A Model for SynbioticActivity Evaluation: Static and Continuous Co-Culture Fermentation of BifidobacteriumAdolescentis ATCC 15703 and Bacillus Cereus ATCC 9634

In this study, the ability of a probioticstrain (BifidobacteriumadolescentisATCC 15703) to inhibit the growth of the common food contaminantBacilluscereusATCC 9634was studied, both individually and as part of a synbiotic with FOS during batch or continuous fermentation (flow fermentation). The conditions of the flow fermentation corresponded to the parameters of the human large intestine: maintaining a pH of 6.8; anaerobiosis; and a medium flow rate of 0.04 h−1. Bifidobacteria and bacilli were co-cultivated on a prebiotic carbohydrate substrate (10 g/L) and the prebiotic was replaced with glucose (10 g/L).The results of the batch and flow fermentation were compared.The synbiotic efficacy of the probioticBif. adolescentisand the prebiotic FOSagainst the common food contaminantBac. cereuswas shown for all conditions. Fermentation of a pure culture of bifidobacteria with varying prebiotic concentrations (2, 5, 10, 15 and 20 g/L) was carried out to study the state of dynamic balance. It was demonstrated that 48 hours is enough to achieve stable dynamic balance.Prebiotics were co-cultivated with varying carbohydrate concentrations of 5, 10, and 15 g/L.The results showed that increasing the prebiotic concentration increased the duration of the lag-phase and reduced the final number of bacilli. Keywords: probiotics, prebiotics, synbiotics, gastrointestinal tract modeling, antagonism, co-culture fermentation

The Effect of Gibberellic Acid on the Production Characteristics and Biochemical Parameters of Tetraselmis Suecica in an Enrichment Culture

The use of gibberellic acid as a stimulator of microalgae growth has beensubstantiatedexperimentally.This research aimed to assess the effect of exposure to a wide range of gibberellic acid concentrations on the growth dynamics ofthe microalgaTetraselmissuecicain an enrichment culture. The duration of the experiments was 14 days. It has been shown that gibberellic acid,atconcentrations of 0.39–3.20× 10−8M, stimulates algaegrowth. In this research, the exposure to gibberellic acid at concentrations of 0.39–3.20 × 10−8M was accompanied by a variation in the pattern of growth curves: the maximum number of cells was recorded on day seven of the experiment. A higher concentration of the phytohormone (3.84 × 10−8М) inhibited the increase inculture density. The growth of theT. suecicaculture in the control group was 332%;the growth of the culture exposed to gibberellic acid at a concentration of 0.39 × 10−8M was1136%. The values of the specific growth rate ofT. suecicawere estimated for different periods of cultivation. On day14 of the experiment, the biochemical composition of microalgae biomass was analyzed.According to the results, gibberellic acid stimulated the accumulation of carbohydrates, proteins, and chlorophyll. Nevertheless, the phytohormone had no effect on lipidaccumulation. An assumption was made thatexposure to low concentrations of phytohormone stimulates the growth of microalgae by reducing the lag phase of growth. Keywords: gibberellic acid, microalga, cultivation, lipids, carbohydrates, proteins

The Legionella Lqs-LvbR regulatory network controls temperature-dependent growth onset and bacterial cell density

Legionella species are facultative intracellular pathogens, which cause a life-threatening pneumonia termed Legionnaires’ disease. Legionella pneumophila employs the Legionella quorum sensing (Lqs)-LvbR network to regulate virulence and motility, but its role for growth in media is ill-defined. Here we report that compared to the parental L. pneumophila strain JR32, a Δ lqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the Δ lqsA , Δ lqsS and Δ lqsT mutants showed a longer lag phase and reached only a lower cell density. A Δ lvbR mutant resumed growth like the parental strain at 30°C, but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in AYE medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C the P lqsR , P lqsA , P lqsS and P lvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32, and possibly also of environmental and clinical L. pneumophila isolates. Importance Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires’ disease, the incidence of which is worldwide on the rise. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a similar mechanism as in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical or biological means will reduce the incidence of Legionnaires’ disease. Here we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is not only determined by the temperature and nutrient availability, but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size ~7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

A possible predictive mathematical model for the growth of a periphytic alga

Algae are photosynthetic organisms and have qualities that are very attractive for cultivation and industrial development for commercial purposes. When algal growth is analyzed for the production of biomass usually only the exponential phase of the growth curve is considered and the other phases are ignored. The objective of the work is to present a possible predictive mathematical model that allows a better understanding of the kinetic behavior of a periphytic microalgae by means of the use of the Smoluchowski discrete equation, with special emphasis on the lag phase. More specifically, unknown connection between the discrete Smoluchowski equation and the deterministic Baranyi model is shown in the present study. Analysis of this connection leads to a possible predictive mathematical model about of the kinetic behavior of a periphytic microalgae.

Characterization of Butachlor Degradation by A Molybdenum-Reducing and Aniline-degrading Pseudomonas sp.

Butachlor is a chloroacetanilide herbicide that is selective in action and commonly used for pre-emergence control of weeds. It is believed to have range of toxicity from acute to chronic and also can cause DNA strand breaks and chromosomal aberrations in humans. This study was aimed at characterizing the potential of previously isolated bacteria for butachlor degradation. The isolates from culture collection, labelled A-F were screened for butachlor degradation on Bushnell Hass agar media with butachlor as a sole carbon source. Isolate A (molybdenum-reducing and aniline-degrading Pseudomonas sp.) was observed to grow best and tolerated the highest concentration of butachlor supplemented in the media after 72 h of incubation at 37 ℃. Characterization study revealed that the Pseudomonas sp. can utilize and grow with butachlor at optimum pH between 6.0 - 6.5, temperature between 30 – 37 ℃ and can tolerate up to 600 mg/L butachlor concentration with increase in growth as inoculum size increases. Additionally, this bacterial strain shows no lag phase regardless of the concentration of the herbicide used and reach its maximum growth after 24 h of incubation. The ability of this isolate to tolerate and utilize butachlor as sole carbon source makes it suitable for future bioremediation of this toxicant.

Calmodulin and Its Interactive Proteins Participate in Regulating the Explosive Growth of Alexandrium pacificum (Dinoflagellate)

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.

The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Predicting the growth of Listeria monocytogenes in cooked sliced deli turkey breast as function of clean label antimicrobial, pH, moisture and salt

The use of antimicrobials in formulations of ready-to-eat meat and poultry products has been identified as a major strategy to control Listeria monocytogenes . The USDA-FSIS recommends no more than 2-logs of Listeria outgrowth over the stated shelf life if antimicrobials are used as a control measure for a product with post-lethality environmental exposure. This study was designed to understand the efficacy of a clean label antimicrobial against the growth of L. monocytogenes as affected by the product attributes. A response surface method-central composite design was used to investigate the effects of product pH, moisture, salt content, and a commercial “clean-label” antimicrobial on the growth of L. monocytogenes in a model turkey deli meat formulation. Thirty treatment combinations of pH (6.3, 6.5, and 6.7), moisture (72, 75, and 78%), salt (1.0, 1.5, and 2.0%), and antimicrobial (0.75, 1.375, and 2.0%) with six replicated center points and eight design star points were evaluated. Treatments were surface inoculated with a 3 log 10 CFU/g target of a five-strain L. monocytogenes cocktail, vacuum packaged, and stored at 5°C for up to 16 weeks. Populations of L. monocytogenes were enumerated from triplicate samples every week until the stationary growth phase was reached. The enumeration data was fitted to a Baranyi and Roberts growth curve to calculate the lag time and maximum growth rate for each treatment. Linear least-squares regression of the lag-time and growth-rate against the full quadratic, including the second order interaction terms, design matrix was performed. Both lag time and maximum growth rate were significantly affected ( p <0.01) by the antimicrobial concentration and product pH. Product moisture and salt content affected ( p <0.05) lag phase and maximum growth rate, respectively. The availability of a validated growth model assists meat scientists and processors with faster product development and commercialization.