Novel Functional beverages based on Polysaccharide additives of algal origin

Alginates are for the moment the only polysaccharides extracted from seaweed that are used in beverages as stabilizers and flavourings. Laminarans and Fucoidans are two other types of polysaccharides which have been described and are subjects of considerable interest because of the various reported bioactive properties they possess, including anti-bacterial properties. The aims of the present project are as follows:

- To produce and mix bioactive polysaccharide extracts with different beverage types

- Identify the best "additive" and beverage combinations and ratios to maximize the claimed benefits

- In carefully planned laboratory studies to generate valid pre-clinical data, study the in vitro and in vivo toxicity and effects on gut microflora and known human pathogens of the selected functional beverages

At the end of this project, one or more of the functional beverages will have been identified as the most active and the best candidate(s) for the future commercial development and clinical trials, prior to a market launch. The overall goal of this project is therefore to set up a reliable and appropriate scientific basis for the development of innovative Irish functional beverages.

Non-destructive extraction protocols using non-chemical methods, column separation, gel permeation chromatography (GPC), and ultra-filtration will be combined and adapted for the production of the polysaccharide-rich extracts. Analytical methods using spectrophotometry, high performance liquid chromatography (HPLC), gas chromatography coupled with mass spectrometry (GC-MS), ESI-Q-TOF MS and biological assays will be used throughout the project to provide a quantitative compositional analysis of the extracts.

The effects of the functional beverages/ingredients at laboratory scale, will be investigated by studying the behaviour of target bacteria (selected human pathogens and organisms associated with the GIT), by using statistically relevant, NCLSS-approved microbiological screening techniques, challenge tests and methods to evaluate pathogen resistance.

A series of pre-clinical trials will be carried out to meet the current European legislation governing FBs. Studies to evaluate the in vitro and in vivo toxicological effects of the target FBs will be conducted by AIT to meet the strictest EU/International standard. AIT is a recognized centre for Cellular and Molecular Toxicology.

The protocols for each "pre-clinical" evaluation and the number of animals required for the in vivo toxicology trials, will be carefully selected to validate the results for eventual clinical trials. A combination of Immunological techniques, including (ELISA), Flow Cytometry, Microscopy, and real time PCR will be used in these studies.

Additional trials will be conducted to evaluate the effects of the target FBs on cytokine expression by specific cell types of immune origin in culture (MGBG, NUI, Galway). Technology currently in use in MGBG, NUI, Galway to evaluate the effects of laminarans and fucoidans on human cells in culture (and the associated RT-PCR and biochemical assays for biomarker expression) will be applied in this project.

Available Macrophage and Jurkat cell lines will be grown in culture and treated with a range of concentrations of the beverages, of known composition. Cell viability will be investigated by Trypan blue staining and MTT tests. Cell morphology will be monitored and specific biomarkers of cell activity (inflammatory and non-inflammatory cytokines) and cell death (caspases and other markers of cell death) will be analyzed by RT-PCR, gel electrophoresis and Western blotting techniques (i.e. combined genomic and proteomic approach). Where studies indicate significant bioactive effects, a microarray analysis will be conducted to obtain a global overview of the cellular and molecular effects of the novel FB.