriate lactic acid bacterium strain to be used for food and feed fermentation, information that is of crucial importance to obtain a high-quality fermented product.

Antimalarial Activity of Croton macrostachyus Stem Bark Extracts against Plasmodium berghei In Vivo.

There is an increasing need for innovative drug and prophylaxis discovery against malaria. The aim of the present study was to test in vivo antiplasmodial activity of Croton macrostachyus H. (Euphorbiaceae) stem bark extracts from Kenyan folkloric medicine. Inbred Balb/c mice were inoculated with erythrocytes parasitized with Plasmodium berghei (ANKA). Different doses (500, 250, and 100 mg/kg) of C. macrostachyus ethyl acetate, methanol, aqueous, and isobutanol extracts were administrated either after inoculation (Peters' 4-day suppressive test) or before inoculation (chemoprotective test) of the parasitized erythrocytes. All the extracts showed significant suppression of parasitemia compared to control (p < 0.001): for the ethyl acetate extract in the range of 58-82%, for the methanol extract in the range of 27-68%, for the aqueous extract in the range of 24-72%, and for the isobutanol extract in the range of 61-80%. Chemoprotective effect was significant (p < 0.001) and the suppression caused by the ethyl acetate extract was between 74 and 100%, by the methanol extract between 57 and 83%, and by the isobutanol extract between 86-92%. The study showed that it is possible to inhibit the growth of the parasites by various stem bark extracts of C. macrostachyus in Balb/c mice supporting the folkloric use of the plant against malaria.

Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations.

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium highly adapted to a wide variety of environments and widely used in food and feed fermentations. Proteomes of two strains of L. plantarum, one isolated from spontaneously fermented cereal-based feed (strain REB1), and the other from white cabbage (strain MLBPL1), were studied to elucidate the strain-specific variation and the physiological changes occurring between the growth (lag, early-exponential, late-exponential and early-stationary) phases of this bacterium when cultivated in a standard rich medium. A total of 231 protein spots were identified by LC-MS/MS. These proteins showed that strain MLBPL1 had more proteins with growth phase-dependent expression than REB1, which possesses a more constant expression profile. The proteins with growth phase-dependent expression in REB1 and MLBPL1 were mainly associated with energy metabolism (glycolysis, phosphoketolase pathway and ribose metabolism), all having preferential expression in the early-exponential phase, confirming the use of different carbohydrates simultaneously. Indication of energy production was also seen in lag and early-stationary phases.

Tetracycline susceptibility of the ingested Lactobacillus acidophilus LaCH-5 and Bifidobacterium animalis subsp. lactis Bb-12 strains during antibiotic/probiotic intervention.

We investigated the effects of oral therapy with doxycycline, a tetracycline group antibiotic, on the gastrointestinal (GI) survival and tetracycline susceptibility of probiotic strains Lactobacillus acidophilus LaCH-5 and Bifidobacterium animalis subsp. lactis Bb-12. In addition, the influence of doxycycline therapy on the diversity of the predominant faecal microbiota was evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Faecal samples from the antibiotic group (receiving antibiotics and probiotics) and the control group (receiving probiotics only) were analysed for anaerobically and aerobically growing bacteria, bifidobacteria and lactic acid bacteria as well as for the dominant microbiota. Although doxycycline consumption did not have a large impact on GI survival of the probiotics, it had a detrimental effect on the bifidobacteria and on the diversity of the dominant faecal microbiota. A higher proportion of tetracycline-resistant anaerobically growing bacteria and bifidobacteria was detected in the antibiotic group than in the control group. Several antibiotic group subjects had faecal B. animalis subsp. lactis Bb-12-like isolates with reduced tetracycline susceptibility. This was unlikely to be due to the acquisition of novel tetracycline resistance determinants, since only tet(W), which is also present in the ingested B. animalis subsp. lactis Bb-12, was found in the resistant isolates. Thus, concomitant ingestion of probiotic L. acidophilus LaCH-5 and B. animalis subsp. lactis Bb-12 with the antibiotic did not generate a safety risk regarding the possible GI transfer of tetracycline resistance genes to the ingested strains.

Toxicological profile of cereulide, the Bacillus cereus emetic toxin, in functional assays with human, animal and bacterial cells.

Some strains of the endospore-forming bacterium Bacillus cereus produce a heat-stable ionophoric peptide, cereulide, of high human toxicity. We assessed cell toxicity of cereulide by measuring the toxicities of crude extracts of cereulide producing and non-producing strains of B. cereus, and of pure cereulide, using cells of human, animal and bacterial origins. Hepatic cell lines and boar sperm, with cytotoxicity and sperm motility, respectively, as the end points, were inhibited by 1 nM of cereulide present as B. cereus extract. RNA synthesis and cell proliferation in HepG2 cells was inhibited by 2 nM of cereulide. These toxic effects were explainable by the action of cereulide as a high-affinity mobile K+ carrier. Exposure to cereulide containing extracts of B. cereus caused neither activation of CYP1A1 nor genotoxicity (comet assay, micronucleus test) at concentrations below those that were cytotoxic (0.6 nM cereulide). Salmonella typhimurium reverse mutation (Ames) test was negative. Exposure of Vibrio fischeri to extracts of B. cereus caused stimulated luminescence up to 600%, independent on the presence of cereulide, but purified cereulide inhibited the luminescence with an IC(50% (30 min)) of 170 nM. Thus the luminescence-stimulating B. cereus substance(s) masked the toxicity of cereulide in B. cereus extracts to V. fischeri.

Antimicrobial and cytotoxic knotwood extracts and related pure compounds and their effects on food-associated microorganisms.

Knotwood or bark extracts prepared from 30 species of hard and soft wood trees as well as selected pure compounds (lignans, stilbenes and flavonoids) were assayed for their antimicrobial activity against a battery of both gram positive and negative bacteria, yeasts, and filamentous fungi (Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Lactobacillus plantarum, Escherichia coli, Salmonella infantis, Pseudomonas fluorescens, Candida albicans, Saccharomyces cerevisiae, Aspergillus fumigatus and Penicillium brevicompactum). By far the most consistent antibacterial and antifungal properties were associated with extracts of Pinus species. These extracts showed also cytotoxicity against a mouse hepatoma cell line. Both antimicrobial and cytotoxic properties correlated with the stilbene content of the extracts. Purified stilbenes showed the most consistent antimicrobial and cytotoxic activities, while purified lignans had marginal effects, only. The results suggest that stilbenes account both for the antimicrobial and cytotoxic properties of Pinus knotwood extracts.

Development of a fermented quinoa-based beverage.

Quinoa is a crop that originated from the Andes. It has high nutritional value, outstanding agro-ecological adaptability, and low water requirements. Quinoa is an excellent crop alternative to help overcome food shortages, and it can also have a role in the prevention of developed world lifestyle diseases, such as type-2 diabetes, cardiovascular diseases, osteoporosis, inflammatory and autoimmune diseases, etc. In order to expand the traditional uses of quinoa and to provide new, healthier and more nutritious food products, a fermented quinoa-based beverage was developed. Two quinoa varieties (Rosada de Huancayo and Pasankalla) were studied. The fermentation process, viscosity, acidity, and metabolic activity during the preparation and storage of the drink were monitored, as well as the preliminary organoleptic acceptability of the product. The drink had viable and stable microbiota during the storage time and the fermentation proved to be mostly homolactic. Both quinoa varieties were suitable as base for fermented products; Pasankalla, however, has the advantage due to higher protein content, lower saponin concentration, and lower loss of viscosity during the fermentation process. These results suggest that the differences between quinoa varieties may have substantial effects on food processes and on the properties of final products. This is a factor that should be taken into account when planning novel products based on this grain.

Pulsed-field gel electrophoresis typing of Listeria monocytogenes isolated in two Finnish fish farms.

The aim of this study was to find sources of Listeria monocytogenes contamination in fish products from a fish farm. The occurrence of L. monocytogenes also was compared in two freshwater fish farms with different types of fishponds. Samples collected from chilled rainbow trout (Oncorhynchus mykiss) and the slaughterhouse environment did not contain L. monocytogenes, but Listeria innocua was found in two samples from the slaughterhouses. Ten isolates of L. monocytogenes were discovered in sediment and water samples from farming tanks and earth ponds. Further characterization by serovar revealed the same serovar (1/2a) for all the isolates. Pulsed-field gel electrophoresis was used to divide the isolates into five different pulsotypes, three of which have been identified previously in fish products on the retail market. This finding supports the assumption that the primary production, and probably the raw fish, is a source of Listeria contamination in fish products. Some of the isolates were associated with a certain type of fishpond, indicating the need for hygienic analysis of the suitability of different types of farming ponds.

Antimicrobial Activity of Croton macrostachyus Stem Bark Extracts against Several Human Pathogenic Bacteria.

In Kenya, leaves and roots from Croton macrostachyus are used as a traditional medicine for infectious diseases such as typhoid and measles, but reports on possible antimicrobial activity of stem bark do not exist. In this study, the antibacterial and antifungal effects of methanol, ethyl acetate and butanol extracts, and purified lupeol of C. macrostachyus stem bark were determined against important human gram-negative pathogens Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, and Enterobacter aerogenes, gram-positive Listeria monocytogenes, and a fungus Candida albicans. The most promising broad scale antimicrobial activity against all the studied pathogens was shown by the ethyl acetate extract. The ethyl acetate extract induced the zone of inhibition between 10.1 ± 0.6 mm and 16.0 ± 1.2 mm against S. typhi, E. coli, K. pneumoniae, E. aerogenes, and L. monocytogenes with weaker antimicrobial activity against C. albicans (zone of inhibition: 5.6 ± 1.0 mm). The antibiotic controls (amoxicillin, ciprofloxacin, ampicillin, benzylpenicillin, clotrimazole, and cefotaxime) showed antimicrobial activity with zones of inhibition within 13.4 ± 0.7-22.1 ± 0.9 mm. The ethyl acetate extract had MIC in the range of 125-250 mg/mL against all the studied bacteria and against C. albicans MIC was 500 mg/mL. The present results give scientific evidence and support the traditional use of C. macrostachyus stem bark as a source for antimicrobials. We show that C. macrostachyus stem bark lupeol is a promising antimicrobial agent against several important human pathogens.

Regulating the safety of probiotics--the European approach.

This article reviews pertinent legislation regulating the safety of probiotics within the European Union (EU). Currently available probiotic organisms and known issues regarding their safety are briefly summarised. While most of the species and genera, particularly lactobacilli and bifidobacteria are apparently safe, certain micro-organisms may be problematic; particularly the enterococci, which are associated with nosocomial infections and harbour transmissible antibiotic resistance determinants. At present, probiotic human foods are not governed under specific EU regulatory frameworks, although the Novel Food Regulation EU 258/97, could be relevant in some specific cases. However, microbial feed additives (regulated by Council Directive 70/254/EEC and in accordance with guidelines of the Scientific Committee on Animal Nutrition (SCAN)) are subjected to detailed safety assessment with the intention of ensuring that they are innocuous to target animals, users and consumers. Particular attention is focused on the presence of transmissible antibiotic resistance markers, and to the potential for production of harmful metabolites. The guidelines do not differentiate between species and strains with long histories of safe use and other micro-organisms. This has caused some concern regarding overregulation, if the same principles are to be applied to probiotics or starter cultures intended for human food use. Accordingly, SCAN has launched an initiative towards a "Qualified Perception of Safety" (QPS) concept, which would allow strains with established safety status to enter the market without extensive testing requirements. It is likely that the European Food Safety Authority (EFSA) will play a central role in the regulation of both human and animal probiotics.

Genome Sequence of Lactococcus lactis subsp. cremoris Mast36, a Strain Isolated from Bovine Mastitis.

The genome sequence of Lactococcus lactis subsp. cremoris Mast36, isolated from bovine mastitis, is reported here. This strain was shown to be able to grow in milk and still possess genes of vegetable origin. The genome also contains a cluster of genes associated with pathogenicity.

Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems.

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.

Surfactins in natto: the surfactin production capacity of the starter strains and the actual surfactin contents in the products.

Surfactin-type lipopeptides are suspected of being implicated in the rare food poisonings caused by Bacillus species outside the Bacillus cereus cluster. In order to get information on surfactin levels in actual human foods, bacilli from three commercial samples of a Japanese traditional bean product, natto, were isolated in order to clarify their potential to produce the suspect lipopeptides. The isolated bacilli were characterized as Bacillus subtilis. They were ß-hemolytic and gave a positive signal in the PCR screen for genes associated with surfactin production, and their culture extracts were cytotoxic to boar sperm cells. Organic extracts of both Bacillus cultures and the natto samples were analyzed for their surfactin content using ultrahigh-performance liquid chromatography with high-resolution mass spectrometry. All the strains proved to be surfactin producers (15 to 39 µg/ml culture medium); the natto samples contained as much as 2.2 mg g(-1) of surfactins. This means that consumers can ingest at least approximately 80 to 100 mg of surfactins per single 50-g natto serving apparently without suffering any ill effects, indicating a very low human toxicity.

Characterization of Lactococcus lactis isolates from bovine mastitis.

Lactococcus lactis is a widely used mesophilic dairy starter and has been included in the Qualified Presumption of Safety (QPS) list of the European Food Safety Authority. However, it is increasingly found as the cause of human or animal infections, such as bovine mastitis, probably due to the improvement of the identification of the infective microorganisms. Since there are some grounds to suspect that at least certain variants of L. lactis may cause animal or human diseases, it is important to properly identify the differences between the strains associated with infections and the safe starter strains. Bovine mastitis isolates and dairy starter strains were genotypically characterized and clustered by the 16S rRNA gene sequence and RAPD-PCR fingerprint patterns, and phenotypically characterized by their tolerance to different stress conditions typically found in the intestinal tract of mammals, the carbohydrate- and antibiotic resistance profile, as well as the in vitro adhesion capacity to udder epithelial cells. Genotypically, there were no differences between the mastitis isolates and the dairy starter strains. However, there were clear phenotypic distinctions between mastitis isolates and typical starter strains, the former showing an increased tolerance to temperature, lysozyme, bile salts, pH and antibiotics, as well as improved carbohydrate fermentation capacity, and in vitro adhesion to udder epithelial cells. Although these differences might not be considered as actual virulence factors, they improve the ability of the strain to survive in the body of homeothermic animals and, interestingly, are also typical properties associated with potential probiotic strains.

The antimicrobial effects of wood-associated polyphenols on food pathogens and spoilage organisms.

The antimicrobial effects of the wood-associated polyphenolic compounds pinosylvin, pinosylvin monomethyl ether, astringin, piceatannol, isorhapontin, isorhapontigenin, cycloXMe, dHIMP, ArX, and ArXOH were assessed against both Gram-negative (Salmonella) and Gram-positive bacteria (Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus) and yeasts (Candida tropicalis, Saccharomyces cerevisiae). Particularly the stilbenes pinosylvin, its monomethyl ether and piceatannol demonstrated a clear antimicrobial activity, which in the case of pinosylvin was present also in food matrices like sauerkraut, gravlax and berry jam, but not in milk. The destabilization of the outer membrane of Gram-negative microorganisms, as well as interactions with the cell membrane, as indicated by the NPN uptake and LIVE/DEAD viability staining experiments, can be one of the specific mechanisms behind the antibacterial action. L. monocytogenes was particularly sensitive to pinosylvin, and this effect was also seen in L. monocytogenes internalized in intestinal Caco2 cells at non-cytotoxic pinosylvin concentrations. In general, the antimicrobial effects of pinosylvin were even more prominent than those of a related stilbene, resveratrol, well known for its various bioactivities. According to our results, pinosylvin could have potential as a natural disinfectant or biocide in some targeted applications.