DEGUMMING METHOD

Abstract

A method for producing membrane polysaccharides from an organism selected from micro-organisms, unicellular organisms and filamentous fungi, the method including at least one step of extracting the membrane polysaccharides as well as a smaller-scale extraction of the soluble proteins, by mechanical treatment of the organism in a ball mill or by physical treatment of the organism by means of ultrasounds.

Claims

1-17. (canceled)

18. A process for obtaining membrane polysaccharides from an organism selected from microorganisms, unicellular organisms and filamentous fungi, wherein the process comprises at least one step of extracting the membrane polysaccharides, accompanied by a reduced extraction of the soluble proteins, by mechanical treatment of the organism in a ball mill or by physical treatment of the organism by means of ultrasound.

19. The process as claimed in claim 18, wherein the mechanical treatment uses a ball mill equipped with glass balls under the following conditions: the mean diameter of the balls (d.sub.GM) ranges from 1.5×10.sup.−3 to 2.5×10.sup.−3 m, the stirring speed at the blade end (ν) ranges from 5 to 20 m.Math.sec.sup.−1.

20. The process as claimed in claim 18, wherein the mechanical treatment in a ball mill is carried out for a period ranging from 2 to 50 minutes.

21. The process as claimed in claim 18 wherein the ball filling rate of the mill ranges from 60% to 80% volume/volume.

22. The process as claimed in claim 18, wherein the physical treatment uses an ultrasound reactor under the following conditions: the energy ranges from 500 to 5000 J, the power ranges from 200 W to 400 W, the feed flow rate of the reactor ranges from 0.150 l/min to 3 l/min.

23. The process as claimed in claim 22, wherein the ultrasound treatment is carried out for a period ranging from 5 to 30 seconds.

24. The process as claimed in claim 18, wherein the mechanical or physical treatment step is followed by a step of measuring the amount of membrane polysaccharides extracted.

25. The process as claimed in claim 18, wherein at least one treatment step is followed by a step of measuring the amount of membrane polysaccharides that have been extracted.

26. The process as claimed in claim 18, wherein the organism is selected from cyanobacteria, microalgae, bacteria and filamentous fungi.

27. The process as claimed in claim 18, wherein the organism is selected from: the division Rhodophyta and from the genera Porphyridium and Rhodella, Spirulina and Dunaliella.

28. The process as claimed in claim 18, wherein it comprises a step consisting in extracting the soluble polysaccharide fraction from the medium.

29. The process as claimed in claim 18, wherein the membrane polysaccharide extraction step is followed by a step of purifying the polysaccharides.

30. A method wherein the soluble polysaccharide fraction obtained as claimed in claim 28 is used in chemical, food, cosmetic or pharmaceutical compositions.

31. A method wherein the soluble polysaccharide fraction obtained as claimed in claim 28, is used as thickeners, gelling agents, surfactants or else stabilizers of compositions.

32. A method wherein the soluble polysaccharide fraction obtained as claimed in claim 28 is used in or as phytosanitary products or, in as adhesives.

Description

DESCRIPTION OF THE FIGURES

[0103] FIG. 1 presents the principle of the circuit mode detailed above.

[0104] FIG. 2 represents the extraction factor as a function of the mean ball size.

[0105] Represented in FIG. 3 are, on the one hand, the curves of the factors of dissolution of the proteins and of the polysaccharides and, on the other hand, the purity index (IP) of the sugars, as a function of the mean ball diameter.

[0106] FIG. 4 represents the ultrasound extraction factor on a young biomass as a function of the number of passes at 2000 J (200 W).

[0107] FIG. 5 represents the ultrasound extraction factor on an intermediate-aged biomass as a function of the number of passes at 2000 J (200 W).

[0108] FIG. 6 represents the ultrasound extraction factor on a mature biomass as a function of the number of passes at 2000 J (400 W).

[0109] FIG. 7 represents the ultrasound extraction factor on a mature biomass as a function of the number of passes at 2000 J (200 W).

[0110] The examples which follow illustrate the invention without limiting the scope thereof.

EXAMPLES

[0111] For all these examples, a Porphyridium cruentum culture medium is used. This biomass was cultured in a 10 l tubular photobioreactor.

[0112] The proteins (denoted “prot” in the figures) are measured by absorbance at 280 nm, said measurement being optionally supplemented by a protein assay carried out according to the BCA protocol in order to verify the accuracy of the spectrophotometric measurements.

[0113] The quantification of the pigments: total chlorophylls and total carotenoids (denoted respectively Chi and PPC in the figures) is carried out by spectrometry respectively at 678 nm and 416 nm.

[0114] The B-Phycoerythrin (denoted B-PE in the figures) is assayed by the Bermejo protocol.

1. Process Using a Ball Mill

1.1 Example: Determination of the Extraction Factor as a Function of the Mean Bead Diameter

[0115] The Porphyridium cruentum culture medium was treated by means of a glass ball mill (DynoMill Mutlilab, WAB, Switzerland) under the following conditions:

[0116] Q: feed flow rate in ml/min: 170

[0117] φ: mill filling rate: 75%

[0118] n: stirring speed: 2389 min.sup.−1.fwdarw.ν: speed at stirrer blade end 8 m.Math.sec.sup.−1.

[0119] By way of reference, a High Pressure mill (2700b) enables cell destruction and consequently the release of all (100%) the metabolites in solution/stable suspension, thus of all the polysaccharides, is carried out. The unit of measurement for each metabolite will be the extraction factor, the maximum (100%) of which corresponds to the response obtained with the HP milled material.

[0120] After the milling step(s), a centrifugation is carried out (13 400×g, 10 min), and the analyses are carried out on the supernatant constituting the microalgae aqueous extract.

[0121] The results are presented in FIG. 2.

[0122] Three milling treatments were carried out with balls having a different mean diameter:

[0123] a) with balls having a mean diameter of 0.625×10.sup.−3 m, deconstruction of the cells is observed, the protein selectivity is at the maximum. The balls are not suitable for recovering the polysaccharides. During the deconstruction of the cells, the cell compartmentalization is respected, the release of the metabolites is generally selective;

[0124] b) with balls having a mean diameter of 1.3×10.sup.−3 m, destruction which is similar to cell disintegration is observed (the cell is completely destructured, the debris is fine), and is accompanied by release of the proteins, of the polysaccharides and also of the pigments in a homogeneous manner. The homogeneous release of all the pigments reflects an extreme destruction of the cell; this process is less selective between proteins and polysaccharides, it does not allow selective extraction of the polysaccharides.

[0125] c) the balls having a mean diameter of 2.15×10.sup.−3 m are the most suitable for the selective extraction of the sugars compared with the cell metabolites; this process is the one which results in the least cell destruction.

1.2 Determination of the Dissolution Factors and Purity Index

[0126] On the basis of the results obtained at the end of the three milling operations above, the curves of the dissolution factors of the proteins and of the polysaccharides were plotted, as a function of the mean ball diameter, in FIG. 3 and on the same graph, the purity index of the sugars was plotted.

[0127] It emerges from this graph that, starting from a mean ball diameter of 1.3×10.sup.−3 m, the dissolution factor of the polysaccharides is greater than the dissolution factor of the proteins and that the purity index of the polysaccharides increases with the size of the balls.

[0128] Selecting balls having a mean diameter greater than 1.5×10.sup.−3 m provides conditions under which the degree of dissolution of the membrane polysaccharides is greater than that of the other cell compounds, in particular the proteins.

[0129] Selecting balls having a mean diameter equal to 2.15×10.sup.−3 m provides conditions under which the degree of dissolution of the membrane polysaccharides is low, but under which the purity index is higher.

[0130] By performing several passes of the medium to be treated through the ball mill, with balls having a mean diameter of greater than 1.5×10.sup.−3 m, dissolutions of the order of 80% are successfully obtained.

2. Process Using Ultrasound

[0131] The technology used is the Sonitube® Type SM 35/3, supplied by Synetude It is an ultrasound tunnel which has a maximum power of 400 W, the operating frequency of which is 35 kHz.

[0132] The suspension is pumped through the Sonitube, in which it will be subjected to the ultrasound treatment (volume of 70 ml of treatment volume). The operating parameters are thus the feed flow rate and also the ultrasound power (200 W-400 W). During operation, a significant part of the energy supplied to the fluid is dissipated in the form of heat. As a result, the temperature of the suspension will be monitored, so that it does not exceed 30° C.

[0133] During each experiment, the HP milling carried out at 2700b defined in the preceding paragraph serves as a reference.

[0134] Following the sonication step, a centrifugation is carried out (13 000×g, 10 min).

[0135] All the analyses will be carried out on the supernatant, constituting the microalgae aqueous extract.

[0136] For the ultrasound, batchwise tests were carried out.

[0137] Biochemical protein assays were carried out in order to dispense with the potential bias that the extraction factor calculated from the A.sub.280 might have.

[0138] On the basis of the finding that, in the stationary phase, the level of production of membrane polysaccharides is greater than the level of dissolution in the culture medium, it was decided to carry out the ultrasound treatment on suspensions of Porphyridium cruentum of which the physiological state differs.

[0139] This is because, in the young physiological state, the level of production of membrane polysaccharides is still low and in any event lower than the level of dissolution in the culture medium.

[0140] a. Young Physiological State

[0141] The culture was at 3.7 g solids/l, including 1.5 g/l of bound membrane polysaccharides and 0.3 g/l of dissolved membrane polysaccharides. The monitoring during the successive passes is presented in FIG. 4.

[0142] A certain selectivity is observed. The ultrasound treatment makes it possible to achieve a release of more than 50% of the bound membrane polysaccharides, with a protein release of the order of 40%. Nevertheless, the physiological state of the cells means that the degumming takes place on a relatively small amount of the bound membrane polysaccharides (1.5 g/l).

[0143] This physiological state may also explain the high level of cell destruction.

[0144] b. Intermediate Physiological State, Beginning of Stationary Phase

[0145] The culture was at 4.5 g solids/l, including 1.7 g/l of bound membrane polysaccharides and 0.4 g/l of dissolved membrane polysaccharides. The monitoring during the successive passes is presented in FIG. 5.

[0146] As the culture ages, the amount of bound EPSs increases. The selective degumming phenomenon appears to be promoted with the increase in bound membrane polysaccharides. Indeed, it is possible to achieve a very satisfactory membrane polysaccharide extraction factor (close to 80%) while limiting the release of soluble proteins (<50%). The thickness of the cell wall of Porphyridium cruentum during its maturation thus appears to make it possible to detach a greater part thereof without however systematically lysing the cells.

[0147] c. Mature Physiological State

[0148] The culture was at 6.4 g solids/l, including 3.2 g/l of bound membrane polysaccharides and 0.44 g/l of dissolved membrane polysaccharides. For this experiment, the minimum and maximum operating powers were tested, namely 200 W and 400 W. The monitorings during the successive passes are presented in FIGS. 6 and 7.

[0149] A very weak cell lysis is observed (<5%) on this biomass. While the reliability of A.sub.280 may be contested (some values around 0%), the B-PE assay is a method acknowledged to be reliable and may thus serve as an indicator of (extreme) lysis.

[0150] In this context, it would appear that the treatment at 400 W is more selective than that at 200 W. Indeed, the ratio of the extraction factors [polysaccharides (%)]/[B-PE (%)] is 3.5 at 200 W and 8 at 400 W. Furthermore, a more extensive degumming is observed at 400 W (close to 20%).

[0151] It emerges from these experiments that ultrasound under appropriate conditions enables degumming of the microalga.