Consumption of apple-boysenberry beverage decreases salivary Actinomyces naeslundii and their adhesion in a multi-species biofilm model.

We hypothesised that consumption of beverage rich in both fibre and polyphenols, rather than each bioactive alone, will modulate populations of selected salivary bacteria, and their adhesion characteristics and that some of these effects may be due to the anti-microbial activity of the beverage bioactives. We investigated the effect of 4 weeks' consumption of beverages, rich in apple fibre, boysenberry polyphenols, or both on salivary bacteria in healthy subjects. In this placebo-controlled crossover study, saliva samples were collected at the beginning and end of each treatment period, and used for qPCR quantitation of Lactobacillus spp., Actinomyces naeslundii and Streptococcus mutans. The counts of salivary A. naeslundii decreased after the consumption of the apple-boysenberry beverage (P<0.05, Student's t-test). We also examined the effect of the subjects' saliva on bacterial adhesion using a mixed species biofilm model. The salivary pellicles prepared before and after each treatment were inoculated with laboratory strains of A. naeslundii, Lactobacillus rhamnosus and S. mutans and tested for biofilm formation. The post appleboysenberry beverage salivary pellicle significantly decreased the adhesion of A. naeslundii at the end of both 3 and 24 h, in the in vitro biofilm. A 1/16 dilution of the apple-boysenberry beverage itself decreased the proliferation of test strains of A. naeslundii and S. mutans by 51 and 55%, respectively (P<0.005), indicating the antimicrobial activity of its bioactives. This study demonstrated that consumption of apple-boysenberry beverage, rather than apple or the boysenberry beverage alone or the placebo, decreased salivary A. naeslundii and their adhesion under laboratory conditions. These changes are factors that influence oral microecology and potentially oral health.

Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel.

AIMS: To determine the mechanism for both the removal and inactivation of 18-h biofilms of a thermophilic Bacillus species that optimally grows at 55 degrees C on stainless steel. METHODS AND RESULTS: The cleaning strategies tested were based on biofilm biochemistry and physiology, and focused on the chemistry of the cleaners, the duration and temperature of the cleaning process and a combination of various cleaners. The success of the cleaning regimes was determined based on the removal of cells and organic debris and the elimination of viable cells. The results confirmed that a caustic (75 degrees C for 30 min) and acid (75 degrees C for 30 min) wash, relied upon heavily in most food processing industries for cleaning-in-place systems, was successful in removing these biofilms. However, any changes in the concentrations of these cleaners or the temperature of cleaning drastically affected the overall outcome. Alternative cleaning agents based on enzymatic or nonenzymatic breakdown of cellular proteins or polysaccharides, surfactant action, use of oxidative attack and free radicals varied in degrees of their success. Combining proteolytic action with surfactants increased wetability and therefore enhanced the cleaning efficiency. CONCLUSIONS: Several procedures, including caustic/acid and enzyme based cleaners, will be satisfactory, provided that the correct process parameters are observed i.e. concentration, time, temperature and kinetic energy (flow). Confirmation of these results should be carried out in a pilot plant through several use/clean cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Confidence in standard and alternative cleaning procedures for food manufacturing plant to prevent contamination with thermophilic bacilli that threaten product quality.

Physiology of biofilms of thermophilic bacilli-potential consequences for cleaning.

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10(6.0) cells/cm2 in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 degrees C developed a mature biofilm of >10(6.0) cells/cm2 within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 microm thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.

Factors influencing attachment of thermophilic bacilli to stainless steel.

AIMS: This project aimed to investigate the mechanism of attachment of the vegetative cells and spores of thermophilic bacilli to stainless steel with a view to devising strategies to limit biofilm development and survival. METHODS AND RESULTS: Spores and vegetative cells of bacterial isolates were exposed to protein denaturing agents (sodium dodecyl sulphate (SDS) and trypsin) and polysaccharide removing agents (sodium metaperiodate, trichloroacetic acid (TCA) and lysozyme). Treatment with sodium metaperiodate, TCA and lysozyme increased the number of vegetative cells attaching in many of the strains studied, while SDS and trypsin decreased attachment. Spores attached to stainless steel in greater numbers than vegetative cells, and the various treatments had less effect on this attachment than for vegetative cells. Viability of the cells or spores was not an important factor in attachment, as cells and spores rendered non-viable also attached to stainless steel in similar numbers. Coating the stainless steel with skim milk proteins decreased the attachment of both vegetative cells and spores. There was no correlation between the degree of attachment and the amount of extracellular polysaccharide (EPS) produced by each strain, surface hydrophobicity or zeta potential of vegetative cells or spores, though spores were found to be more hydrophobic than vegetative cells. CONCLUSIONS: The results suggest that biofilm formation by these thermophilic bacilli is probably a multifactorial process, and that cell-surface proteins play a very important role in the initial process of attachment during the formation of biofilms by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved cleaning systems to control biofilms of thermophilic bacilli in dairy manufacturing plants.

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

Regulation of follicular maturation by human ovarian follicular fluid peptide.

Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, recent evidence indicates that local factors are also responsible for the regulation of folliculogenesis. In addition to their endocrine action on pituitary gonadotrophins, inhibin, activin and follistatin also have a paracrine role in follicular maturation. An ovarian follicular fluid peptide (OFFP) has been identified from sheep and humans. Purification of OFFP has been achieved by ultrafiltration and gel chromatography with further purification by fast performance liquid chromatography and reversed phase-high pressure liquid chromatography. OFFP is a small (< 5 kDa) peptide that competes with FSH in binding to granulosa cells in vitro and inhibits progesterone secretion from granulosa cells in culture. Immunohistochemical localization revealed the presence of OFFP mainly in granulosa cells of ovarian follicles. Furthermore, the peptide caused apoptosis in granulosa cells and induced follicular atresia. OFFP may act indirectly on oocytes via its effect on granulosa cells. The peptide from ovarian follicular fluid appears to have an important autocrine or paracrine role in the regulation of folliculogenesis.