Novel aqueous oil-in-water emulsions containing extracts of natural coniferous resins are strongly antimicrobial against enterobacteria, staphylococci and yeasts, as well as on bacterial biofilms.

AIMS: The aim of this study was to examine the antimicrobial properties of novel aqueous natural rapeseed oil/saline emulsions containing different soluble components of spruce resin. METHODS AND RESULTS: The composition of aqueous resin emulsions was analysed by GC-MS and their antimicrobial properties were studied with challenge tests and with turbidometric assays. The emulsions were strongly antimicrobial against common Gram-positive and Gram-negative bacteria (including MRSA) as well as common yeasts. Furthermore, they inhibited the biofilm formation and eradicated the microbial biofilms on tested microbes. Characteristic for the emulsions was the presence of oxidized resin acids. Other main components present in emulsions, such as lignans and coumaric acids, were not antimicrobial, when tested separately. CONCLUSIONS: The results indicated that the oxidized resin acids were the antimicrobial components in the emulsions. Also, there appears to be a stoichiometric relationship between the number of resin acid molecules and the number microbe cells in the antimicrobial action. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that these solutions do not contain abietic acid, which is the main allergenic compound in resins, suggests that these solutions would be suitable, well-tolerated antimicrobials for various medical applications. The aqueous formulation will also allow the expansion of the use of these emulsions in from medical applications to the food preservatives and disinfectants.

Pseudomonas M162 confers protection against rainbow trout fry syndrome by stimulating immunity.

AIMS: To evaluate the antagonistic effect of Pseudomonas M162 against Flavobacterium psychrophilum. METHODS AND RESULTS: The antagonistic activity of M162 was tested in vivo and in vitro, and its mode of action examined by siderophore production and immunological responses of rainbow trout (Oncorhynchus mykiss) fry. Pseudomonas M162 inhibited the growth of Fl. psychrophilum in vitro and increased the resistance of the fish against the pathogen, resulting in a relative per cent survival (RPS) of 39·2%. However, the siderophores produced by M162 did not have an inhibitory effect on Fl. psychrophilum. In fish fed with M162, the probiotic colonized the gastrointestinal tract and stimulated peripheral blood leucocyte counts, serum lysozyme activity and total serum immunoglobulin levels after 3 weeks from the start of feeding. CONCLUSIONS: This study showed the potential of Pseudomonas M162 as a probiotic by reducing the mortalities that occurred during an experimental Fl. psychrophilum infection, resulting mainly through the immunostimulatory effects of the bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: Rainbow trout fry syndrome (RTFS) causes high mortalities during the early life stages of the fish's life cycle, partly because their adaptive immunity has not yet fully developed. Thus, immunomodulation by probiotics could be an effective prophylactic method against RTFS.

Pseudomonas sp. M174 inhibits the fish pathogen Flavobacterium psychrophilum.

AIMS: To study the antagonic affect of probiotic Pseudomonas M174 on the fish pathogen Flavobacterium psychrophilum. METHODS AND RESULTS: The ability of Pseudomonas M174 to inhibit the growth of Fl. psychrophilum was examined in iron-sufficient and -deficient media. Possible siderophore production was also investigated. Antagonistic activity was confirmed in disease challenge experiments using a rainbow trout (Oncorhynchus mykiss) model. Adhesion of Pseudomonas M174 to fish surfaces and its ability to stimulate innate immunity was also investigated in vivo. Pseudomonas M174 antagonized Fl. psychrophilum and produced siderophores in vitro. In challenge experiments with Fl. psychrophilum, fish fed with Pseudomonas M174 had lower levels of mortalities than the controls. It was possible to find Pseudomonas M174 in the intestinal content of these fish after feeding and bathing with the probiotic, but probiotic was obtained from the gills only after feeding. Respiratory burst activity was also found to be enhanced in the M174 fed fish. CONCLUSIONS: These results suggest that M174 is a potential probiotic against Fl. psychrophilum and has several modes of action. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotics are a promising alternative to the use of antibiotics in aquaculture and could be a more sustainable disease control method.

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

Fermented pig liquid feed: nutritional, safety and regulatory aspects.

Fermented liquid feed has been lately much investigated in order to compensate the use of antibiotics in pig production. The fermentation process has been claimed to be the reason of the benefits associated with this type of feeding. However, contradictory results have been obtained in feeding trials due to the variable conditions in each experiment. This review focuses on the different factors that would ensure a proper fermentation with all its beneficial effects. In particular, while fermenting a liquid diet with lactic acid bacteria has been shown to improve the quality of feed and to be beneficial to the health of the animals, spontaneously fermented liquid feed appears to be unsafe for the pigs and eventually affects the consumers' safety. Consequently, the use of specific starters or inoculants to ensure the proper fermentation could be a practical solution. The regulatory status of fermented liquid feed in the EU is still unclear, but the use of specific inoculants could be considered as a special case of microbial feed additives.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Characterization of dominant cultivable lactobacilli and their antibiotic resistance profiles from faecal samples of weaning piglets.

AIMS: To examine the lactic acid bacteria flora of weaning piglets, to define the distribution of different lactobacilli species in piglet faecal samples, and to determine the susceptibility phenotype to 11 antibiotic of different families. METHODS AND RESULTS: The faecal samples were taken from piglets with good herd status at 11 and 28 days after weaning. The Lactobacillus isolates (n = 129) from 78 animals housed in pairs in 39 pens were preliminarily identified by their morphology and biochemical characteristics. Partial 16S ribosomal DNA (16S rDNA) was used to identify the isolates to the species level, and RAPD (randomly amplified polymorphism DNA) profiles to differentiate Lactobacillus isolates to the strain level. Based on these studies, 67 strains were selected for antibiotic resistant tests. The most numerous Lactobacillus species found in the piglets was Lactobacillus reuteri (n = 43). Other lactobacilli were L. salivarius (n = 15), L. agilis (n = 4), L. johnsonii (n = 2), L. vaginalis (n = 1), L. mucosae (n = 1) and L. gallinarum (n = 1). All the strains were susceptible to chloramphenicol, ampicillin and gentamicin. Two L. salivarius isolates and two L. reuteri isolates were found to be multiresistant. CONCLUSIONS: This study indicates that the faecal Lactobacillus flora in piglets consists mainly of L. reuteri, L. salivarius and L. acidophilus group lactobacilli, and the distribution of lactobacilli is similar between individuals of the same age and with the same diet. Most of the Lactobacillus isolates tested were sensitive to the antibiotics used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable information on Lactobacillus species distribution and their antibiotic resistance profiles in piglets is obtained.

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project.

An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.

Occurrence of virulence factors among human intestinal enterococcal isolates.

AIMS: The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. METHODS AND RESULTS: The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. CONCLUSIONS: Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Lactobacillus crispatus and its nonaggregating mutant in human colonization trials.

A wild-type Lactobacillus crispatus, showing a cell aggregation phenotype and its spontaneous nonaggregating mutant were compared for their in vitro adhesion properties to human ileal mucus and to a cultured human colonic cell line (Caco2) and for their in vivo colonization and adhesion potential with colonoscopy patients as volunteers in feeding trials. The wild-type strain adhered better to mucus or to Caco2 cells than did the mutant. Altogether, three human trials with the wild type and two with the mutant strain were performed. In two of the trials, the wild type could be recovered from either fecal samples or biopsies taken from the colon, while the mutant strain could not be demonstrated in either of the trials where it was used. The L. crispatus colonies recovered from the trials were often mixed, and several enterococci and lactobacillus strains coaggregating with L. crispatus wild type could be isolated. The results indicate that the surface-mediated properties, such as aggregation, of lactobacilli can have a role in adhesion and colonization.

Fish quality assessment with ion mobility based gas detector.

Measurement of the mixture of extractable volatile compounds of the gills of vendace was used in the fish freshness evaluation (Coregonus albula L.). MGD-1 gas detector was used for the detection of volatile compound profiles of hexane extracts of gills. The measuring principle of MGD-1 is ion mobility distribution and is based on IMCELL technique. A 2.5 microl sample of hexane extract was heated on a ceramic plate and the vapour was carried with a constant flow (2.5 I/min) of active carbon filtered air to the detector. The ion mobility distribution spectrum was measured every 1 second for 2 minutes. For reference, the microbial counts of gills, ATP-breakdown products (K-value) of fish flesh, electrical properties (Torrymeter scores) of fish skin and sensory evaluation scores were measured. The ion mobility distribution profiles of gills changed significantly during the 7 days storage period The findings related to freshness evaluation were supported by the reference methods. There is, however, a seasonal difference in the freshness probably due to seasonal variation of microbial flora. Besides the evaluation of fish quality, ion mobility distribution based technique provides a new analytical tool for food quality assessment.

Physiology and taxonomy of thiobacillus strain TJ330, which oxidizes carbon disulphide (CS2).

A bacterium (strain TJ330) capable of using carbon disulphide (CS2) as its sole energy source in an acidic environment was isolated from a peat biofilter used in experiments to remove CS2 and hydrogen sulphide (H2S) from air. Its physiology and taxonomy are described here. The strain oxidized CS2, H2S and elemental sulphur to sulphate chemolithotrophically. The rate of sulphate production was highest at pH 2. The maximum growth rate constant (micromax) using CS2 as a substrate was 3.9 x 10(-2) h(-1) (generation time 18 h) and the Monod constant (Ks) was 0.97-2.6 micromol l(-1) CS2 (74-198 microg l(-1)), corresponding to an equilibrium with 15-40 ppm CS2 in the headspace. The optimum growth temperature using elemental sulphur as a substrate was 28 degrees C. The strain bears morphological and physiological similarities to Thiobacillus thiooxidans, but the latter is incapable of oxidizing CS2. The strain TJ330 (DSM 8985) showed only 44.2 + 11.8% DNA homology with the type strain T. thiooxidans ATCC 19377, while its homology with T. ferrooxidans ATCC 23270 was 17.1 + 3.4%. The strain TJ 330 represents a high-affinity bacterium which can effectively remove low CS2 concentrations in an acid environment. These properties can be utilized in biotechnological purification applications.

Probiotic bacteria and intestinal health: new methods of investigation.

This paper highlights some new methods in the probiotic research based on the use of colonic biopsies and molecular biological techniques for strain identification.

Probiotics: established effects and open questions.

Human intestinal microflora is a complex ecosystem with hundreds of bacterial species. Its metabolic functions and interactions with the host probably affect the human health and well being, but these effects are extremely difficult to study. However, for about 100 years, the idea of modifying the composition of colonic flora by consuming viable bacteria in order to improve the quality of life and to prevent and treat intestinal disorders has had some popularity. Solid clinical data have usually been lacking to support the health claims associated with these so-called probiotics. The situation, however, is rapidly changing. Competent clinical studies are accumulating, showing that specific probiotic microbes, mainly lactic acid bacteria and bifidobacteria, can alleviate or prevent diverse intestinal disorders and reduce the risk of some intestinal diseases. Some indication of the mechanisms of action can also be deduced from the data available, while rapidly developing molecular biological methods offer new tools to verify the survival of the probiotics in the gut and the subsequent adhesion to mucosae. While development of foods containing probiotic bacteria has a great potential for the food industry and can be expected to positively affect the general health of the population, safety considerations have to be taken into account while introducing new species or strains without a previous history of safe food use.

Toxicity of selected plant volatiles in microbial and mammalian short-term assays.

In this study, several short-term microbial and mammalian in vitro assays were used to evaluate cytotoxicity and genotoxicity of four plant volatiles showing antifungal activity: cinnamaldehyde, carvacrol, thymol and S(+)-carvone. All inhibited viability and proliferation of Hep-2 cells in a dose-dependent manner. IC50 ranged from 0.3 mM (cinnamaldehyde) to 0.7 mM (thymol) in viability tests and from 0.2 mM (carvacrol) to 0.9 mM (carvone) in the proliferation test. The morphological analysis suggested an involvement of apoptosis in the cases of carvone, carvacrol and cinnamaldehyde. At nontoxic doses, carvacrol and thymol increased the number of revertants in the Ames test by 1.5-1.7 times, regardless of metabolic activation. In the SOS-chromotest, none of the four plant volatiles caused DNA damage at non-toxic doses. In the DNA repair test, a marked dose-dependent differential toxicity was observed with carvone and, to a lesser extent, with cinnamaldehyde, while with thymol and carvacrol, this effect was less pronounced. In conclusion, the considered in vitro cytotoxicity assays have shown to be sensitive enough to highlight a variety of toxic effects at the cellular level, which can be rather different between chemically closely related compounds, such as isomers.

The effect of lactose derivatives on intestinal lactic acid bacteria.

Nine strains of lactic acid bacteria were studied for growth and fermentation end products on lactulose, lactitol, and lactobionic acid. In addition, human fecal and biopsy isolates were screened for new potential by probiotic strains utilizing lactose derivatives, and one new isolate of Lactobacillus rhamnosus was enriched. The utilization of lactose derivatives and the effect on the fermentation end products were dependent on strain. Typical mixed-acid fermentations were observed with Lb. rhamnosus and Lactococcus lactis. Microbiota enriched from fecal and biopsy samples using modified MRS medium consisted mainly of enterococci and streptococci. The adhesion of tested strains to Caco-2 cells was not dependent on carbon source. The new Lb. rhamnosus strain VTT E-97800 has potential for further probiotic studies.

The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME).

The aim of the present work was to study five potential probiotic strains (Lactobacillus plantarum, two strains of L. paracasei subsp. paracasei, L. rhamnosus and Bifidobacterium sp.) comparatively in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) in vitro model, and to evaluate this model as a tool in the screening and selection of probiotic bacteria. The impact of the strains on the composition of microbiota and its metabolic activities (production of lactic acid and short-chain fatty acids) was studied. Changes in composition of the microbiota become apparent as a result of probiotic treatment. A marked, but temporary, increase was noted in the number of lactic acid bacteria and bifidobacteria. The profiles of D(-) and L(+) isomers of lactic acid detected in the SHIME after addition of probiotic strains corresponded well to those that are produced in pure culture conditions. The numbers of enterobacteriaceae decreased markedly and those of clostridia detectably during the intervention, while the enterococci tended to increase after the treatment. This pattern was similar in the reactors representing both the small and large intestine in the model. The changes in short-chain fatty acids were small, and no definite trend was observed.

Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption.

Lactobacillus rhamnosus GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption. L. rhamnosus GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.