Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese.

A total of 114 lactic acid bacteria were isolated at one and 21 days of ripening from a traditional raw cow's milk cheese without the addition of starter culture, produced by three artisanal cheese-makers in Azores Island (Pico, Portugal). Identification to species and strain level was accomplished by16S rRNA gene and PFGE analysis. Carbohydrate utilization profiles were obtained with the relevant API kits. Isolates were evaluated according to safety and technological criteria. The most frequently observed genus identified by 16S rRNA sequencing analysis was Enterococcus, whereas API system mostly identified Lactobacillus. The highest percentages of antibiotic resistance were to nalidixic acid (95%), and aminoglycosides (64-87%). All isolates were sensitive to several beta-lactam antibiotics and negative for histamine and DNase production. Gelatinase activity was detected in 49.1% of isolates, 43% were able to degrade casein and 93% were α-hemolytic. Most enterococci presented virulence genes, such as gelE, asaI, ace. Diacetyl production was found to be species dependent and one strain (Leu. citreum) produced exopolysaccharides. Selected strains were further studied for technological application and were found to be slow acid producers in milk and experimental cheeses, a desirable trait for adjunct cultures. Two strains were selected on the basis of technological and safety application as adjunct cultures in cheese production and presented the best cheese aroma and flavor in consumer preference tests. This is the first effort to characterize Pico cheese LAB isolates for potential application as adjunct cultures; the results suggest the potential of two strains to improve the quality of this traditional raw milk product.

Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models.

BACKGROUND AND AIMS: Increased efficiency of energy harvest, due to alterations in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has been implicated in obesity in mice and humans. However, a causal relationship is unproven and contributory variables include diet, genetics and age. Therefore, we explored the effect of a high-fat (HF) diet and genetically determined obesity (ob/ob) for changes in microbiota and energy harvesting capacity over time. METHODS: Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR); faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal energy content (by bomb calorimetry) and microbial composition (by metagenomic pyrosequencing). RESULTS: A progressive increase in Firmicutes was confirmed in both HF-fed and ob/ob mice reaching statistical significance in the former, but this phylum was unchanged over time in the lean controls. Reductions in Bacteroidetes were also found in ob/ob mice. However, changes in the microbiota were dissociated from markers of energy harvest. Thus, although the faecal energy in the ob/ob mice was significantly decreased at 7 weeks, and caecal SCFA increased, these did not persist and faecal acetate diminished over time in both ob/ob and HF-fed mice, but not in lean controls. Furthermore, the proportion of the major phyla did not correlate with energy harvest markers. CONCLUSION: The relationship between the microbial composition and energy harvesting capacity is more complex than previously considered. While compositional changes in the faecal microbiota were confirmed, this was primarily a feature of high-fat feeding rather than genetically induced obesity. In addition, changes in the proportions of the major phyla were unrelated to markers of energy harvest which changed over time. The possibility of microbial adaptation to diet and time should be considered in future studies.

Impact of beneficial bacteria supplementation on the gut microbiota, colony development and productivity of Apis mellifera L.

Honey bees are important pollinators of several crops and ecosystems, having a great ecological and economic value. In Europe, the restricted use of chemicals and therapeutic agents in the beekeeping sector has stimulated the search for natural alternatives with a special focus on gut symbionts. The modulation of the gut microbiota has been recognised as a practical and successful approach in the entomological field for the management of insect-related problems. To date, only a few studies have investigated the effect of bacterial supplementation on the health status of colonies, colony productivity and gut symbionts. To this purpose, a preparation of sugar syrup containing bifidobacteria and lactobacilli isolated from bee gut was sprayed on the frames of an apiary located in open field once a week for four weeks. Treated and control hives were monitored for two months for brood extension, honey and pollen harvest. The presence of beneficial gut microorganisms within bee gut was investigated with denaturing gradient gel electrophoresis and next generation sequencing. The administered bacteria led to a significant increase of brood population (46.2%), pollen (53.4%) and harvestable honey in honey supers (59.21%). Analysis of the gut microbiota on the new generation of bees in treated hives showed an increase in relative abundance of Acetobacteraceae and Bifidobacterium spp., which are known to be involved in bee nutrition and protection.

Demonstration of production and isolation of three sulfated glycoproteins from the rabbit oviduct.

[35S] sulfate-labeled oviductal fluid and [35S] sulfate-labeled extracts of cultured oviductal epithelium were obtained from rabbit. Fractionations by gel filtration and ion exchange chromatography indicated both of these sources contained 3 high molecular weight (greater than 200,00 daltons) sulfated mucoproteins, SDS electrophoresis indicated subunits of 71,000 daltons and 32,000 daltons, where only the 71,000 dalton component was sulfated. Ouchterlony analysis indicated complete homology between these oviductal components separated by DEAE chromatography and partial cross-reactivity with a component(s) present in blood plasma. This suggests that cross-reactivity with serum may have masked previous results where investigators were seeking oviduct-specific components, a problem which is overcome in these experiments by use of the oviductal epithelium incubations.

Immunocytochemical localization and determination of hormone-induced synthesis of the sulfated oviductal glycoproteins.

Secretory products of the oviduct provide part of the milieu for the critical events of fertilization and embryo development. Past work from this laboratory has indicated that three large sulfated glycoproteins can be isolated from rabbit oviductal fluid and are synthesized by oviductal epithelium incubated in vitro. These three glycoproteins are antigenically similar. This paper presents evidence for their localization within the oviductal tissue and their hormonal control of synthesis. Utilizing goat antiserum to these oviductal glycoproteins and the immunoglobulin-horseradish peroxidase bridge method, these macromolecules have been localized in the ampulla and isthmus of the oviduct. Ten days after ovariectomy an oviduct was removed for immunolocalization. The does were then given estradiol for the next 4 days and the second oviduct was removed. Oviducts treated with estradiol showed immunostaining of virtually all of the secretory granules within the secretory cells of the isthmus. While light level immunocytochemistry suggested the possibility of two populations of secretory granules within the ampulla because some of the granules did not show immunocytochemical staining, the more sensitive immunocytochemistry at the electron microscopic level showed staining of all granules of the ampulla and isthmus. Absorption of the antiserum with pure antigen prevented all staining. After ovariectomy and hormone withdrawal, most of the immunostaining was lost in the isthmus and virtually no staining in the ampulla was observed. Oviductal cell suspensions were made to evaluate incorporation of [35S] sulfate and [3H] leucine as a function of hormonal priming of the tissue. Estrogen-primed oviductal cells incorporated the sulfate and leucine into these specific glycoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)