Safety assessment of Lactobacillus strains: presence of putative risk factors in faecal, blood and probiotic isolates

The widespread use of immunosuppressive therapy and antimicrobial agents as well as novel probiotics without a long history of safe use has increased requirements for safety assessment of probiotic lactobacilli. In this study 44 faecal, 52 blood and 15 probiotic isolates (including 3 dairy strains) of Lactobacillus were assayed for their adhesion properties to extracellular matrix proteins and mucus, hemolysis, ability to avoid the induction of respiratory burst in peripheral blood mononucleocytes (PMN) and resistance to human serum. Among tested strains adhesion to collagen, fibrinogen and mucus was isolate-specific and no statistically significant differences were obtained between faecal, blood and probiotic isolates. However, blood isolates showed a trend for higher adhesion to mucus than probiotic strains (P=0.07). Probiotic strains induced lower respiratory burst in PMN when compared to the blood isolates (P<0.05). Moreover, there was a positive correlation between adhesion to collagen and induction of respiratory burst for faecal isolates (P<0.05). In the determination of serum resistance, probiotic strains showed a trend for lower sensitivity to human serum-mediated killing when compared to the faecal isolates (P=0.07). None of the measurable virulence factors were found to be present at statistically higher level in clinical blood isolates when compared to faecal and/or probiotic isolates indicating that these factors do not cause risk when safety of probiotics is considered. However, the significance of adhesion to mucus, low induction of respiratory burst in PMN and resistance to human serum-mediated killing may need further evaluation in experimental animal models and in epidemiological data.     

Specific Bifidobacterium strains isolated from elderly subjects inhibit growth of Staphylococcus aureus

Cell-free, pH-controlled supernatants of thirty-eight Bifidobacterium strains isolated from healthy elderly subjects were subjected to antimicrobial activity assay. Bioluminescent indicator strains Staphylococcus aureus RN4220, Escherichia coli K-12, and Salmonella enterica serovar Typhimurium ATCC 14028 were used as targets of antimicrobial activity. The effect of nutrient depletion on the inhibition was eliminated with spent-culture controls. Three out of thirty-eight Bifidobacterium strains were capable of inhibiting the growth of S. aureus. The inhibition was equal to 23.2+/-19.1% to 50.4+/-26.7% of the inhibition caused by 50 IU/ml nisin. Reuterin-producing positive strain Lactobacillus reuteri SD2112 was capable of 86.0+/-24.6% inhibition, but Bifidobacterium lactis Bb-12, a known probiotic strain, showed no inhibition. None of the strains was capable of inhibiting the growth of E. coli or S. enterica. The observed inhibition by bifidobacteria was related to hydrogen peroxide formation and possible production of heat-stable proteinaceous compounds. The results suggest that production of antimicrobial substances other than organic acids is not common among Bifidobacterium strains typical of elderly subjects. However, specific strains were identified which showed considerable inhibitory activity against S. aureus.     

Adhesion and aggregation properties of probiotic and pathogen strains

Autoaggregation has been correlated with adhesion, which is known to be a prerequisite for colonization and infection of the gastrointestinal tract by many pathogens. The coaggregation properties of probiotic strains with pathogens as well as their ability to displace pathogens are of importance for therapeutic manipulation of the aberrant intestinal microbiota. Consequently, the ability to aggregate and coaggregate are desirable properties for probiotics in health-promoting foods. Aggregation assays and bacterial adhesion to hydrocarbons (BATH test) demonstrated significant differences in cell surface properties among the tested commercial probiotic strains. Hydrophobicity increased when the cells were heat-inactivated. All probiotic strains tested showed aggregation abilities with the pathogen strains tested, but the results were strain-specific and dependent on time and incubation conditions. Our results indicate that the ability to autoaggregate, together with cell-surface hydrophobicity and coaggregation abilities with pathogen strains can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use.     

Kinetics of adsorption and desorption of aflatoxin B1 by viable and nonviable bacteria

The reactions involved in the binding (adsorption) and release (desorption) of aflatoxin B1 (AFB1) to and from the surface of bacteria were investigated. Viable and heat-killed Lactobacillus rhamnosus GG, L. rhamnosus LC-705, and Propionibacterium freudenreichii subsp. shermanii JS were incubated in phosphate-buffered saline containing variable concentrations (0.0017 to 13.3 microg/ml) of AFB1. The relationship between the bacterial surface hydrophobicity and the AFB1 adsorption affinity was also investigated. A linear relationship was observed between the specific rate of AFB1 adsorption and the AFB1 concentration for all bacteria. The nature of desorption of adsorbed AFB1 was investigated by repetitive aqueous washes. A linear relationship was observed between the natural log value of the concentration of AFB1 adsorbed and the number of washes for all bacteria studied. The desorption constants were strain-dependent and were lower for heat-killed bacteria than for viable bacteria. Heat treatment appears to alter the surface properties of the bacteria rather than expose new adsorption sites. No correlation was found between the hydrophobicity and the AFB1 adsorption affinity.     

Effects of sample drying and storage,and choice of extraction solvent and analysis method on the yield of birch leaf hydrolyzable tannins

In this study, I investigated the effects of different methods of sample drying and storage, and the choice of extraction solvent and analysis method on the concentrations of 14 individual hydrolyzable tannins (HTs), and insoluble ellagitannins in birch (Betula pubescens) leaves. Freeze- and vacuum-drying of birch leaves were found to provide more reliable results than air- or oven-drying. Storage of leaves at –20C for 3 months before freeze-drying did not cause major changes in tannin content, although levels of 1,2,3,4,6-penta-O-galloylglucose and isostrictinin were altered. Storage of dried leaf material at –20C is preferred because 1 year storage of freeze-dried leaves at 4C and at room temperature decreased the concentration of the pedunculagin derivative, one of the main ellagitannins of birch. Furthermore, storage at room temperature increased the levels of isostrictinin and 2,3-(S)-HHDP-glucose, indicating possible HT catabolism. Of the extraction solvents tested, aqueous acetone was superior to pure acetone, or aqueous or pure methanol. The addition of 0.1% ascorbic acid into 70% acetone significantly increased the yield of ellagitannins, presumably by preventing their oxidation. By comparing the conventional rhodanine assay and the HPLC–ESI-MS assay for quantification of leaf galloylglucoses, the former tends to underestimate total concentrations of galloylglucoses in birch leaf extract. On the basis of the outcomes of all the method and solvent comparisons, their suitability for qualitative and quantitative analysis of plant HTs is discussed, emphasizing that each plant species, with its presumably unique HT composition, is likely to have a unique combination of ideal conditions for tissue preservation and extraction.     

Immunological Memory of Mountain Birches: Effects of Phenolics on Performance of the Autumnal Moth Depend on Herbivory History of Trees

Plants have been suggested to have an immunological memory comparable to animals. The evidence for this, however, is scarce. In our study with the mountain birch—Epirrita autumnata system, we demonstrated that birches exposed as long as 5 yr to feeding of E. autumnata larvae (delayed induced resistance, DIR), responded more strongly to a new challenge than trees without an herbivory history. Pupal weights remained lower, and the duration of the larval period was prolonged in the DIR trees, although immunity, measured as an encapsulation rate, was not affected. We further demonstrated that the effects of birch phenolics on performance of E. autumnata were different in the exposed (DIR) trees from naive control trees, although we found only one significant change in chemistry. The quercetin:kaemferol ratio was increased in DIR trees, suggesting that herbivory caused oxidative stress in birches. In DIR trees, phenolics, especially hydrolyzable tannins (HTs), affected pupal weights negatively, whereas in control trees, the effects were either nonsignificant or positive. HTs also prolonged the duration of the larval period of females, whereas peroxidase (POD) activity prolonged that of males. We suggest that the causal explanation for the induced resistance was an enhanced oxidation of phenolic compounds from the DIR trees in the larval digestive tract. Phenolic oxidation produces semiquinones, quinones, free radicals, and ROS, which may have toxic, antinutritive, and/or repellent properties against herbivores.     

BET Inhibition Upregulates SIRT1 and Alleviates Inflammatory Responses

Control of histone acetylation is a part of the epigenetic mechanism that regulates gene expression and chromatin architecture. The members of the bromodomain and extra terminal domain (BET) protein family are a group of epigenetic readers that recognize histone acetylation, whereas histone deacetyl‐ ases such as sirtuin 1 (SIRT1) function as epigenetic erasers. We observed that BET inhibition by the specific inhibitor JQ1 upregulated SIRT1 expression and activated SIRT1. Moreover, we observed that BET inhibition functionally reversed the pro‐inflammatory effect of SIRT1 inhibition in a cellular lung disease model. SIRT1 activation is desirable in many age‐related, metabolic and inflammatory diseases; our results suggest that BET protein inhibition would be beneficial in treatment of those conditions. Most importantly, our findings demonstrate a novel mechanism of SIRT1 activation by inhibition of the BET proteins.     

Carotenoid Composition of Invertebrates Consumed by Two Insectivorous Bird Species

Dietary carotenoids are important pigments, antioxidants, and immune-stimulants for birds. Despite recent interest in carotenoids in bird ecology, we know surprisingly little about the carotenoid content of invertebrates consumed by birds. We compared carotenoid (lutein, β-carotene, and total) concentrations in invertebrates brought to nestlings by two insectivorous passerines, the great tit, Parus major and the pied flycatcher, Ficedula hypoleuca. We also compared carotenoid levels between environments that were either polluted by heavy metals or were not polluted, because the carotenoid-based plumage color of P. major nestlings is affected by environmental pollution. Lepidopterans were the most carotenoid-rich food items and contained the largest proportion of lutein. There were no differences in carotenoid concentrations in the food items of the two bird species but P. major nestlings obtained more carotenoids from their invertebrate diet than F. hypoleuca nestlings because the P. major diet had a higher proportion of lepidopteran larvae. In polluted areas, P. major nestlings consumed lower levels of dietary carotenoids than in unpolluted areas because of temporal differences in caterpillar abundance between polluted and unpolluted sites. Our study suggests that pollution-related difference in nestling plumage color in P. major is related to varying dietary proportion of lutein-rich food items rather than pollution-related variation in insect carotenoid levels.