Method for producing metabolites from plants cultivated in soil-less medium

#The invention relates to a method of producing metabolites, continuously or semi-continuously, from plants cultivated in a soil-less medium, without losing the viability of said plants, and to an installation for carrying out this method.

#Plants produce a large number of the molecules which man uses as drugs, colorants, flavourings, comestible additives or pesticides. These compounds are often typical of a given plant family, genus or species. They are classified as secondary metabolites because they do not always appear to be essential to the survival of the unstressed plant (Bentley 1999, Bourgaud et al 1999, Gontier 1993). Many of these molecules of plant origin serve or have served as a model for chemical synthesis for commercial use. Sometimes, the molecules are complex and extraction from wild plants, plants cultivated in a field or in vitro remain the best sources of supply (Herman 1993).

#In particular, cell or tissue culture in bioreactors have been developed for molecules having a high commercial value (price per kilogramme and market volume) (Herman 1993). In this case, the cultures have to be axenic, in other words free from micro-organisms (bacteria and fungi) of which the presence could be harmful to the growth of the plant material and to the production of metabolites thereof. The maintenance of axenic conditions over time is sometimes difficult and leads to significant costs (Gontier 1993). Furthermore, the use of sophisticated systems for in vitro culture (fermenters) adds a cost which is sometimes prohibitory, making the system economically unviable. In vitro biomass production is generally expensive. To make the system cost-efficient, an attempt will be made to produce the maximum of molecules by this biomass without destroying it and while reusing it to the maximum as biocatalyst (Herman 1993).

#The plants used originate from seeds provided by the Institut für Pflanzengenetik und Kultulpflanzenforschung (D-06466 Gatersleben, Corrensstr. 3, Germany). These seeds were scarified in concentrated sulphuric acid for 10 minutes at ambient temperature. After abundant rinsing in running water, they were caused to germinate in pots containing moist soil at a temperature of 25° C.+/−1° C. with a photoperiod of 16 h and hygrometry of 60 to 70%. After two months, when the plants had reached a size of 15 to 20 cm, in other words a stage of 8 to 10 leaves, they were depotted and transferred into glass pots of the type known as “baby food jars” (Sigma-Aldrich Inc., France) of 175 ml. The plants were held by the pot lids, previously perforated with three holes (one for the stem, one for the air bubbling and one for topping up the liquid, see FIG. 1). An MS type nutrient solution diluted four fold (Murashige and Skoog, 1962) was added to the pot. Air could be bubbled into the nutrient solution through silicone tubes in order to maintain good oxygenation of the plant root system and also to avoid the excessive development of populations of micro-organisms. Sixty plants were thus installed. They were cultivated for two weeks under the same conditions of light, temperature and hygrometry as mentioned above.

#Increasing doses (0, 1, 2, 3 or 5% V/V—volume by volume) of surfactant (of the type known by the name Tween 20: polyoxyethylenesorbitan monolaurate) were added to the nutrient solution. Permeabilisation using Tween 20 (sold by the company Prolabo) was carried out in the absence of air bubbling (to avoid the formation of foam) for 0, 12, 24 or 48 hours. The nutrient solution of each plant was reharvested, filtered and analysed by gaseous phase chromatography. The average total concentration of hyoscyamine and scopolamine increases gradually with the surfactant concentration. It passes from 7 mg/l without surfactant to 45 mg/l with 5% of Tween 20. The concentration of hyoscyamine and of scopolamine increases with the duration of the treatment.

#All the permeabilisation solutions were recovered and the molecules which they contained were taken up in chloroform after addition of ammonia to pH 9. The chloroform was evaporated and the dry residue contained both the two hyoscyamine and scopolamine molecules.