The group in the office of AC&H Consultores

Lisboa, Oct 10th, 1998

The yearly general meeting of EEC Project CT 97 3039 was held in Lisboa, hosted by AC&H Consultores.
The group met at 9:00 am in the office of AC&H Consultores.
Following a brief introduction by the Coordinator Claudio Galli (University of Milan), the discussion among the participants included a presentation by each partner, starting with partners involved in Task 1 of the project.
It was initially pointed out by the coordinator that, due to the seasonal availability of olives for the production of waste waters, i.e. at the end of wintertime, the preparation of waste water extracts and the subsequent experiments on the biological activities of the extracts started several months after the official start of the project.

Presentations by partners can be summarized as follows:

Task I

Partner 8 (Tecnoalimenti)

Tecnoalimenti (Raffaello Prugger and Marco De Vito) has optimized the conditions for the collection and shipment of olives from the different locations to Partner 4 for waste water production. Approaches emerging from the activities of Partner 5 (Task 2) will allow, in the future, the simplification of the preparation of phenolic extracts of the shipment because of the use of proper sorbents that will trap bioactive compounds in the waste waters thus reducing the overall volume.

Partner 4 (University of Florence)

Patrizia Pinelli, on behalf of Partner 4 (FF Vinceri), reported on the different extraction methods of waste waters and on the quali/quantitative analysis of the final extracts. It appears that the content of phenols and the composition of the phenolic fraction depend on the cultivar. The phenolic content and composition of waste waters obtained from olives collected in Spain, France, and Italy is very similar, whereas olives from Portugal appear to yield a product with a much lower phenolic content, possibly because of degradation processes that occurred as a consequence of an incorrect storage. Commercial waste waters from ltaly had a much higher content of phenols, due to the fact that these waters were subjected to recycling during malaxation. PG Pifferi (Partned 5) commented that from 1 Lt of waste waters the recovery of total phenols is about 4-6 gr, of which 10% is made of o-diphenols, ie the compounds endowed with more potent biological activities.

Parter 1 (University of Milan)

Francesco Visioli (University of Milan), illustrated the results of the tests on the antioxidant activities, obtained in a model of oxidation of low density lipoprotein (LDL) of the Extracts. It appears that Extract l, the less purified one, has a slightly stronger antioxidant capacity, as compared to the others. Conversely, the antiplatelet activity of the Extracts was nil, regardless of the aggregant employed. During the discussion it was commented that the compounds may not be able to enter the cells or that a higher degree of purity of the phenolic components in the fractions is required in order to disclose their biological activity.

Partner 3 (University of Montpellier)

Claude Leger reported on the inhibition of superoxide production affordedby the Extracts. In a model of THP-1 monocytes, differentiated to macrophages, there was a very weak dose-dependent relationship, but the extracts, even if removed from the medium were able to at least partially prevent superoxide formation from macrophages. Thus, the effects on the cells were retained in the absence of the products. One problem was the interference of the vehicle, since ethanol per se is able to induce bioluminescence and thus can create artifactual readings. Experiments are in progress with the aim of optimizing the conditions for the study of the effects of the extracts on cells while keeping the compounds in the reaction medium.

Partner 2 (University of Granada)

MA Dolores Suarez is still to finalize the tests, which employ H35 hepatoma cells incubated with hydrogen peroxyde to check for a protective effect on antioxidant enzymes, namely glutathione tranferase. Exporiments using intact cells and homogenates, or purified enzyme preparations will be completed within few weeks.

Task 2

Partner 5 (University of Bologna)

Pier Giorgio Pifferi and Leonardo Setti have tackled the problem of preparing purified fractions from waste waters on a large scale in order to set up an industrial application. This approach is somewhat different from that employed by the University of Florence, which was sized to an analytical scale. The different methods are based on the use of sorbents that are able to selectively trap total phenols from the waste waters. This approach appears to be advantageous: it results in a rapid recovery of phenols, leaving behind unwanted materia (simple and complex sugars, proteins etc.). lt also results in the rapid reduction of the remarkable volume of the waste waters, since the addition of about 5% W/w of soluble sorbent to waste waters allows the reduction of over 90% of the weight and, therefore, it simplifies the transport of waste waters from the site of production to the processing plant. Out of the 5 alternative procedures that have been tested, one appears to be more convenient from many points of view and the fractions obtained in this way will be shortly tested for their biological activities.

Partner 6 (AC&H Consultores)

Joao Bordado elucidated the composition of waste waters and their recovery and stated that they can prepare both hydrophylic and hydrophobic fractions from a complex extract.

After a break, the group rejoined at 3:00 pm to draw conclusions and set shortand long-term goals (Tasks for 2nd year).

As far as Task is concerned, it could be concluded that the fractions prepared by Partner 5 so far are active in vitro as antioxidants with respect to LDL oxidation. They also inhibit free radical generation in monocytes even after they have been removed from the medium. The conditions for testing the activities on monocytes and on liver cells are being oprimized, while a greater purification of the phenolic fractions, obtained with the aid of Partner 4, should result in a higher activity of the extracts.

Regarding Task 2, the development and application of procedures based on the use of selected sorbents for phenols recovery (Partner 4) and the associated development of procedures aimed at differentially recover hydrophylic and hydrophobic components (Partner 6) will result in an enhanced efficacy of waste water extracts and will allow more diversified approaches to separately test lipid-and water-based antioxidant activities.

Partner 8 has also initiated the preparation of an Internet Web site that will allow access to information about FAIR CT 97-3039 and the preliminary outline was briefly showed to the other partners.

The Coordinator insisted on the timely preparation of the yearly report and urged all the partners to conclude their activities on time. Further, it was proposed that all partners will exchange information by fax or e-mail in order to provide an intermediate report within 6 months and regroup for the next yearly meeting (proposed location: Granada) to thoroughly discuss the advancement of their activities.

The meeting ended at 6:00 pm.

Participants
Partner 1 - Claudio Galli, Francesco Visioli
Partner 2 - MA Dolores Suàrez
Partner 3 - Claude Léger
Partner 4 - Patrizia Pinelli
Partner 5 - PierGiorgio Pifferi, Leonardo Setti
Partner 6 - Joào Bordado, M Lavique Santos
Partner 8 - Raffaello Prugger, Marco De Vito
Partner 7 - Jose Queralto