FR2604182A1 - PROCESS FOR TREATING CELLS USING FROZEN PARTICLES OF A SUSPENSION CONTAINING CELLS - Google Patents

Abstract

CELL TREATMENT PROCESS. A SUSPENSION CONTAINING CELLS AND NITROGEN GAS IS SPRAYED BY A NOZZLE 4, SO AS TO OBTAIN FROZEN PARTICLES WHICH ARE THEN PROJECTED ON A PLATE 92 TO BREAK THE SURFACE OF THE CELLS. ORGANIC INDUSTRY.

Description

Method of treating cells using frozen particles

  a suspension containing cells. The present invention relates to a method of treating cells to facilitate the extraction of useful substances. In particular, it aims

  a method of treating cells which consists of

  throw frozen particles from a suspension containing

  cells, so that they enter into colli-

  sion and thereby give energy from the shock to the sur-

faces of the cells.

  It is known that one can efficiently extract substances from a cell of a microorganism / such as yeasts or bacteria, by freezing and

grinding the cell.

  For example, the demand for

  The unexamined method, published in Japan No. 29983/78, discloses a method of freezing cells such as yeasts by contacting them with

  a liquefied gas to form particles having a di-

  meter from 1 to 5 mm, then grind the frozen cells obtained by a grinder such as a screw extractor, at a very low temperature, in a gaseous atmosphere at low temperature. But this process can not efficiently break or damage the cells, since it freezes liquid entities within the cells and the particles have to reach a diameter of 1 to 5 mm in order to be reduced in size. powder

  by the grinder mentioned above.

  Japanese Patent Application No. 869-83 discloses a method of freezing a suspension which

  contains a microorganism in a certain concentration

  to form particles having a diameter of about 10 mm, or a volume of 1 cm3, and then to powder the frozen particles obtained such as

  what are. But since this process uses

  relatively large frozen eggs, as described above.

  above, grinders that can be used are limited, for example to hammer mills, ball mills, etc. In addition, it is difficult to surrender

  grinding master suitable for extracting

  pure cells, when using these grinders.

  As the heat entrained during their operation modifies the properties of the substances to be extracted, it can occur a pollution of fragments of balls and these mills often crush so excessively the

  cells that there may be pollution of the fragments

  sprays of cell membranes and / or

  constituents of cells within the cell.

  The invention relates to a method for treating cells, the operation of which is easy to control and which breaks and damages, in a continuous manner, the

  cells to facilitate the extraction of substances

  these useful in a high purity, without modifying the properties of these useful substances by heat, as well as without pollution of cell membranes or

  of crushed cell constituents.

  The disadvantages of the processes can be overcome

  classics mentioned above by projecting

  frozen cells of a suspension containing cells so as to obtain a collision and to give a

energy due to shock to the cell.

  The invention therefore relates to a method for treating cells so as to facilitate the extraction of useful substances from their breast, characterized in that it consists in projecting frozen particles containing cells so as to bring the particles into collision and to give them energy due to shock

  in order to damage the surface of the cells.

  According to another of its aspects, the invention relates to a method for treating cells so as to facilitate the extraction of useful substances from their breast, characterized in that it consists in producing microfine liquid droplets having a diameter of

  500 microns or less than this value, a suspen-

  sion containing cells, to put the droplets

  microfines of liquid in contact with a refrigerant

  to project frozen particles having a diameter of substantially 500 microns or less, and then to project the frozen particles to collide and give them shock energy to damage the

cell surface.

  There is no limitation to cells that can

  can be processed by the process according to the invention.

  Examples of these cells are those delevures,

  of actinomycetes, fungi, bacteria,

  tozarians, protists, algae, plant cells

  and animal or their cell lines.

  According to the invention, it is possible to break or damage

  cell surfaces such as cell walls and cell membranes so as to efficiently extract useful substances, such as proteins, including peptides and enzymes,

  alkaloids, steroids, saponins, anti-

  biotics, carcinostatic, unsaturated higher fatty acids, vitamins, natural sweeteners and other raw constituents as medicaments. It is also possible to damage algal cell surfaces, such as chlorella, spirillina, etc., so as to effectively digest them. There is no limitation to the size and shape of the frozen particles containing cells that can be processed by the process according to the invention, as long as they can be sprayed from a nozzle. But, preferably, the frozen particles containing cells have a diameter of substantially 500 microns or less, since a large projection energy is required to remove particles having a diameter of more than 500 microns. Preferably the frozen cell-containing particles are dry, have a particle diameter of 500 microns or less, and have an internal temperature of -15 ° C or less, preferably -50 ° C or less. , and are frozen

  by one of the following two processes, or by a combination

  These two processes are a first liquid-liquid contact method and a second gas-liquid contact method. In the first method, microfine droplets of liquid from a suspension containing

  cells with a diameter of 500 microns or a diameter

  below this value, drop by drop into a liquid refrigerant consisting of a liquefied gas on the surface of which is preferably generated

  wrinkles. In the second process, micro-droplets

  fines of a suspension containing

  having a diameter of 500 microns or an inferior diameter

  at this value, drop into a gaseous cooling region in which

  a low temperature gas evaporated from a refrigerator

manager of liquefied gas.

  In the present invention, the distance between the position where frozen microfine droplets of liquid containing cells are projected and the position where shock energy is given to them, and which is hereinafter referred to as the point of impact, depends

  the speed and quantity of the particles

  b. But the distance above, which is designated by projection distance, is preferably 10 to 100 mm, since a longer distance makes the surface of the point of impact increases with corresponding increase.

  loss of energy due to shock.

  The frozen particle containing cells,

  used in the present invention, preferably contains

  from 0.005 to 0.5 g of cell per ml. If the

  It contains an excessively large amount of

  lules, not only the medium in which the cells are suspended, but also the liquid species

  inside the cells are frozen and the cells

  are damaged so excessively that it

  pollution of membranous fragments may occur.

  cells and / or constituents of cells within the cell, and that the yield of useful substances to extract from the breast of cells decreases. If the particle contains an excessively small amount of cells, the membranes of the cells are not broken or endangled sufficiently to extract the substances

useful from the breast cells.

  There is no limitation to the medium in which the cells are suspended, as long as it does not affect the properties of the cells or cells.

  useful substances to extract. In general, we can use

  to be conveniently and satisfactorily, as a medium, aqueous media, such as water, a solution

  of saline, a buffer solution, a solution of

  isoosmolar solution, saline solution, etc. We can

  also use aqueous media containing soil

  organic solvents which contain, with the aqueous media mentioned above, hydrophilic organic solvents, such as lower alkanols, such as methanol, ethanol and propanol, and lower alkyl ketones such as acetone, methyl ethyl ketone and diethyl ketone. Organic solvents such as ethylene chloride (mp 83.degree.-35.3.degree. C.), tertiary butyl chloride (mp = -28.5.degree. 51.0 C), chloride

  methylene (m.p.-96 ° C, 41.6 ° C), chloral (mp.

  -57.5 C, eb. 98 C), chloroform (mp 63.5 ° C, eb.

  61.2 ° C) and carbon tetrachloride (mp 22.6 ° C, eb 76.7 ° C), and mixtures thereof. The above-mentioned organic solvents can be mixed with lower esters of fatty acids, such as methyl acetate, methyl propionate and the like. and / or hydrocarbon solvents, such as cyclohexane, cyclopentane, pentane, heptane, benzene, etc.

  There is no limitation to the refrigerant used

  to freeze the medium, provided that it has a boiling point of 15 ° C or less, and preferably -50 ° C or less when used in an aqueous medium. When using an aqueous medium containing an organic solvent, or when using an organic solvent, the refrigerant

  must be easy to evaporate and have a boiling point

  below the melting point of the medium or soil

  efore. In general, refrigerants such as liquid nitrogen, liquid helium, liquid argon, liquid air, etc., which have a temperature of -150 ° C. or less, and, more preferably, from the practical point of view, from -150 ° C. to -200 ° C. When an aqueous medium is used, it is possible to use liquefied carbon dioxide or a liquefied gas such as methane, ethane, propane, etc., as refrigerant. The refrigerant can be used in the form of a liquid, in which the microfine droplets of liquid from the suspension containing cells are poured directly drop by drop and thus frozen by a liquid-liquid contact, yes-in the form of a gas which constitutes a gaseous cooling region, in which droplets. microfines of liquid from

  the suspension containing cells are poured

  drip and are thus frozen by a gas-

  liquid. In both cases, the refrigerant is maintained

  at a temperature below the melting point of

  place used. If the refrigerant temperature is higher than

  below -15 C, the frozen particles of the suspension

  containing cells will melt on their surface and

  thus will agglomerate to each other. It's for-

  the refrigerant must have a temperature of -15 C, or lower than this value and, preferably,

  from -50 C or below this value. If the temperature

  re is -15 C or less than -15 C, frozen particles are obtained which flow easily. If the temperature is -50 C or lower than this

  value, you can freeze gouttelet-

  your microfine liquid suspension containing

cells.

  To prepare frozen particles having a preferred diameter of 500 microns or less, by driping microfine droplets of liquid from the cell-containing suspension into a liquid refrigerant, it is preferred to generate wrinkles having a height of 5 microns. at

mm on the surface of the refrigerant.

  Figure 1 is a flow diagram of an installation suitable for performing a method according to the invention;

  FIG. 2 is a flow diagram of the unit

  manufacturing unit for the frozen particles of FIG. 1;

  Figures 3 and 4 are cutaway views, respectively

  in plan and elevation, illustrating the layout

  of the bubbling duct 3a used as a means for

  wrinkle wrinkles in Figure 2; Figs. 5 (A), 5 (B), 5 (C) and 5 (D) are cross-sectional and elevational views of other examples of

  means for generating wrinkles, which can be used

  instead of the bubbler 3a of Figure 3; FIG. 6 is a diagram illustrating the function of the bubbles generated by the bubble duct 3a of FIG. 3;

  Figure 7 is a sectional and elevational view of

  an exemplary projection means of FIG. 2; Fig. 8 is a sectional and elevational view of another example of feed means 6, which may be used for the unit of Fig. 2; Fig. 9 is a diagram showing a suitable embodiment for practicing the present invention, using two projection means or several projection means;

  Figures 10 (A) and 10 (B) are views, res-

  in perspective and in elevation, schematically, means for recovering destroyed particles; Figure 11 is a sectional elevational view of another apparatus, adapted to allow the implementation of a method according to the invention; Figure 12 is a sectional view in elevation of a variant of the apparatus of Figure 11;

  FIG. 13 is a sectional view, to a larger extent

  scale, of the apparatus of Figure 12; FIGS. 14 to 17 are sectional and elevational views of variants of the apparatus of FIG. 11, and FIG. 18 is a graph illustrating the variations of the evaporation refrigerant generation rate in the cooling gas region of FIG.

  the apparatus of FIGS. 11 to 17, according to the

partition of temperatures.

  Referring now to FIGS. 1-10, a suitable installation will be described.

perform the present invention.

  Figure 1 schematically represents an

  appropriate for the implementation of the procedure

  according to the invention by freezing microfine droplets sprayed with a suspension containing cells, by dropping them drop by drop in

a liquid refrigerant.

  In Figure 1, the reference number 50 indicates

  that, in general, a unit to make

  frozen particles in which particles are

  Jellies, preferably having a diameter of 500 microns or less, are made from a suspension containing cells. We describe

  unit 50 in detail below. The freezing particles

  made by the unit 50 are withdrawn from the bottom of the unit 50 and are sent via a conduit.

  having an elevator 60, on a belt conveyor 70.

  The belt conveyor 70 brings them to a hopper on its sieve screen belt. Air is

  drawn into the space defined inside the courtyard.

  by a suction pump, which is not

  sented. A conduit 81 communicates with the bottom of the

  hopper 81, nitrogen gas or low temperature air

  This can be fed to the hopper via this conduit so as to fluidize the frozen particles therein. Frozen particles in the hopper

  are sent via a conduit 82 to the lateral entrance

  the projection device 91 which is mounted on the recovery box 90. Air or nitrogen gas at low temperature, hereinafter called entrainment gas, is sent to the central input of the projection device 91, through a conduit 93, to accelerate and project the frozen particles to a point of impact. In the box 90, the point of impact is formed by the surface of an obstacle such as a plate 92 hard. Although the distance between the projection device 91 and the point of impact 92 can be determined according to the shape and

  the size of the frozen particles, it is generally

  ral of 10 to 100 mm. The frozen particles are

  thrown by the projection device 91 in

  their own temperature, which is lower than

  their melting point. Then, the projected particles

  tees collide with the 92 hard tray on

which they crash.

  The hard tray 92 described above is only

  an example of the obstacles

  of impact to frozen particles that are projected.

  The obstacles may have a curved surface instead of the flat surface of the hard tray 92. Although the materials, in which these obstacles such as the hard tray 92 are formed, can be chosen

  depending on the hardness and speed of the particles

  the frozen ones that are projected, it is preferable

metals and ceramics.

  Alternatively, the point of impact can be formed by projecting frozen particles from two or more projection devices 91, so that the frozen particles collide with each other.

  with each other, as shown in Figure 9.

  In this case, although there is no limitation to

  angle between each projection device

  this angle is preferably between 60 and

    approximately when distances D1 and D2 between

  positive projection and the point of impact are 10

  at 100 mm. In addition, two points of

  pact by fixing an obstacle such that the plate 92 incur-

  behind the point where the frozen particles collide with one another as shown

in Figure 9.

  It is better, according to the invention, to project

  frozen particles inside the housing

  shown in Figure 10, which is specifically

  to recover the destroyed particles, because otherwise the frozen particles that are projected are sent upwards, when they are destroyed at the point of impact. The case has a diameter of 500 mm and a height of 110 mm. One, two or more projection devices 91 can be mounted in the center of the

  upper surface of the case. When particles

  jellies are projected into the casing, the particles

  destroyed are distributed in a sensitive direction

  horizontally in the bin, while being exposed to

  less pressure, and de-accelerated, due to

  baffles 94 so that they can be collected

lie easily.

  According to the invention, the particles thus

  picked can be destroyed again, if it is

  wish, after being melted and refrozen.

  Referring to FIG.

  detail unit 50 of Figure 1 for manufacturing

frozen particles.

  Fig. 2 is a flow diagram of the frozen particle manufacturing unit 50 of Fig. 1. In Fig. 2, reference numeral 1 indicates a liquid refrigerant storage tank, reference numeral 2 indicates the refrigerant liquid stored in tank 1, reference number 3 indicates

  means for generating wrinkles on the surface of the

  frigating by giving kinetic energy to the area

  refrigerant, the reference number 4 indi-

  that spraying means that mix a suspension

  containing single-gas cells and atomize the suspension

  Commission, reference number 5 indicates ways of

  refrigerant level setting, the order number

  indicates ways to evacuate the freezing particles

  the reference number 7 indicates means of

  pre-cooling of the suspension containing

  intended to be frozen and gas intended to be mixed with the suspension, reference number 8

  indicates a source of refrigerant, the reference number

  reference 9 indicates a source of suspension containing the

  cells and the reference number 10 indicates a source

  of gas intended to be mixed with the suspension

holding cells.

  The storage tank 1 comprises a casing

  square cross-section and a pyra-shaped bottom

  Inverted mide which is stainless steel (SUS 304). The tank 1 has a width of 400 mm, a depth of 400 mm and a height of 200 mm. On the outer wall of the tank 1 is provided an insulating vacuum layer

who is not represented.

  The storage tank 1 contains a liquid refrigerant 2 consisting of liquid nitrogen which is supplied from the source 8 via a pipe 11. The level indicated in L of the refrigerant 2 stored in the tank 1 is maintained

  about 500 mm from the bottom of the tank.

  The level L is set at a predetermined height by using refrigerant level control means, which means includes a level detector 5a, a level control panel 5b, a control valve 5c, and the like.

  The means for generating wrinkles 3 comprise

  a splash duct 3a, a valve 3b for com-

  spear, flow meter 3c, etc. which give kinetic energy to the refrigerant, so that

  wrinkles occur on the surface of the refrigerant 2.

  The splash duct 3a has the shape of a square handle, as shown in FIG. 3, and is fixed horizontally at a level of 40 to 150 mm below the surface of the coolant, as shown in FIG. 3a is set at a lower level, the efficiency of the bubbling decreases, because the bubbling gas from the duct 3a can be excessively cooled and because a more

  large amount of refrigerant and bubbling gas.

  It is therefore desirable that the level of

  bubbler 3a at 100 mm below the

  refrigerant 2 side or at a lower distance

  100 mm from the surface of the refrigerant 2.

  The sparging duct 3a is provided, on the inside, with several orifices 3d from which gas is sent in the form of bubbles, the orifices

being spaced 50 to 100 mm apart.

  The gaseous refrigerant is sent from the gaseous region of the refrigerant source 8, via the conduit 12 and the control valve 3b, and is then sent into the liquid refrigerant 2 through the orifices 3d.

  The amount of gaseous refrigerant that is

  channel 3 is advantageously comprised of

  be 200 and 400 liters / m2.minute. In this range, there is an upward flow of bubbles in the vicinity of the surface of the refrigerant 2. The flow of the bubbles

  13 gives kinetic energy to the surface of the refrigerator.

  manager 2, generating wrinkles W having a ripple height of 5 to 20 mm, as shown in FIG.

  Figure 6. Wrinkles vibrate each freezing particle

  which falls into the refrigerant 2 and thus prevent any agglomeration of the frozen particles. The presence of the bubbles 13 flowing in the refrigerant causes the density of the liquid to decrease in the vicinity of the surface of the refrigerant 2 and thus facilitates the fall of the frozen particles into the refrigerant.

the refrigerant 2.

  The optimal height of the undulations of the wrinkles is about 5 to 10 mm. If the height of the undulations is greater than 20 mm, the wrinkles stir the refrigerant close to its surface and thus influence the

drop of frozen particles.

  In FIG. 2, the splash duct 3a is

  used as a means of generating wrinkles,

  gaseous manager being sent from the gaseous region of the source 8 of the refrigerant. Alternatively, the gaseous refrigerant can be sent to the sparging conduit 3a by another source provided for storing the refrigerant

  gaseous regardless of source 8.

  The gas sent to the bubbler 3a is not necessarily the gaseous refrigerant. It can be any other gas that has a low dew point or that contains CO2 gas. We can use,

  for example, decarbonated air instead of refrigerant

gaseous manager.

  The following means can be used as means for generating wrinkles, instead of

3a bubbling described above.

  (a) Means for generating liquid spray-type ripples: In this case, a duct which has a structure similar to the bubble duct 3, in FIG. 3, is fixed below the refrigerant 2. This duct projects liquid refrigerant from a source 8 by

  its orifices. This conduit does not require the presence of

* duit 111but requires the presence of a recovery duct

  refrigerant unit 1, so as to maintain the surface L refrigerant to a predetermined level. (b) Means for generating oscillation-type wrinkles: In this case, oscillators 20 are mounted which are reciprocated or which rotate at a given speed on the inner wall of the vessel

  1, as shown in FIGS. 5 (A) and 5 (B). These oscillations

  The latters 20 are driven by motors 21 mounted on the outer wall of the vessel 1 and give vibrational kinetic energy to the refrigerant 2, which generates

  wrinkles on the surface L of the refrigerant 2.

  (c) Means for generating acoustic-type wrinkles: In this case, acoustic vibrators 22 are mounted which generate vibrations having a frequency determined in advance in the wall of the vessel 1, as shown in FIG. ). The acoustic vibrators 22 give acoustic energy to the refrigerant 2, this

  which generates wrinkles on the surface L of the refrigerant 2.

  (d) Means for generating spray-type wrinkles:

  In this case, spraying devices

  above the surface of the refrigerant 2. The spraying devices project liquid refrigerant or a gas having a relatively low dew point

  such as gaseous refrigerant and decar-

  on the surface of the coolant and thereby give the kinetic energy to the coolant 2, which

causes wrinkles on the surface.

  (e) Means for generating shake type wrinkles:

  In this case, a tank 1 for refrigeration storage

  Rant is mounted on a shaking table 22 and is imparted kinetic force by shaking by means of cams 23 and springs 24, so as to shake refrigerant 2 and generate wrinkles at the same time.

surface of the refrigerant 2.

  The spraying means 4 mix a sus-

  pension containing single-gas cells and produce microfine droplets of liquid from the mixture of the

  suspension and gas. The microfine droplets must

  have a diameter of 500 microns or a diameter

less than this value.

  Figure 7 shows an example of the spraying means 4, which comprises a body 4a having an inlet 4b for liquid at the posterior end and an inlet 4c for the gas on the posterior side. The body 4a is provided, on the side of the rear part, with two passages 4b 'and 4c' coaxial and, on the side of the part

  previous, a throttle 4d where the suspension contains

  cells are mixed with gas. The mixture of gas and liquid thus produced is then sent to a mixing chamber 4e where the mixture is stirred and

  dispersed by a guide pallet 4f, then is ato-

  put and projected by the orifice of the nozzle 4g. Well

  stretched, the spray device according to the figure

  7 may be replaced by another spray device

  with a different structure or configuration

  ferent, provided that its function of mixing gas with liquid and its atomizing and

  Spraying the gas-liquid mixture is ensured.

  As can be seen best in Figure 2, a

  suspension containing cells to be frozen is

  at the inlet for the liquid 4b spraying means 4, under a feed pressure of 1.0

  at 2.0 kg / cm2 from a source 9, through

  A pressure reducing valve 15, a control valve 16, and a cooling means 7 are provided with a pump 144.

  will be described later. Relatively inert gas

  in the liquid is sent, under a pressure of 1.0 to 2.0 kg / cm 2, from a source 10 to the inlet for the gas 4c of the spraying means 4, through a reduction valve of the pressure 17, a flow meter 18, a control valve 19 and cooling means 7. Alternatively, the gas at the inlet 4c can be introduced from the gas region of

  source 8 of the refrigerant, instead of using the

this 10.

  The ratio of the suspension containing the

  the gas in the spraying means 4 is optimally between 0.5 and 1.5, calculated by the following equation: Liquid (liter / hour) Gas (normal liters / minute)

  By varying the ratio in the range mentioned above

  above, it is possible to modify the diameter of the particles

  jellies of maximum diameter up to one-tenth of that-

  ci, even when the supply pressures, both gas and suspension, are fixed. Although

  more advantageous than the orifice of the nozzle 4c has a di-

  small meter, the orifice 4g preferably has a diameter of about 0.3 to 1.0 mm so as not to have problems

manufacturing and congestion.

  The mixture of gas and liquid which is produced in the throttled portion 4d of the mixing chamber 4e, is uniformly dispersed outside the guide pallet 4f and is then projected downwards by the nozzle 4g onto the surface refrigerant. When he passes

  in the nozzle 4g, the gas is at the same time

  inside and outside the atomic liquid particles

  sé. After passing through the nozzle 4c, the gas inside the atomized liquid particles expands itself and causes the atomized particles to divide into smaller particles, while the gas outside the particles of atomized liquid forces the particles to diffuse in a vigorous manner and thus forces the

  particles to collide with each other

  very much and divide into smaller particles.

  The microfine droplets of liquid thus produced by the spraying means thus become spherical particles because of the surface tension when they fall into the tank 1 and

  then reach the surface of the refrigerant.

  The distance between the projection means 4 and

  the surface of the refrigerant and the temperature inside the

  in the tank 1 have a great influence on the di-

  meter and the shape of the particles that will be

  in the refrigerant 2. The distance must be

  between 500 and 1500 mm. The temperature must be between -15 C or lower than this value and, preferably, be -20 C or be less than

  this value. It goes without saying that distance is decompo-

  between the top of the wrinkles and the means of spraying

  4, although the level of the refrigerant surface

always varies due to wrinkles.

  Means 5 to adjust the level of the surface

  of the refrigerant serve the purpose of maintaining

  the distance between the surface of the

  refrigerant L and the spraying means 4. The means 5 are a combination of the known devices, such as a liquid surface detector 5a, a device for controlling the surface of the liquid 5b,

  a control valve 5c, provided with a feed duct

  refrigerant 5c, etc. The cooling means 7 lower the temperature of the suspension containing cells and a gas before they are introduced into the spraying means 4 and make it possible to save refrigerant. The cooling means 7 comprise a cooling device 7a for liquid and a

  cooling device 7b for gas. Refrigeration

  evaporated manager is introduced from tank 1 to both

  positive cooling 7a and 7b so as to pre-

cool the suspension and the gas.

  In the installation of FIG. 2, the liquid and the gas are pre-cooled separately and are then mixed and atomized in the spraying means 4. Alternatively, the liquid can be mixed first and

  gas in gas and liquid mixing chambers

  which would be provided independently of the means of

  spraying 4 then cool the mixture in the cooling means

  7 and spray it by means of spraying.

4.

  Each droplet K of suspension containing the cells, which has been projected by the spraying means 4, falls into the liquid refrigerant and

  freezes, then can lower independently of

  because the wrinkles generated on the surface of the

  refrigerant prevent frozen particles from

  Mérer. Frozen particles, which have fallen to the bottom of the tank 1, are withdrawn through a pipe 1

having evacuation means 60.

  The evacuation means 60 in FIG.

  take an elevator 60 of pneumatic conveyor type

  somebody sends the frozen particles from unit 1 to

  conveyor 70, passing through a conduit which

  binds. In unit 1 of FIG. 2, a lift of the screw conveyor type, as represented in FIG.

  can be used in place of the elevator 60 and

  takes a guide duct 6a inserted in the tank 1, a rotating screw 6b which is provided inside the guide duct 6a and a drive motor 6c which drives the screw 6b. Belt conveyor type evacuation means and other types, for example an apparatus for discharging frozen particles described in Japanese Patent Application No. 173317/86, may also be used. bottom of a tank, are drawn at the same time

  time that an upward flow of refrigerant bubbling

  through a conduit and then separated from the refrigerant.

  The frozen particles thus withdrawn can

  can be projected by a projection device,

as described above.

  Referring to Figures 11 to 18, we will describe

  other devices that are suitable for

  a process according to the invention.

  Fig. 11 is a diagram of an apparatus suitable for carrying out the process according to the present invention, frozen particles of a suspension containing cells being produced in a gaseous cooling region by gas-liquid contact, against current, a rising gas of evaporated refrigerant and microfine droplets of liquid from the suspension containing cells falling

  drop, the frozen particles being then projected

  in such a way as to collide. This device,

  according to Figure 1, is a variant to the unit of

  Frozen Particle Filling 1 of FIG.

  In Figure 11, the reference number 111

  a tank for making frozen particles, the reference number 112 for collecting the frozen particles, the reference number 113 for the projection means, the reference number 114,

  means for generating refrigerant vapor.

  The tank 111, to make particles con

  jelly, is a tank that is insulated and is sealed and has a square horizontal section, each side having 400 mm. The tank 111 is provided, at the upper end of one of its side walls, with a

  11la output for cooling gas.

  The means 112 for collecting frozen particles comprise a sieve 115 in the form of a pyramid

  inverted and an outlet tube 116, which is vertically

  in the center of the tank 111, below the bottom of the screen 115. The screen 115 is fixed along the inner wall of the tank 111 and divides the interior of the tank 111 into a region for the cooling gas

  117a at the upper part of the tank 111 and in one

  to generate refrigerant vapor 117b at the bottom of the tank 111. The screen 115 is

  wire grid that withstands a tempera-

  It has a low mesh size and has a mesh size such that only refrigerant in the form of steam can pass through it, for example a sieve having a mesh size of 0.8 mm and manufactured in SUS 304. The output 116 leads to

  the outside of the bottom of the tank 111 and is provided with

  near the bottom of a valve 116a-such as a gate valve

  to extract frozen particles. Although the angle e between the sieve 115 and a horizontal line

  should vary according to the amount of refrigerant

  poré, the diameter of the frozen particles and, if one uses scrapers or not, it is typically 450 when one

  do not use squeegee. The sieve can be inclined di-

  from a side wall to the side wall op-

  posed, as shown in Figure 14.

  The projection means 113 comprise a projection device 118 which is mounted in the center of

260418Z

  the upper wall of the tank 111, a conduit 119

  which is connected to the projection device 118, of

  to feed it in suspension containing

  cells, and a conduit 120 which communicates with the

  projection positive 118, so as to supply the driving gas such as nitrogen gas cooled and put under a suitable pressure. By the projection means 113, a suspension containing cells

  is projected downwards by the nozzle 118a provided at

  puts projection device 118, because of the

  pressure of the driving gas. The size of the droplets

  microfine liquid particles, i.e., particles containing cells 124a, may be modified depending on the diameter of the orifice of the

  nozzle and the pressure of the driving gas.

  The means 114, for generating refrigerant vapor, comprise a duct 121 which sends a predetermined amount of refrigerant such as liquid nitrogen 122 to the region o is generated from

  the refrigerant vapor 117b and a bubble duct

  ge 123 which is arranged in the coolant 122 and which is supplied with gas such as nitrogen gas, argon gas and dry air for bubbling steam

of refrigerant 122a.

  In the particle production unit con-

  jelly of FIG. 11, evaporated refrigerant 122a

  passes from region 117b generating evaporative refrigerant

  pored, to the region 117a of cooling gas, passing through the sieve 115 and ascends into the region

  117a to exit 111a. In other words, refrigeration

  Evaporated manager 122a gradually rises to the outlet 11la as its density changes by exchanging heat with droplets of

  suspension containing the cells 124a which are projected

  Tees. On the other hand, as the droplets

  letters 124a containing cells fall into the

  117a of the cooling gas, they exchange

  of heat with the refrigerant 122a evaporated ascending

  by counter-current gas-liquid contact and freeze. Since the droplets 124a are free

  in region 117a in the gaseous phase of refrigeration

  evaporated, they are shaped into

  spherical jellies, because of their own surface tension. Since the droplets 124a only come into contact with the ascending evaporated refrigerant, no agglomeration of droplets occurs.

  124a, the droplets 124a are frozen independently.

  from each other and thus obtain parti-

  frozen eggs having a uniform diameter.

  In the device of Figure 11, the

  frozen capsules thus obtained fall on the

  115. As the sieve 115 is inclined, the particles

  the frozen 124 are collected easily without accumulating on the screen 115. The evaporated refrigerant,

  which passes upwards, through the sieve 115,

  holds the temperature of the frozen particles that have

  reaches the sieve 115 and fluidizes the particles

  frosts. Refrigerant also prevents the

  frozen cules on sieve 115 see their hardness

  decrease and they agglomerate.

  An experiment carried out on the device of

  Figure 11 shows that the effective height of the

  Cooling gas 117a, i.e. the distance H between the nozzle 118a and the screen 115, can be decreased to about 1 meter, to obtain high quality frozen particles 124b, provided that the three the following parameters are satisfied: a) the speed of the suspension containing cells

  from the nozzle 118a is 0.2 liter / minute (12

  hour / hour), which is sufficient to maintain the average temperature at -80 C or below -80 0C in the lower side of the region

  117a of the cooling gas, i.e. close to

  mity of the upper part of the sieve 115; (b) the

  projected particles 124a have a diameter of 300 microns or a diameter less than this value; and (c) the projection pressure of the suspension containing

cells is 4 kg / cm2.

  In the device of FIG. 11, there is a close relationship between the temperature of the region of the

  cooling gas and the rate at which the

  Evaporated refrigerant 122a is generated. Figure 18 illus-

  the distribution in a horizontal plane of the temperature

  erosion of the 117a region of the cooling gas

  when the speed at which the gas is generated at

  liquid nitrogen is 20, 40 and 60 m3 normal / hour, while the suspension containing the cells is projected at a rate of 0.2 liter / minute, that the space charge is 75 kg / m2h, and that the charge of the cooling gas is 250 m3 normal / m2h when the surface in horizontal section of the gas region

cooling is 0.16 m2.

  Figure 18 shows that 40 normal m3 / h of evaporated refrigerant is required to maintain

  the average temperature of the region of the cooling gas

  at -80 C or lower, if the

  The horizontal straight line is 0.16 m2, if the suspension containing cells is projected at a flow rate of 0.2 liters / minute (12 liters / hour), and if the projected microfine droplets of the suspension containing cells have a diameter. 300 microns, or a diameter smaller than this value. In this case, the distance that

  required to obtain sufficient time to

  freeze the projected microfine droplets of suspen-

  containing cells is about 1 meter.

  As the effective distance H of the region 117a of the cooling gas can be decreased to about 1 meter in the manner mentioned above,

  the entire unit can be designed in a very

tite, just like the tank 111.

  It is possible to project the suspension

  holding cells in the vertical direction, as shown in Figure 11, if the projection pressure

  is low or if the projected droplets or

  Frozen ticles have a small diameter. Otherwise, the tank 118 should be large enough to provide a certain amount of contact time between the projected droplets 124a and the evaporated coolant 122a if a suspension containing the cells is projected from the nozzle 118a in the vertical direction, as shown in FIG. But, if the suspension is projected in the horizontal direction, as shown in Figure 14,

  the duration of bringing the droplets 124 into contact with each other

  evaporated gas 122a can be ensured without

a large tank.

  The frozen particles 124b that have fallen on the sieve 115 move automatically along the sieve 115 and are collected in the evacuation conduit 116, since the sieve 115 is inclined toward the sieve 115.

  down to the evacuation conduit 116, and then

  evaporated refrigerant 122a passes up through the screen 115. The frozen particles thus collected are discharged out of the chamber.

  tank 111 through the evacuation valve 116, and are then projected

  on the tray 128 of hard material.

  The conditions of particle production con-

  jelly, from microfine droplets of suspen-

  containing cells that are projected, can be determined experimentally by the specialist and in particular, for example, the projection rate of the suspension containing cells, the temperature of the

  region of the cooling gas where the particles are

  the frozen, the rate of formation of the evaporated refrigerant which is necessary to maintain constant the temperature of the region of the cooling gas, the particle diameter which is necessary there, the falling speed of the frozen particles, the contact time between the particles frozen and refrigerant gas, etc. Collecting effectively frozen particles 124b in the evacuation conduit 116 depends

  the angle and inclination of the sieve 115, the temperature

  sieve 115, the evaporated refrigerant velocity

  passing through the sieve 115 and the diameter of the

  frozen eggs. Nevertheless, whatever these parameters

  very, we can collect in an efficient way the

  frozen particles 124b in the evacuation conduit 116

  by giving the screen 115 a vibration, an oscilla-

  continuous or intermittent shaking, using a device such as a vibrator, which is not

not represented.

  As shown in FIGS. 12, 13 and 15 to 17, the means 112 for collecting frozen particles may be of the type which sends the produced frozen particles 124b into a tank 111 directly to means 125 which are intended to project the frozen particles and which are provided outside the

tank 111.

  Referring to FIG. 12, the tank 111 for making frozen particles is provided, on the

  superior, of a chamber 126 of recovery of the

  their perduequi communicates with the tank 111 by an exit 1lla for gas. A projection device, which projects frozen particles, is mounted at the bottom of the chamber 126. In the device of the

  FIG. 12, means 112 for recovering particles

  the frozen ones comprise a conduit 102 for discharging the frozen particles which directly puts the bottom of the

  sieve 115 in communication with the projection device

  125. The projection device 125 communicates

  also with a conduit 127 for a driving gas

  is lying. As entrainment gas, air is introduced

  under pressure, nitrogen gas, etc., in the

  projection 125 through line 127, the particles

  the frozen 124b being sucked by the conduit 116 to the projection device 125, by virtue of the ejection effect, then are projected onto a tray

128 made of hard material.

  The conduit 127 has, within the chamber 126, a portion 129 which serves as a heat exchanger for exchanging heat between the stripping gas and the evaporated refrigerant 122a entering the chamber 126

  by the exit 111a. Part 129 exchanging

  it is shaped like a serpentine, with thick walls,

* with a number of fins 129a, as

  Figure 13, to increase the contact area with the evaporated refrigerant and effectively conduct heat and also to increase heat build-up. Part 129 is preferably made of alloy

  copper having a high thermal conductivity.

  It is thus possible to pre-cool sufficiently gas

  to keep the particles 124b freezing

  to a sufficient hardness and to prevent any

  agglomeration of the frozen particles 124b in the

  positive projection 125 and thus project the particles

  to break or effectively damage cell membranes. As the 129 part has a thick wall with a number

  129a of the fins, it is possible to pre-cool the entrainment gas

  because of heat build-up, even though

  evaporated gas 122a is not generated during operation.

  intermittent operation of the manufacturing unit of

  frozen particles. This heat exchanger can pre-

  cooling the driving gas to the temperature of the evaporated refrigerant from the outlet 111a. But, if necessary, it is possible to cool the gas

  at a much lower temperature in the

  flowing a condenser to the coil such as liquid nitrogen from a nozzle 130 mounted on the upper part of the heat recovery chamber 126.

  As shown in Figure 15, we can use

  ser, instead of the inclined sieve 115 shown in Figures 11, 12 and 14, a sieve plane 115 associated with a squeegee 132 reciprocated above the sieve

  by a jack 131, so as to send the particles

  the frozen 124a accumulating on the sieve in a hopper 133a. As a driving gas is introduced into the spraying device 125, frozen particles 124b collected in the hopper 133a are sucked to the spraying device 125.

  then are projected onto the plate 128 made of hard material.

  In addition, the means to collect

  frozen eggs may include a sieve which

  place from the inside to the outside of the tank 111. A

  example is given in Figure 16 and includes a trans-

  carrier 135 having a sieve runner 115 ', an end portion of the conveyor 135 being disposed in the

  chamber 133 for recovering frozen particles.

  The transporter 135 subdivides the interior of the tank 111 into two regions 117a and 117b and is driven into

  the direction indicated by an arrow in Figure 16,

  seeing frozen particles 124b accumulating on the sieve chute 115 ', from the interior of the tank 111 to a hopper 133a of the chamber 133. Another example is given in Figure 17 and includes a sieve-shaped tray 115 "which rotates about a vertical axis by being driven by a suitable drive mechanism 136. In this case, frozen particles 124b accumulating on the tray 115" of the tank 111 are sent to the chamber. recovery 133 and collected

  in the hopper 133a using a doctor blade 137 suitable for

  nable. In FIGS. 11 to 17, the spraying means

  118 may be the spray device

  shown in Figure 7; the distance between the spraying means 125 and the plate 124 and the material and

  The shape of this plate can be chosen as men-

  mentioned above with respect to the device of FIG. 1; and we can perform the projection and recovery

  frozen particles in the manner illustrated in FIGS.

res 9 and 10.

Example

  We will now explain a production process

  frozen particles containing cells following

  the present invention with reference to the examples

  following, to which the invention is not limited.

Example 1

  A yeast is suspended in water

  living bread preparation, available in the com

  made by Toyo Jozo Co., for

  prepare a yeast suspension at about 0.1 g / ml.

  Then we perform an experiment using

  the device shown in FIG. 1 and described above.

  above. A tank having a square body of

  400 mm x 400 mm cross section and 900 mm high

  and a 300 mm inverted pyramid-shaped

  height, as storage tank 1 of the refrigerated

  liquid of said device. Next, liquid nitrogen is charged as liquid refrigerant 2 until

  the height of 500 mm from the bottom of the tank,

  shown in Figure 2. In other words, the distance between the top of the tank and the surface L of the

  Liquid refrigerant is 700 mm.

  In addition, there is disposed horizontally 50 mm below the surface of the liquid, a conduit 3 of

  bubbler shaped like a square loop (350 mm x 350 mm). The conduit 3 is sent nitrogen gas from

  the gas phase region of a liquid nitrogen source 8

  at a rate of 300 liters / m2; minute and it is projected

  through the openings 3d inside the tank 1 for

  generate wrinkles of medium ripple height

  8 mm on the outside surface of liquid nitrogen.

  The yeast suspension is introduced under the

  form of a liquid to be frozen and azo

  gas at 25 C, stored in a tank 10 high

  pressure in the form of gas, in means of

  4, at flow rates of 0.2 liter / minute and.

  1 liter / minute respectively, so that

  in the means of spraying 4. The means of

  4 are placed at about 700 mm above

  above the L surface of liquid nitrogen and have a

  nozzle orifice with a diameter of 0.7 mm. Then we

  throws the mixture of gas and liquid on the surface

  liquid under a projection pressure of 4 kg / cm 2.

  The highest temperature of the upper space of

the tank 1 is -20 C.

  On the other hand, to maintain the surface of the liquid nitrogen 2 in the vessel 1 at a constant level,

  we send liquid nitrogen from the conduit II to the inte-

  the tank at a rate of 20 liters / hour. Of the-

  bit of nitrogen gas leaving the tank is 20 x 0.65 m3 normal / h, in order to precool the yeast suspension and the nitrogen gas to be mixed by

the cooling means 7.

  When the mixture of the yeast suspension and the nitrogen gas is continuously sprayed by the spraying means 4 in about 10 minutes under the conditions mentioned above, about 1000 ml of frozen particles containing cells are obtained. baker's yeast in the form of ice spheres. The average dimension of the spheres is

between 50 and 100 microns.

  The ice spheres are stored by means of an elevator 60 and a conveyor 70 in a storage device to which nitrogen gas is supplied at a pressure of 2 kg / cm 2 and at a temperature of 70 ° C. in order to fluidize the ice spheres from the bottom and to maintain the temperature of the ice spheres at -70 C. Then they are projected, by a device

  projection 91 having a nozzle of 8.3 mm in diameter.

  The gaseous nitrogen gas introduced into the central inlet duct of the ejection device 91 is under an acceleration pressure of 4.7 to 5 kg / cm 2 and at a temperature of -70 ° C., while the temperature Spheres of ice, after having been thrown by the nozzle, is -70 ° C. Spheres of ice are sprayed under the above conditions, while fixing the projection distance D at 20 mm, and at 50 mm, then the respective destroyed particles containing cells are collected

yeast.

  Suspended in water, as a sample, respectively yeast suspension, ice pheromone containing yeast cells and destroyed particles containing cells.

  yeast. Then they are separated by centrifugation to obtain

  a fraction of yeast cells and a fraction

  supernatant for each sample.

  The proteins for supernatant fractions are quantitatively determined by the Lowry method, and enzymatic activity by means of

  alcohol dehydrogenase. The results obtained are considered

Table 1.

(Test method).

  The alcohol dehydrogenase activity is measured by

  absorptiometry using 1.00 ml of a reaction mixture

  liquid solution comprising 0.63 ml of 0-0.05 M barbital buffer (pH 8.6), 0.10 ml of a 0.25% aqueous solution of nitrotetrazolium blue (NTB),

  made by Dojin Kagaku Institute, 0.02 ml albumin

  10% mine (bovine serum), that is Fraction V Powder manufactured by Armor Pharmaceutical Co., 0.10 ml of 10 mM NAD, ie NAD + manufactured by Oriental Kobo Co., 0.05 ml of diaphrasis (DI) at 100 U / ml, manufactured by Toyo Jozo Co.,

and 0.10 ml of ethanol 1.M.

  On the one hand, after loading 1.00 ml of the

  liquid reaction ge in a glass cell and

  have heated it preliminarily at 37 C for 5 minutes

  10 ml of the samples described above are poured into the liquid reaction mixture and left at 37 ° C. for 10 minutes. Then add 2 ml of acid

  0.1 N hydrochloric acid to complete the reaction and

  terminates the absorbance As of the sample to a length

wave of 550 microns.

  On the other hand, after loading 1 ml of:

  In a glass cell and left at 37 ° C. for 15 minutes, 2 ml of 0.1 N HCl and 10 microliters of the sample are poured into the liquid reaction mixture. Absorbance Ab is then determined as a control

wave of 550 microns.

  From the absorbance As of the sample

  and the absorbance Ab of the control, the activity is determined.

  Alcohol dehydrogenase fraction of the sample by the following equation: (activity) = (As-Ab) / 3.01

16.4 0.01

  wherein: 16.4 is the molar extinction coefficient of reduced type NTB (cm2 / Pmole) is the reaction time (minute) 3.01 is the finally dissolved amount (ml) 0.01 is the amount of solution of enzyme (ml). The quantitative determination of the

  proteins by the method of Lowry as described above.

  below. After intimately mixing 0.5 ml of

  the sample to 5 ml of reagent A prepared by the

  described below, in a glass cell, and after leaving the mixture at room temperature for 10 minutes, is added rapidly to the mixture.

  0.5 ml of Folin reagent prepared by diluting

  commercially available (manufactured by the

  tee Wako Seiyaku Co.) twice in the water, then

  leave it for 30 minutes. We then measure the ab-

  sorbance at 750 microns by an absorpti

  and determine the quantitative determination with respect to the calibration curve of serum albumin

beef.

  Reagent A is an alkaline reagent prepared by mixing a reagent (1) obtained by dissolving NaCO3 in 1N NaOH in a concentration of 2%, and a reagent (2) obtained by dissolving CuSO4,5H20 in 1% sodium citrate, in a concentration of 0.5% just before use.

Example 2

  Using the same yeast suspension as

  Example 1, prepared frozen particles containing

  yeast cells under the same conditions

  than in Example 1, except that the flow rate of the

  yeast pension is 0.2 ml / minute, the nitrogen gas flow rate of 1 liter / minute, the nozzle diameter of 0.5 mm and the spray pressure is

between 2 and 2.5 kg / cm.

  Ice spheres containing yeast cells having a diameter of 200 to 300 are obtained

microns.

  In the same way as in Example 1, the ice spheres are sprayed, then the destroyed particles containing yeast cells are collected. We perform

  then an analysis in the same way as in Example 1.

  The result obtained is shown in Table 2.

Example 3

  We perform an experiment in the same way

  than in Example 1, except that a suspen-

  yeast containing 0.0583 g / ml cells

  yeast per ml. The experiment is carried out with a distance

  this projection D of 20 mm only.

  The result obtained is shown in Table 3.

  Comparative Example Yeast cell bodies were powdered using a commercially available cell sprayer, that is,

  Dyno-Mill KDL manufactured by Willy A Bachofen Maschinen

  Fabrik. At 2000 revolutions per minute

  a 0.6 ml volume grinding mill for use in the Dyno-Mill and filled with glass beads of the MK-2G type having a particle size corresponding to diameters between 0.25 and 0.5 mm . To carry out the destruction of the yeast cells, a suspension containing yeast cells which has been added to the container at a rate of 100 ml / minute is continuously added to the container.

  prepared by placing 0.23 g / ml of live yeast

  born in bread making and made by Toyo

Jozo Co. in water.

  The result obtained is shown in Table 4.

Example 4

  An experiment is carried out in the device shown in FIG. 12, using solutions diluted half and quarter of the yeast suspension.

  at 0.1 g / ml. The production vessel of the freezing particles

  in the device consists of a tank having a square body portion of 400 mm x 400 mm of straight section and 1400 mm in height and provided at the bottom with a metal screen element 115

  SUS 303, with a mesh size of 0.105 mm and

  at an angle 6 of 60. Liquid nitrogen is charged as a liquid refrigerant to a height of about 100 mm from the bottom of the vessel. The distance between the highest part of the sieve element and the surface of the liquid nitrogen is about 300 mm

  and the distance between the highest part of the

  sieve and the nozzle is approximately 1000 mm.

  In addition, an upward gas is generated under

  of refrigerant, at a flow rate of normal m3 / hour, to maintain at -80 C approximately the temperature inside the manufacturing tank

frozen particles.

  Then, using the yeast suspension! as the liquid to be frozen and the nitrogen gas at 25 ° C., as the mixing gas, they are sent to spraying means 118 situated approximately 1000 mm above the highest part of the screen-shaped element, and having a nozzle orifice of a diameter of

  0,5 mm at a flow rate of 0,2 liter / minute for the suspension

  yeast and at a rate of 5 liters / minute for nitrogen gas. They are mixed in the means of

2604 182

  spraying 118, then the mixture is sprayed onto the

  under a spray pressure after the

age of 3.5 kg / cm 2.

  After continuously blowing the yeast suspension and nitrogen gas for about 10 minutes under the conditions mentioned above, about 1000 ml of ice spheres containing yeast cells are obtained. The average size of spheres

is about 100 microns.

  The ice spheres thus obtained are then projected continuously, the projection device 125, which has a nozzle of 8.3 mm in diameter, while maintaining the temperature of the product at -70 C. In this case, the nitrogen * The gaseous feed gas delivered by the central inlet duct of the projection device 125 is under an acceleration pressure of 4 kg / cm 2 and has a temperature of -70 ° C. The temperature of the ice spheres after

  projection of the nozzle, is -70 C. In addition, the dis-

  between the nozzle and the plate 128 of hard material

is 20 mm.

  After the screening, particles are collected

  the destruction of ice spheres containing

  the same analyzes as in Example 1. The results indicated in FIG.

table 5.

Example 5

  The experiment is carried out under the same conditions

  than in Example 4, except that only

  the yeast suspension at 0.05 g / ml, the flow rate of the yeast suspension at 0.2 liter / minute, the flow rate of nitrogen gas at 1 liter / minute and

  the spraying pressure of the spraying means

  118 to 25 kg / cm 2 after mixing. Ice spheres containing yeast and having a diameter of about 20 microns in the unit are thus obtained.

2 6 0 4 1 82

  manufacturing frozen particles.

  After the screening, the particles are collected

  the destroyed ones containing yeast, and they are

  apply the same analyzes as in example 1. It is

  keep the results shown in Table 6.

Table 1

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g: :) (mg / g: :) Yeast suspension (0.1 g / ml) Ice spheres containing 101 108 yeast cells Particles I destroyed containing cells of 99 158 yeast (projection distance 20mm) Particles II I destroyed containing cells of 1058 yeast (projection distance 1 50mm) ::: g indicates the weight of the yeast cell

Table 2

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g: :) (mg / g :) Yeast suspension (0.1 g / ml) 3 Ice spheres containing 55 69 yeast cells

Particles I destroyed

  containing yeast cells 65 107

(projection distance

20 mm)

Particles II destroyed

  containing yeast cells 73 121

(projection distance

50 mm)

Table 3

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g: :) (mg / g: :) 3 yeast suspension (0.0583 g / ml)

Ice spheres con-

  holding 115 155 cells of yeast Particles destroyed

containing cells

  yeast (spraying distance (20 mm)

Table 4

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g ':) (mg / g: :) Yeast suspension 3 (0.23 g / ml) Solution treated at 245 165 Dn-il245 165 Dyno-Mill

Table 5

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g ') (mg / g:') Yeast suspension 1 (0.025 g / ml) Ice spheres containing 189 87 yeast cells Particles destroyed

containing cells

  Yeast Yeast (Projection Dispersion mm) Yeast Suspension 26 (0.05 g / ml)

Ice spheres contained

  217 102 Yeast cells destroyed

containing cells

  yeast (projection 469 mm mm)

Table 6

  Alcohol activity Dehydrogenase content: protein: supernatant supernatant fraction (U / g: :) (mg / g: :) Yeast suspension 26 (0.05 g / ml) Ice spheres containing cells yeast Particles destroyed

containing cells

  yeast (20 mm spraying distance)

Claims (14)

  1. A method of treating cells in a manner   it facilitates the extraction of useful substances from their breast, which consists in projecting frozen particles containing cells in such a way as to cause the particles to collide and to give them shock energy so as to damage them the cell surface.   2. Process according to claim 1, characterized   rised in that it consists in giving energy due to   shock by bringing the frozen particles into read with each other.   3. Process according to claim 1, characterized   it is to give the particles the energy of the shock by bringing in the particles   frozen in collision with obstacles.   4. Method according to one of claims 1   at 3, characterized in that the frozen particles have   a diameter of 500 microns or less than this value.   5. Method according to one of claims 1   to 4, characterized in that it consists in projecting the   frozen particles by a projection device.   6. Process for treating cells so as to facilitate the extraction of useful substances from their breast, characterized in that it consists in producing microfine droplets of liquid having a diameter   500 microns or less than this value, of a   pension containing cells, to put the droplets   microfine liquids in contact with a refrigerant to produce frozen particles having a diameter of substantially 500 microns or less, and then to spray the frozen particles in a colliding manner   and give them energy due to the shock, in order to damage the surface of the cells.   7. A process according to claim 6, characterized   risky in that it consists in producing the particles   frozen by dropping the droplets   microfine bottles of liquid, with a diameter of 500   or less than this value, in a refrigerant consisting of a liquefied gas, so as to freeze the microfine droplets of liquid by a contact   liquid-liquid with the refrigerant.   8. A process according to claim 7, characterized   rised in that it consists in dropping   the microfine droplets of liquid, having a diameter of 500 microns or less than this value, in a refrigerant on the surface of which wrinkles are generated.   9. A process according to claim 6, characterized   risky in that it consists in producing the particles   frozen by dropping the droplets   microfine liquids, having a diameter of 500 microns or less, in a cooling gaseous region, with a refrigerant in the form of   ascending vapor in order to freeze the droplets   microfine liquids by a gas-liquid contact with the refrigerant.   10. Process according to one of the claims 6   at 9, characterized in that it consists in giving energy due to the shock by bringing in the particles   frozen collide with each other.   11. Method according to one of claims 6   at 9, characterized in that it consists in giving energy due to the shock by bringing the frozen particles into collision with obstacles.   12. Process according to one of the claims 6   at 11, characterized in that the suspension contains 0.005 g to 0.5 g of cell per ml.   13. Process according to one of the claims 6   at 12, characterized in that it consists in producing microfine droplets of liquid using a sprayer.   14. Process according to one of the claims 6   13, characterized in that it consists in projecting the   frozen particles by a projection device.