WO2001052992A1

WO2001052992A1 - Improved grinding method and related device

Info

Publication number: WO2001052992A1
Application number: PCT/FR2001/000211
Authority: WO
Inventors: Jean-Paul Maisonneuve; Alain Murigneux
Original assignee: Limagrain Genetics Grandes Cultures
Priority date: 2000-01-24
Filing date: 2001-01-23
Publication date: 2001-07-26
Also published as: FR2804047B1; AU2001235584A1; FR2804047A1

Abstract

The invention concerns a method for grinding at least a substance, in particular a vegetable sample, comprising a stirring step along a reference direction (Z) of at least a closed vessel containing the substance(s) and at least a solid ball. The invention is characterised in that said reference direction is the vertical direction, each closed vessel is an elongated test tube, the direction of the test tube elongation being parallel to the reference direction (Z), and the stirring of each tube is carried out along a direction parallel to said reference direction. The invention also concerns a device for implementing said method.

Description

IMPROVED CRUSHING PROCESS AND ASSOCIATED DEVICE

The present invention relates to methods and devices for crushing crushing and / or mixing of materials.

More specifically, the invention relates to a method and a device enabling a large number of material samples to be ground quickly and efficiently. A particularly advantageous application of the invention which will be described in this text by way of example is the grinding of samples of plant material; however, the invention can also be applied to any type of material capable of being ground for experimental or industrial applications (in particular in the biological and chemical fields: grinding of bones, living or inert tissues, but also of materials plastics, food, minerals ...)

A first known method for grinding materials consists in using a mortar and a pestle.

This process is still very widely used for numerous applications, including the grinding of plant samples with a view to their study in the laboratory or their industrial exploitation, which is, as we have said, one of the preferred applications of the invention ( for example for a DNA extraction protocol).

This conventional process, if it allows to crush relatively large samples (of the order of 5 to 10 grams of plant material), is however ill-suited to the grinding of samples of smaller size, because in this case the associated losses decrease the yield and distort the quantities obtained.

In addition, with this process it is necessary to transfer the ground material to another container for its subsequent use (analysis, dosing in an experimental or industrial mixture, etc.), which creates the risk of contamination and dosing errors. . Finally, this process being carried out manually, its yield is intrinsically very limited (of the order of only 10 samples per person per hour). There are certainly devices to improve the results of this procodé (see in particular the mortar of US 4,397,425), but they do not make it possible to respond effectively to the drawbacks mentioned above.

An improvement of this process, used in particular for biological or chemical applications, consists in manually grinding samples placed directly in a conventional test tube with a ground glass rod. However, this technique only allows the crushing of relatively small samples (500 milligrams maximum) and its yield remains limited (100 samples per person per hour).

There is also known from US 3 847 363 a more sophisticated grinding device, but in this case again the yield remains limited, and this device is not freed from the significant drawback linked to the risks of contamination and dosing errors. , due to the need to transfer the crushed material to another container for its subsequent operation.

Furthermore, an automatic device is known which transmits to an elongated stainless steel bowl an alternating movement in a horizontal direction parallel to the lengthening axis of the bowl. To grind material, the material is placed with one or more balls in the closed bowl, the agitation of the bowl causing the movement of the balls which crush the material.

This device, if it represents a certain progress compared to the grinding means mentioned above, however only allows the grinding of a very limited quantity of material, and this grinding is carried out in the horizontal direction which is a configuration in which the grinding yield is not optimal. In addition, the implementation of this device almost always requires the transfer of the ground material to a test tube or other container for further treatment, which leads to the risks of contamination and associated dosage error mentioned above.

Finally, in the case where it is desired to grind frozen material (which is commonly the case, in particular for applications in plant biology), it is necessary to associate with this mill a complex refrigeration device, which increases the time for carrying out the grinding and decreases its yield.

The object of the invention is to make it possible to grind materials of various kinds, in particular plant, biological or chemical materials, said materials being able to be in the form of fresh material or having undergone a preliminary treatment such as a lyophilization or a freezing. , by overcoming the drawbacks mentioned above.

In order to achieve this aim, the invention proposes, according to a first aspect, a process for grinding at least one material, in particular a plant sample, comprising a stirring step in a reference direction of at least one closed container which contains the material or materials and at least one solid ball, characterized in that said reference direction is the vertical direction, each closed container is an elongated test tube, the direction of elongation of the tube being parallel to the reference direction, and in that the agitation of each tube takes place in a direction parallel to said reference direction.

Preferred, but non-limiting aspects of the process according to the invention are the following: the material or materials comprise a plant sample and the process comprises the harvesting of the plant sample and the placing of the plant sample in a tube and the tight sealing of each tube, immediately after harvesting the plant sample.

• during the stirring step, the tubes are placed on at least one rack.

• the process comprises prior to the stirring step a step of freezing the tube or tubes and during the stirring step, the tube or tubes are placed on at least one isothermal rack.

• the agitation of the tube or tubes is carried out by a reciprocating movement of given frequency which can be controlled.

• said frequency is variable. • the process includes, prior to the stirring step, treatment of the material or materials such as freeze-drying.

According to a second aspect, the invention provides a device for implementing the stirring step of a method according to one of the aspects mentioned above.

Preferred, but non-limiting aspects of the device according to the invention are the following:

• it comprises a base intended to receive fixedly at least one rack P on which the test tubes are placed, and means for imparting to the base a reciprocating movement of vertical translation of given controlled frequency.

• It is associated with means for controlling the movement of the base.

• the means for imparting to the base an alternating movement of vertical translation of a given controlled frequency comprise a piston, a connecting rod and a crankshaft.

Other aspects, aims and advantages of the invention will appear better on reading the following description of a preferred embodiment of the invention referring to the drawing of the single appended figure which schematically shows in side elevation a device allowing to implement the method according to the invention.

In this single figure, there is shown a base 100 on which are fixed two brackets 101 and 102 which face each other, each bracket carrying two rails 103 (a single rail of each bracket appearing in the figure), so that the rails of each set square are facing each other covering the same vertical stroke.

A base 104 which extends horizontally is mounted on a support 105 which is integral with four bearings 106, which can cooperate with the rails 103 of the brackets, the base 104 thus being able to move only in vertical translation (the vertical direction corresponding to the Z axis of the figure). The base 104 is intended to receive on its upper face a rack P such as those used in known manner for carrying a series of test tubes. Means are also provided for fixing the rack to the base; in the embodiment described here, these means include four studs 1041 secured to the base, which pass through a clamping plate 107, a quick tightening bolt 108 being associated with each stud to tighten the clamping plate on top of the rack and make it integral with the base 104.

The base 104 and the rack can thus move in the vertical direction, a stop 109 being provided on at least one of the rails of the device in order to limit the stroke of the base upwards.

The device also comprises a piston 110 fixed under the underside of the base and the lower end of which is articulated by a pin 111 on a first upper end of a connecting rod 112, the lower end of the connecting rod being articulated by a axis 113 on a crankshaft 114 which is driven by an electric motor (not shown in the figure).

The assembly is covered by a cover 115 which can be made of sheet metal, fixed on the edges of the base 100. The device is associated with a control console 20 which is connected by electric cables C to the motor in order to control its supply, the console 20 comprising a button 21 for starting the engine, a button 22 for stopping the engine, a button 23 for operating the engine discontinuously (electrical supply by square or sinusoidal pulses for example), a button 24 d emergency stop and a rotary button 25 for controlling the speed of rotation of the motor by potentiometer.

According to the invention, samples of material, for example plant material, are placed in conventional test tubes into which are also introduced one or more balls which may be made of stainless steel or not, or of any other material (for example example in agate, glass, corundum, zirconium oxide, polystyrene, ...) depending on the desired result. Immediately after the introduction of the material to be ground into the tube, the tube is sealed and placed on the rack P (the clamping plate 107 does not yet cover it). It is thus possible to place a plurality of tubes on the rack. The Applicant has thus sized prototypes whose rack can carry one of the following configurations: microtiter plates of 96 wells, 121 individual tubes of 1.5 ml,

384 tubes of 1 ml in 96 format plate (called micron tubes), • 121 tubes of 5 ml, 81 tubes of 20 ml, 36 tubes of 30 ml.

The tubes are elongated tubes such as those conventionally used in chemistry or biology; they can be for single use or not. Several racks can also be used on the base 104.

Advantageously, the quantity of material introduced into the containers represents approximately from 5 to 80% of the volume of the container, and particularly advantageously, one third of the volume of the container. Advantageously, at the same time as the material to be ground, additives, solvents or reagents can be introduced into the container.

Thus, compared with existing devices, the invention makes it possible to grind samples whose size can describe a very wide range.

The rack is then covered by the flange which keeps it fixed relative to the base 104, and the motor power is controlled by these control buttons. Under the effect of the engine drive, the crankshaft drives via the connecting rod 112 the piston 110 in an alternating vertical reciprocating movement which is transmitted to the base 104, to the rack P and to the bridles he wears. The ball or balls contained in each tube are driven by this movement and cause the material contained in the tube to be crushed. It will be noted that the crankshaft can be dimensioned so as to counterbalance all of the elements in movement (base 104, rack and its tubes, clamping plate 107), in order to avoid vibrations of the device.

It will also be noted that, depending on the size of the tubes used and the nature of the material to be ground, the diameter of the balls can be optimized in order to improve the grinding.

The Applicant has conducted studies demonstrating that the duration of grinding of a sample rack can vary between approximately two and six minutes. For example, for 1.5 ml tubes, it is thus possible to grind 726 samples per hour and per person; it is thus found that the invention also greatly increases the quantitative yield of grinding (the grinding protocols developed by the Applicant allow in particular the rapid grinding of samples varying from 50 milligrams to 5 grams).

Since the grinding is carried out directly in a test tube, no transfer of material is necessary to carry out subsequent stages of dilution / dosing of the ground material for exploitation and / or experimentation, these subsequent stages being able to be made in the same tube. In this way, the risks of contamination and handling error are considerably reduced. In addition, the fact that the grinding takes place in an individual closed tube further reduces the risk of contamination. Finally, in order to further reduce this risk, it is entirely possible to collect the plant samples directly on the field inside the tubes and to re-seal them tightly at the time of harvest. According to another important characteristic of the invention, the racks are made isothermal by any means known per se such as an envelope of insulating material, which allows the grinding of materials or fresh tissue, but also lyophilized or frozen. In practice, it often turns out to be advantageous to freeze fresh samples by immersion (for example in liquid nitrogen), before placing them on the racks P, because the crushing of frozen materials or tissues proves to be often more effective. Under the effect of the engine drive, the crankshaft 114 and the connecting rod 1 12 drive the piston 110, the base 104 and the rack P in a vertical reciprocating movement the frequency of which can be controlled using button 25 of the dimmer on the control console. It is also possible to control by the button 23 a pulsed operation of the motor ("blow by blow", sinusoidal variations of the speed ...), which can prove to be advantageous in certain grinding protocols.

It is thus possible to adjust the characteristics of the grinding to adapt the speed and the operation of the engine to the nature of the material to be ground, and to the desired result.

This is particularly advantageous in the treatment of materials of varying hardness (leaves, leaf tissue, stems, roots, seeds ...). By way of example, the Applicant has thus proceeded to the grinding of

2500 samples of 2 grams each, each sample being placed in a 30 ml tube with two 15 mm diameter beads. After freezing in liquid nitrogen, the tubes were placed on the isothermal rack, 36 tubes taking place on the rack. After 4 minutes of stirring, the grinding result is of higher quality than that obtained by grinding in a mortar because it is more homogeneous.

It is thus possible to vary the frequency of agitation of the rack, for example between 0 and 900 vertical back and forth movements per minute.

An important characteristic of the invention resides in the fact that the materials to be ground are placed in test tubes which contain one or more balls, and to which an alternating movement is printed in a direction parallel to the longitudinal direction of the tube; this arrangement makes it possible both to carry out the grinding without requiring transfer to another container, and to guarantee a very good quality of said grinding.

In addition, the fact that the stirring of the tubes takes place in the vertical direction makes it possible to combine the effects of the entrainment of the tube and the or balls by the motor, to that of gravity which also acts on the ball or balls of the tube.

The invention can be applied for the grinding of various materials, a preferred application being, as has been said, the grinding of plant samples; it is also possible to grind any type of material, the invention also being able to be implemented for the rupture or the disintegration of cells (bacteria, yeasts or others).

In addition, the invention makes it possible not only to grind materials but also to homogeneously mix materials of diverse nature.

Of course, the invention is not limited to the particular embodiment described above; in particular, provision may be made to use any known means for imparting a vertical reciprocating movement back and forth to the rack P.

Claims

1. Method for grinding at least one material, in particular a plant sample, comprising a step of stirring in a reference direction (Z) of at least one closed container which contains the material or materials and at least a solid ball, characterized in that said reference direction is the vertical direction, each closed container is an elongated test tube, the direction of elongation of the tube being parallel to the reference direction (Z), and in that that the agitation of each tube takes place in a direction parallel to said reference direction.

2. Method according to claim 1, characterized in that the material or materials comprise a plant sample and the method comprises the harvesting of the plant sample and the placing of the plant sample in a tube and the tight sealing of each tube, immediately after harvesting the plant sample.

3. Method according to one of the preceding claims, characterized in that during the stirring step, the tubes are placed on at least one rack (P).

4. Method according to one of the preceding claims, characterized in that the method comprises, prior to the agitation step, a step of freezing the tube or tubes and during the agitation step, the tube or tubes are placed on at least one isothermal rack.

5. Method according to one of the preceding claims, characterized in that the agitation of the tube or tubes is carried out by a back-and-forth movement of given frequency which can be controlled.

6. Method according to claim 5, characterized in that said frequency is variable.

7. Method according to one of the preceding claims, characterized in that the method comprises, prior to the stirring step, a treatment of the material or materials such as lyophilization.

8. Device (10) for implementing the stirring step of a method according to one of the preceding claims, characterized in that it comprises a base (104) intended to receive fixedly at least one rack (P) on which the test tubes are placed, and means (110-114) for imparting to the base an alternating movement of vertical translation of controlled frequency.

9. Device according to claim 8, characterized in that it is associated with control means (20) of the movement of the base.

10. Device according to claim 8 or 9, characterized in that the means for imparting to the base an alternating movement of vertical translation of controlled frequency includes a piston (110), a connecting rod (112) and a crankshaft (114) .