GB2196018A - Process for treating cells by use of frozen particles of cells-containing suspension - Google Patents

Description

GB2196018A 1

SPECIFICATION

Process for treating cells by use of frozen particles of cells-containing suspension This invention relates to a process for treating cells so as to facilitate the extraction of useful 5 substances from the cells. More specifically, it relates to a process for treating cells which comprises ejecting frozen particles of cells-containing suspension so as to collide and thus imparting an impact energy to cell surfaces.

It has been known that substances in a cell of such a microorganism as yeasts or bacterium can be efficiently extracted by freezing and milling the cell. 10 For example, Japanese published unexamined patent application No.29983/78 discloses a process wherein cells such as yeasts are frozen by contacting them with a liquefied gas to form particles having a diameter of 1-5 mm and then the resulting frozen cells are milled by such a mill as a screw feeder at a very low temperature under a low-temperature gas atmosphere.

However, this process cannot efficiently break or damage the cells since it freezes liquid moiet- 15 ies inside of the cells and such large. particles having a diameter of 1- 5 mm are required to be pulverized by the aforementioned mill.

Japanese patent publication No. 869/83 discloses a process wherein a suspension which contains a microorganism at a certain concentration is frozen to form particles having a diameter of about 5-10 mm or a volume of 1 cm3 and then the resulting frozen particles are pulverized 20 as they are. However, since this process employs relatively large frozen particles as described above, the mills to be employed are limited, for example, to hammer mills, ball mills and the like. Further, it is difficult to control a milling condition suiable for extracting pure substances from cells when these mills are used. Because heat generated during their operation changes the properties of substances to be extracted, contamination of fragments of balls may occur, and 25 these mills often so excessively grind cells that contamination of pulverized fragments of cell membrances and of cell constituents inside of the cell may occur.

It is an object of the present invention to provide a process for treating cells which is easy to control an operation and continuously breaks or damages the cells so as to facilitate the extraction of useful substances in high purity without property changes of the useful substances 30 by heat as well as without contamination of grinded cell membranes or cell constituents.

As a result of the intensive research, the present inventors have found out that the above defects of the conventional processes can be eliminated by ejecting frozen particles of cells containing suspension to make a collision so tht an impact energy can be imparted to the cell.

According to one aspect of the present invention, there is provided a process for treating cells 35 so as to facilitate the extraction of useful substances from the inside of the cells which comprises ejecting frozen particles containing cells therein so as to collide and thus imparting an impact energy to the particles so as to break or damage the surfaces of the cells.

According to another aspect of the present invention, there is provided a process for treating cells so as to facilitate the extraction of useful substances from the inside of the cells which 40 comprises producing liquid microfine droplets having diameter of 500 microns or less of cells containing suspension, and contacting the liquid microfine droplets with a coolant so as to produce frozen particles having diameter of substantially 500 microns or less, and then ejecting the frozen particles so as to collide and thus imparting an impact energy to the frozen particles so as to break or damage the surface of the cells. 45 Cells which can be treated by the present invention are not limited. Examples of the cells are those of yeast, actinomycetes, fungi, bacteria, protozoan, protista, algae, and plant cells and animal cells or their cell lines.

According to the present invention, cell surfaces such as cell walls and cell membranes can be broken or damaged so as to efficiently extract substances such as proteins including peptide and 50 enzyme, alkaloid, steroid, saponin, antibiotics, carcinostatics, unsaturated higher fatty acids, vitamins, natural sweetening, and other crude drug co,ponents. Also, cell surfaces of algae such as chlorella, spirillina, etc. may be damaged so as to be efficiently digested.

The frozen cell-containing particles of the present invention are not limited in size and in shape as long as they can be ejected from a known nozzle. However, preferably, the frozen cells- 55 containing particles substantially have diameter of 500 microns or less since a large amount of ejecting energy is required to blast out particles having diametr of more than 500 microns. More preferably, the frozen cells-containing particles are those being dry, having particle diameter of 500 microns or less and having their own temperature of -15'C or lower, preferably -50'C or lower, which are frozen by either or the combination of the following two methods: A first 60 liquid-liquid contact method and a second gas-liquid contact method. In the first method, liquid microfine droplets of cells-containing suspension having a diameter of 500 microns or less are dropped into a liquid coolant of a liquefied gas on the surface of which ripples are generated preferably. In the second method, liquid microfine droplets of cells- containing suspension having diameter of 500 microns or less are dropped within a cool gas region where a low-temperature 65 2 GB2196018A 2 gas which vaporizes from a coolant of a liquefied gas is ascending.

In the present invention, the distance between the position where frozen cells-containing liquid microfine droplets are ejected and the position where an impact energy are imparted to the particles which is hereinafter referred to as a impact point, can be varied depending upon velocity and quantity of particles to be ejected. However, the above distance which is referred 5 to as eject distance is preferably 10 to 100 mm since the longer distance casuses the area of the impct point to be expanded and thus loss of the impact energy increases.

The frozen cells-containing particle used in the present invention preferably contains 0.005-0.5 grams/mi. of cells. If the particle contains an excessively large amount of cells, not only medium in which cells are suspended but also liquid moieties inside of the cells are frozen and thus the 10 cells are so excessively damaged that the contamination of fragments of cell membranes and of cell constituents inside of the cells may occur and thereby yield of useful substances to be extracted from the inside of the cells is decreased. And if the particle contains an excessively small amount of cells, membranes of cells are not broken or damaged enough to extract useful substances from the inside of the cells. 15 The medium used in the present invention in which cells are suspended is not limited as long as it does not affect properties of cells or useful substances to be extracted. Generally, aqueous mediums such as w ater, physiological salt solution, buffer solution, isoosmolar solution, saline solution, etc. can be used as a medium conveniently and satisfactorily. Organic-solvent contain ing aqueous mediums which contain with the above aqueous mediums hydrophilic organic sol- 20 vents such as lower alkyl alcohols including methanol, ethanol and propanol and lower alkyl ketones including acetone, ethyl methyl ketaone and diethyl ketone, may also be used. Further, organic solvents such as ethylene chloride (mp -35.3'C, bp 83.7'C), tert- butyl chloride (mp -28.5'C, bp 51.0'C), methylene chloride (mp -96'C, bp 41.6'C), chloral (mp -57.5'C, bp 98'C), chloroform (mp -63.5'C, bp 61.2 'C) and carbon tetrachloride (mp - 22.6'C, bp 763'C), 25 and the mixture thereof, may also be used as a medium. The above organic solvents may be mixed with lower fatty acid esters such as methyl acetate, methyl propionate etc. and/or hydrocarbon solvents such as cyclohexane, cyclopentane, pentane, heptane, benzene, etc.

The coolant used in the present invention in order to freeze the above medium is not limited as long as it has a boing point of - 15'C or lower, preferably -50'C or lower when an aqueous 30 medium is used. When organic-solvent containing aqueous medium or an organic solvent is used, the coolant should be easy to vaporize and have a boiling point lower than the melting point of the medium or the solvent. Generally, preferred are such coolants as liquid nitrogen, liquid helium, liquid argon, liquid air, etc. which have a temperature of - 150'C or lower, more preferably from the practical viewpoint, - 150'C to - 200'C. When an aqueous medium is used, 35 liquefied carbon dioxide gas or such liquefied gas as methane, ethane, propane, etc. may be used as a coolant.

The coolant may be used in the form of liquid in which liquid microfine droplets of cells- containing suspension are directly dropped and thus frozen by liquid- liquid contact, or in the form of gas which constitutes a cool gas region in which liquid microfine droplets of cellscontaining suspension are dropped and thus frozen by gas-liquid contact. In both cases, the coolant is held at a temperature lower than the melting point of the medium used. If the temperature of the coolant is higher than -15'C, frozen particles of cells-containing suspension will melt on their surface and thus will aggregate each other. Therefore, the coolant should have a temperature of - 15'C or lower, preferably -50C or lower. If the temperature is - 15'C or 45 lower, easy-to-flow frozen particles are obtained. If the temperature is - 50C or lower, liquid microfine droplets of cells-containing suspension can be instantly frozen.

In order to produce frozen particles having preferred diameter of 500 microns or less by dropping liquid microfine droplets of cells-containing suspension into a liquid coolant, it is preferred to generate ripples having a height of 5-20 mm on the surface of the coolant. 50 Figure 1 is a flow diagram of an apparatus suitable to conduct a process according to the present invention; Figure 2 is a flow sheet of frozen particles making unit of Fig. 1; Figures 3 and 4 are respectively plan and elevational views in section which show an arrange- ment of bubbling pipe 3a used as a ripple generating means in Fig. 2; 55 Figure 5 (A), (8), (C) and (D) are elevational views in section of other examplwes of ripple generating means which may be used in place of the bubbling pipe 3a of Fig. 3; Figure 6 is a schematic illustration which shows a function of bubbles generated from the bubbling pipe 3a of Fig. 3; Figure 7 is an elevational view in section of an example of spraying means 4 of Fig. 2; 60 Figure 8 is an elevational view in section of another examoles of discharging means 6 which may be used for the unit of Fig. 2; Figure 9 is a schematic illustration which shows an embodiment suitable to conduct the present invention using two or more spraying means; Figures 10 (A) and (B) are resectively a schematic perspective view and an elevational view in 65 3 GB2196018A 3 section of meanws for recovering destroyed particles; Figure 11 is an elevational view in section of another apparatus suiable to conduct a process according to the present invention; Figure 12 is an elevational view in section of a modification of the apparatus of Fig. 11; Figure 13 is an expanded sectional view of a part used in the apparatus of Fig. 12; 5 Figures 14 to 17 are elevational views in section of other modifications of the apparatus of Fig. 11; and Figure 18 is a graph which shows relationships between a temperature distribution and a generation rate of vaporized coolant in the cool gas region of the apparatus of Figs. 11 to 17.

Referring now to Figs. 1 to 10, an apparatus which is suitable for conducting the present 10 invention will be set forth hereinafter.

Fig. 1 diagrammatically shows an apparatus suitable for one aspect of the process accorcing to the present invention wherein sprayed microfine droplets of cells- containing suspension are frozen by dropping them into a liquid coolant.

In Fig. 1, the numeral 50 generally. indicates an unit for making frozen particles wherein frozen 15 particles preferably having particle diameter of 500 microns or less are made from cells-contain ing suspension. The unit 50 will be described below in detail. The frozen particles made by the unit 50 are discharged from the bottom of the unit 50 and transferred through a conduit having a lifter 60 onto a belt conveyor 70. The belt conveyor 70 transfers to a hopper 80 the frozen particles on its screen-like mesh belt. Air is sucked from the inside of the belt by a suction 20 pump not shown. A conduit 81 is connected to the bottom of the hopper 80, through which low-temperature nitrogen gas or air is supplied to the hopper 80 so as to fluidize the frozen particles stored therein. The frozen particles in the hopper 80 are led through a conduit 82 to the side inlet of ejecting device 91 which is mounted on the recovering box 90. Low-tempera ture nitrogen gas or air which is hereinafter referred to as drive gas is supplied to the central 25 inlet of the ejecting device 91 through a conduit 93, in order to accelerate and eject the frozen particles to an impact point. In the box 90, the impact point is formed by the surface of such an obdtacle as a hard plate 92. Although the distance between ejecting device 91 and the impact point 92 may be determined depending upon the shape and the size of frozen particles, it is generally 10-100 mm. The frozen particle are ejected from the ejecting device 91, keeping their 30 own temperature lower than their melting point. Then, the ejected particles collide with the hard plate 92 and are crushed.

The hard plate 92 described above is only an example of the obstacles that provide the ejected frozen particles with an impact point. The obstacles may have a curved surface in place of the flat surface of the hard plate 92. Although materials from which such obstacles as the 35 hard plate 92 are made may be selected depending upon the hardness and speed of the ejected frozen particles, they are preferably ceramics and metals.

Alternatively, the impact point may be formed by ejecting frozen particles from two or more ejectors 91 so that the frozen particles collide with each other, as shown in Fig. 9. In this case, although the angle a between each ejector is not limited, it is preferably abont 60 to 1200 when 40 the distances D, and D2 between the ejectors and the impact point are 10 to 100 mm. Further, two impact points may be provided by fixing such an obstacle as the curved plate 92 behind the point where frozen particles collide with each other, as shown in Fig. 9.

It is preferred in the present invention that frozen particles are ejected within the inside of the casing as shown in Fig. 10, which is specifically designed to recover the destroyed particles. 45 Because otherwise the ejected frozen particles are flown up when they are destroyed at the impact point. The size of the casing is 500 mm in diameter and 110 mm in height. One or two or more ejectors 91 may be mounted in the center of the upper surface of the casing. When frozen particles are being ejected inside of the casing, the destroyed particles scatter in a substantially horizontal direction within the casing while they are depressurized and decelerated 50 due to baffles 94 so that they can be readily recovered.

In the present invention, the particles thus recovered may be destryoed again and again, if desired, after being melted once and frozen again.

Reffering to Fig. 2, frozen particles making unit 50 of Fig. 1 is set forth in detail hereinafter.

Fig. 2 shows a flow sheet of frozen particle making unit 50 of Fig. 1. In Fig. 2, the numeral 1 55 indicates a storage tank for liquid coolant, the numeral 2, liquid coolant stored in the tank 1, the numeral 3, means for generating ripples on the surface of the coolant by imparting kinetic energy to the upper region of the coolant, the numeral 4, spraying means which mixes cells containing suspension with a gas and atomizes the suspension, the numeral 5, means for controlling the level of coolant, the numeral 60, means for discharging frozen particles, the 60 numeral 7, means for pre-cooling cells-containing suspension to be frozen and a gas to be mixed with the suspension, the numeral 8, a source of coolant, the numeral 9, a source of cells containing suspension, and the numeral 10, a source of gas to be mixed with the cells containing suspension.

The storage tank 1 comprises a squared body and a reversed pyramid-like bottom, which is 65 4 GB2196018A 4 made from stainless steel (SUS 304). The outer size of the tank 1 is 400 mm in width, 400 mm in depth and 1200 mm in height. The tank 1 has on the exterior wall thereof a vacuum layer for insulation which is not shown.

The storage tank 1 stores therein a liquid coolant 2 of liquid nitrogen which is supplied from the source 8 via a conduit 11. The level indicated at L of the coolant 2 stored in the tank 1 is 5 kept about 500 mm from the bottom of the tank.

The level L is regulated at a predetermined height by use of means for controlling the level of coolant which comprises a level detector 5a, a level control panel 5b, a control valve 5c and the like.

10. The ripple-generating means 3 comprises a bubbling pipe 3a, a valve 3b for controlling 10 bubbling, a flow meter 3c and the like, which impart kinetic energy to the coolant so that ripples occur on the surface of the coolant 2.

The bubbling pipe 3a has a shape of a squared loop as shown in Fig. 3 and is horizontally fixed at a level of 40 to 150 mm below the surface of the coolant as shown in Fig. 4. As the pipe 3a is fixed at a deeper level, efficiency of the bubbling lessenes because bubbling gas from 15 the pipe 3a may excessively be cooled and thus a larger amount of coolant and bubbling gas is required. Therefore, it is desirable that the level of the bubbling pipe 3a is 100 mm or less below the surface of the coolant 2.

The bubbling pipe 3a is provided on the inward side thereof with a plurality of openings 3d from which gas is bubbled, at an inerval of 50-100 mm. 20 The gaseous coolant is supplied from the gas region of the coolant source 8 through the pipe 12 and the control valve 3b, and then is sparged into the liquid coolant 2 through the openings 3d.

The rate of gaseous coolant which is sparged from the pipe 3a is optimally 200-400 liters/M2Min. In this range, an ascending flow of bubbles occurs in the upper neighborhood to 25 the surface of the coolant 2. The flow of the bubbles 13 imparts kinetic energy to the surface of the coolant 2, generating ripples W having the wave height of 5 to 20 mm as shown in Fig.

6. The ripples vibrate each frozen particle which is sinking in the coolant 2 and thereby prevent the aggregation of frozen particles.

The presence of the bubbles 13 flowing in the coolant renders the liquid density decrease near 30 the surface of the coolant 2 and thus makes it easier for the frozen particles to sink in the coolant 2.

Optimum wave height of the ripples is about 5-10 mm. If the wave height is more than 20 mm, the ripples stir the coolant near its surface and thereby interfere the sinking of the frozen particles. 35 In Fig. 2, the bubbling pipe 3a is used as ripple generating means, to which gaseous coolant is supplied from the gas region of the source 8 of the coolant. Alternatively, the gaseous coolant may be supplied to the bubbling pipe 3a from another source which is equipped for storing the gaseous coolant independently of the source 8.

The gas supplied to the bubbling pipe 3a is not limited to the gaseous coolant. The gas may 40 be any other gases which have a low dew point or contains C02 gas. For example, a decarbo nated air can be used in place of the gaseous coolant.

The following means may be used as ripple generating means in place of the bubbling pipe 3a described above.

(a) Liquid sparging type of means for generating ripplesz; 45 According to this type of means, a pipe which has a structure similar to the bubbling pipe 3a of Fig. 3, is fixed below the coolant 2. This pipe sparges liquid coolant which is supplied from a source 8 through the openings thereof. This pipe does not require the pipe 11, but requires a pipe for recovering coolant from the unit 1 so as to keep the surface L of the coolant at a predetermined level. 50 (b) Oscillation type of means for generating ripples:

According to this type of means, oscillators 20 which oscillate, reciprocate or rotate at a given speed, are installed on the inside wall of the tank 1 as shown in Figs. 5 (A) and (B). The oscillators 20 are driven by motors 21 mounted on the outside wall of the tank 1 and impart kinetic vibration energy to the coolant 2, generating ripples on the surface L of the coolant 2. 55 (c) Sonic type of means for generating ripples:

According to this type of means, sonic vibrators 22 which generate vibration having a predetermined frquency, are installed in the wall of the tank 1 as shown in Fig. 5 (C). The sonic vibrators 22 impart sonic energy to the coolant 2, generating ripples on the surface L of the coolant 2. 60 (d) Spray type of means for generating ripples:

According to this type of means, sprayers are equipped above the surface of the coolant 2.

The sprayers eject liquid coolant or such a gas having a relatively high dew point as gaseous coolant and decarbonized air toward the surface of the coolant 2 and thereby impart kinetic energy to the coolant 2, generating ripples on the surface. 65 GB2196018A 5 (e) Shake type of means for generating ripples:

According to this type of means, a coolant storage tank 1 is mounted on an shaking table 22 and is provided with kinetic shaking force through cams 23 and springs 24, so as to impart shaking to the coolant 2, generating ripples on the surface of the coolant 2.

The spraying means 4 mixes therein cells-containing suspension with a gas and produces 5 liquid microfine droplets of the mixture of the suspension and the gas. The microfine droplets should have a diameter of 500 microns or less.

Fig. 7 shows an example of the spraying means 4 which has a body 4a having an inlet 4b for liquid at a rearward end thereof and an inlet 4b for gas at a rearward side thereof. The body 4a is provided inside of the rearward portion with coaxial two passages 4b' and 4c' and inside of 10 the forward portion with a throat portion 4d where cells-containing suspension is mixed with a gas. The gas-liquid mixture thus produced is further led to a mixing chamber 4e where the mixture is stirred and dispersed by a guide vane 4f, and then is atomized and sprayed out from the nozzle opening 4g. Of course, the sprayer of Fig. 7 may be replaced with another sprayer having a different structure or configuration as long as it has a function of mixing gas with liquid 15 and a function of atomizing and spraying the gas-liquid mixture.

As shown best in Fig. 2, to the liquid inlet 4b of the spraying means 4, cells-containing suspension to be frozen is supplied under a supplying pressure of 1.0 to 2.0 Kg/CM2g from a source 9 via a pump 14, a reducing valve 15, a control valve 16 and a cooling means 7. The cooling means 7 will be described later. To the gas inlet 4c of the spraying means 4, a gas 20 which is relatively insoluble to liquid is supplied under a pressure of 1. 0 to 2.0 Kg/CM2g from a source 10 via a reducing valve 17, a flow meter 18, a control valve 19 and a cooling means 7.

Alternatively, the gas may be introduced to the inlet 4c from the gas region of the source 8 of coolant in place of the source 10.

Ratio of the cells-containing suspension to the gas in the spraying means 4, is optimally 25 between 0.5 to 1.5, which is calculated by the following equation:

Liquid (liter/hour) Gas (N liter/min.) 30 By varying the ratio within the above range, the diameter of frozen particles can be varied from the maximum diameter down to one-tenth thereof even when the supplying pressures of both the gas and the suspension are fixed. Although it is more advantageous that the nozzle opening 4g has a smaller diameter, the opening 4g preferably has a diameter of about 0.3 to about 1.0 35 mm to avoid the problems on fabrication and clogging.

The gas-liquid mixture which is produced in the throat portion 4d and the mixing chamber 4e is uniformly dispersed ouwardly of the guide vane 4f and then is downwardly sprayed from the nozzle 4g to the surface of the coolant. When passing through the nozzle 4g, the gas is present both inside and outside of atomized liquid particles. Once having passed through the nozzle 4g, 40 the gas inside of atomized liquid particles expands itself and then makes the atomized particles to divide into smaller ones whilst the gas outside of atomized liquid particles forces the particles to vigorously diffuse and thereby causes the particles to collide with each other and to divide I - into smaller ones whilst the gas outside of atomized liquid particles forces the particles to vigorously diffuse and thereby causes the particles to collide with each other and to divide into 45 smaller ones.

The liquid microfine droplets thus produced by the spraying means become spherical particles by virtue of surface tension while falling within the tank 1, and then reach the surface of coolant.

Distance between the spraying means 4 and the coolant surface, and temperature inside of the 50 tank 1 have a great influence on the diameter and the shape of the particles which will be frozen in a coolant 2. The distance should be 500 to 1500 mm. The temperature should be - 15'C or lower, desirably -20"C or lower. It should be understood that the distance is between the top of the ripples and the spraying means 4 although the level of the coolant surface is always varying due to ripples. 55 The means 5 for regulating the level of the coolant surface has a function of keeping substantially constant the distance between the coolant surface L and the spraying means 4.

The means 5 is a combination of known devices such as a liquid surface detector 5a, a liquid surface controller 5b, a control valve 5c equipped with a coolant supplying pipe 5c and the like.

The cooling means 7 lowers the temperature of cells-containing suspension and a gas before 60 they are introduced into the spraying means 4, and thereby saves the consumption of coolant.

The cooling means 7 comprises a cooler 7a for liquid and a cooler 7b for gas. Vaporized coolant is introduced from the tank 1 to both coolers 7a and 7b so as to pre-cool the suspension and the gas.

In the apparatus of Fig. 2, liquid and gas are separately pre-cooled, and are then mixed and 65 6 GB2196018A 6 atomized in the spraying means 4. Alternatively, liquid and gas may be first mixed in a gas-liquid mixing chamber which would be provided independently of a spraying means 4, and then the mixture may be cooled in the cooling means 7 and sprayed from the spraying means 4.

Each droplet K of cells-containing suspension which has been sprayed from the spraying means 4, falls into the liquid coolant and freezes, and then can sink independently of each other 5 since ripples generated on the coolant sufface prevent the aggregation of the frozen particles.

Frozen particles which have sunk on the bottom of the tank 1, are discharged through a conduit having a discharge means 60.

The discharge means 60 of Fig. 1 comprises a lifter 60 of the pneumatic conveyor type, which transfers frozen particles from the unit 1 to the conveyor 70 through a conduit connected 10 therebetween. In the unit 1 of Fig. 2, a lifter of the screw conveyor type as shown in Fig. 8 may be used in place of the lifter 60, which comprises a guide pipe 6a which is inserted into the tank 1, a rotatable screw 6b which is provided inside of the guide pipe 6a and a drive motor 6c which drives the screw 6b. Also can be used a belt conveyor type and other types of discharge means, forexample, an apparatus for discharging frozen particles which described in 15 Japanese patent application No. 173317/86 wherein frozne particles which have sunk on the bottom of-a tank are discharged together with ascending bubbled flow of coolant through a conduit and then are separated from the coolant.

The frozen particles thus discharged may be ejected by an ejector as described above.

Hereinafter, referring to Fig. 11 to 18, other apparatus which are suitable to conduct a process 20 of the present invention are set forth.

Fig. 11 diagrammatically shows an apparatus which is suitable to conduct another embodiment of the present invention wherein frozen particles of cells-containing suspension are produced in a cool gas region by counercurrent gas-liquid contact of an ascending gas of vaporized coolant with dropping liquid microfine droplets of the cells-containing suspension, and said frozen par- 25 ticles are then ejected to collide. This apparatus of Fig. 11 is an alternative of the frozen paticle making unit 1 of the apparatus of Fig. 1.

In Fig. 11, the numeral 111 indicates a tank for making frozen particles, the numeral 112, means for recovering frozen particles, the numeral 113, spraying means, and the numeral 114, means for generating vaporized gas of coolant. 30 The tank 111 for making frozen particles is a tank which is insulated and sealed and has a square horizontal section each side of which is 400 mm. The tank 111 is provided on the top end of a side wall thereof with an outlet 111 a for cool gas.

The means 112 for recovering frozen particles comprises an screen 115 shaped like a reversed pyramid and an outlet tube 116 which is vertically positioned at the center of the tank 35 111 below the bottom of the screen 115. The screen 115 is fixed along the interior wall of the tank 111 and divides the inside of the tank 111 into a cool gas region 1 17a of the upper portion of the tank 111 and a vaporized-coolant generating region 1 17b of the lower portion of the tank 111. The screen 115 is made of wire netting which has a resistance to low-tempera ture and a mesh size through which only vaporized coolant can pass, for example, a screen of 40 mesh made from SUS 304. The outlet 116 extends out of the bottom of the tank 111 and is provided adjacent the bottom end of the outlet with a valve 11 6a such as a rotary valve for discharging frozen particles. Although the angle 6 between the screen 115 and ahorizontal line should be varied depending upon an amount of vaporized coolant, diameter of frozen particles and whether scrapers are employed, it is typically about 45' when no scraper is used, The 45 screen may be inclined from a side wall directly toward a opposite wall as shown in Fig. 14.

The spraying means 112 comprises a sprayer 118 which is mounted on the center of the upper wall of the tank 111, a conduit 119 which is connected with the sprayer 118 so as to supply cells-containing suspension to the sprayer 118, and a conduit 120 which is connected with the sprayer 118 so as to supply a drive gas such as a suitably pressurized and cooled 50 nitrogen gas to the sprayer 118. By the spraying means 113, cellscontaining suspension is downwardly sprayed from the nozzle 118a provided on the tip of the sprayer 118 by virtue of the pressure of the drive gas. The size of sprayed liquid microfine droplets, that is, cell containing particles 124a can be varied depending upon the diameter of the nozzle opening and the pressure of the drive gas. 55 The means 114 for generating vaporized gas of coolant comprises a conduit 121 which supplies a predetermined amount of coolant such as liquid nitrogen 122 to the vaporized-coolant generating region 117b and a bubbling pipe 123 which is located in the coolant 122 and is supplied with a gas such as nitrogen gas, argon gas and dry air so as to bubble a vaporized coolant 122a. 60 In the unit for making frozen particles of Fig. 11, vaporized coolant 122a moves from the vaporized-coolant gen ' erating region 117b to the cool gas region 117a, passing through the screen 115, and ascends the region 1 17a toward the outlet 111 a. In other words, vaporized coolant 122a gradually ascends toward the outlet 111 a as it changes its own density by exchanging heat with sprayed droplets of cells-containing suspension 124a. On the other hand, 65 7 GB2196018A 7 as the cells-containing droplets 124a fall within the cool gas region 117a, they exchange heat with the ascending vaporized coolant 122a by countercurrent gas-liquid contact and then freeze.

Since the droplets 124a are frozen within the gas region 117a of vaporized coolant, they are shaped to spherical frozen particles by virtue of their own surface tension. Since the droplets 124a contact only the ascending vaporized coolant, no aggregation of droplets 124a occur, the 5 droplets. 124a are frozen independently of each other, and thus frozen particles having a uniform diameter are obtained.

In the apparatus of Fig. 11, the frozen particles 124b thus obtained fall onto the screen 115.

Since the screen 115 is inclined, the frozen particles 124b are readily recovered without the particles accumulating on the screen 115. The vaporized coolant which upwardly passes through 10 the screen 115 keeps the temperature of the frozen particles which have reached the screen 115, and fluidizes the frozen particles. The coolant also prevents the frozen particles on the screen 115 from decreasing their hardness and aggregating with each other.

An experiment on the apparatus of Fig. 11 by the present inventors reveals that the effective height of the cool gas region 117a, i.e. the distance H between the nozzle 118a and the screen 15 can be down to about 1 meter to obtain high-quality frozen particles 124b, provided that the following three parameters are met: a) the rate of cells-containing suspension from the nozzle 118a is 0.2 liter/min. (12 liters/hour) which is sufficient to keept the average temperature at -80'C or less in the lower side portion of the cool gas region 117a, i. e. in the neighborhood of the upper portion of the screen 115; b) sprayed particles 124a have diameter of 300 microns 20 or less; and c) spraying pressure of cells-containing suspension is 4 Kg/CM2.

In the apparatus of Fig. 11, there is a close relationship between the temperatureof the cool gas region and the generation rate of the vaporized coolant 122a. Fig. 18 shows a temperature distribution in a horizontal cross section of the cool gas region 117a when the gas generation rate from liquid nitrogen varies 20, 40 and 60 NM3/h where cells- containing suspension is 25 sprayed at the rate of 0.2 liter/min., the areal load is 75 Kg/M2h, and cool gas load is 250 NM3/M2h when the horizontal sectional area of the cool gas region is 0.16 M2.

From Fig. 18, 40 Nm3/h of vaporized coolant is required to keep the average temperature of the cool gas region at -80'C or lower if the horizontal cross sectional area is 0.16 M2, cells containing suspension is sprayed at the rate of 0.2 liter/min. (12 liter/hour), and sprayed 30 microfine droplets of the cells-containing suspension have diameter of 300 microns or less. In this case, the distance which is required to obtain time sufficient to freeze the sprayed micrfine droplets of cells-containing suspension is about 1 meter.

As the effective distance H in the cool gas region 117a may be reduced down to about 1 meter in the above way, the whole unit can be designed to be considerably small as well as the 35 tank 111.

It is acceptable to spray cells-containing suspension in a vertical direction as shown in Fig. 11 if spraying pressure is low of if sprayed droplets or frozen particles have small diameter.

Otherwise, the tank 1 18a has to be of large size enough to ensure the time to contact sprayed droplets 124a with vaporized coolant 122a if cells-containing suspension is sprayed from the 40 nozzle 1 18a in a vertical direction as shown in Fig. 11. However, if the suspension is sprayed in a horizontal direction as shown in Fig. 14, the time to contact sprayed droplets 124a with vaporized gas 122a can be ensured without such a large-sized tank.

Frozen particles 124b which have fallen onto the screen 115 automatically move along the screen 115 and collect in the discharge pipe 116 since the screen 115 is inclined downwardly 45 to the discharge pipe 116 and vaporized coolant 122a is upwardly passing through the screen 115. The frozen particles 124b thus collected are discharge outside of the tank 111 by the discharge valve 116a and then ejected to the hard plate 128.

The conxditions to produce frozen particles from sprayed microfine droplets of cells-containing suspension can be experimentally determined by the one skilled in the art, for example, a rate to 50 spray cells-containing suspension, a temperature of the cool gas region where frozen particles are made, a generation rate of vaporized coolant required to keep constant the temperature of the cool gas region, particle diameter required, a failing speed of frozen particles, time to contact frozen particles with coolant gas, etc.

Whether frozen particles 124b can efficiently collect into the discharge pipe 116 or not, 55 depends upon an inclination angle 0 of the screen 115, temperature of the screen 115, a speed of vaporized coolant passing.through the screen 115 and diameter of frozen particles. However, regardless of such parameters, frozen particles 124b can be efficiently collected into the dis charge pipe 116 by providing the screen 115 with continuous or intermittent vibration, oscilla tion or shake by equipment such as a vibrator not shown. 60 As shown in Figs. 12, 13 and 15 to 17, means 112 for recovering frozen particles may be of the type which supplies frozen particles 124b produced in a tank 111 directly to means 125 for ejecting frozen particles provided outside of the tank 111.

Referring to Fig. 12, the tank 111 for making frozen particles is provided on an upper side thereof with a waste heat recovering chamber 126 which is connected with the tank 111 65 8 GB2196018A 8 throu gh an outlet 11 la for gas. An ejector 125 which ejects frozen particles is mounted on the bottom of the chamber 126. In the apparatus of Fig. 12, means 112 for recovering frozen particles comprises a frozen particles-discharging pipe 116 which directly connects the bottom of the screen 115 with the ejector 125. The ejector 125 is also connected. with a conduit 127 for a drive gas. As a drive gas such as suitably pressurized air, nitrogen gas and the like is 5 introduced into the ejector 125 via the conduit 127, frozen particles 124b are sucked through the pipe 116 to the ejector 125 by virtue of the ejection effect and then ejected to a hard plate 128.

The conduit 127 is formed inside of the chamber 126 with a portion 129 which functions as a heat exchanger which heat-exchanges the drive gas with vaporized coolant 122a entering the 10 chamber 126 through the outlet 111 a. The heat-exchanging portion 129 is shaped like a coil pipe having a thick wall with a lot of fins 129a as shown in Fig. 13, in order to increase an area to contact vaporized coolant and efficiently conduct heat and also to enhance a heat accumula tion. The portion 129 is preferably made from a copper alloy having a high heat conductivity.

In this manner, drive gas can be pre-cooled sufficiently to maintain a suitable hardness of 15 frozen particles 124b and to prevent aggregation of frozen particles 124b in the ejector 125 and thereby can eject the particles so as to efficiently break or damage cell membranes. Since the portion 129 has a thick wall with a lot of fins 129a, drive gas can be pre-cooled by virtue of the heat accumulation even when vaporized gas 122a is not generated during an intermittent operation of the unit for making frozen particles. This heat-exchanger can pre-cool the drive gas 20 down to the temperature of vaporized coolant the drive gas down to the temperature of vaporized coolant coming from the outlet 111 a. However, if necessary, it is possible to cool and drive gas to a much lower temperature by spraying toward the coil-like portion a coolant such as liquid nitrogen from a nozzle 130 mounted on the upper portion of the heat recovering chamber 126. 25 As shown in Fig. 15, in place of the inclined screen 115 as shown in Figs. 11, 12 and 14, a flat screen 115 may be used in combination with a scraper 132 which is reciprocated above the screen 115 by a cylinder 131 so as to transfer frozen particles 124a accumulating on the screen into a hopper portion 133a. As a drive gas is introduced into the ejector 125, frozen particles 124b recovered in the hopper portion 133a are sucked to the ejector 125 and then ejected to 30 the hard plate 128.

Furthermore, means for recovering frozen particles may be those which comprise a screen which moves from the inside to the outside of the tank 111. An example is shown in Fig. 16, which comprises a conveyor 135 having an endless mesh belt 115% an end portion of the conveyor 135 being located in the frozen particles-recovering chamber 133. The conveyor 135 35 divides the inside of the tank 111 into two region 1 17a and 1 17b, and is driven in the direction idnciated by an arrow in Fig. 16, transferring frozen particles 124b accumulating on the mesh belt 115' from the inside of the tank 111 to a hopper 133a of the chamber 133. Another example is shown in Fig. 17, which comprises a screen plate 11W which is rotated about a vertical axis driven by a suitable driving mechanism 136. In this case, frozen particles 124b 40 accumulating on the plate 1 1W in the tank 111 are transferred to the recovering chamber 133 and recovered into the hopper 133a by use of a suitable scraper 137.

In Figs. 11 to 17, spraying means 118 may be the sprayer shown in Fig. 7; the distance between spraying means 125 and hard plate 125, and the material and shape of the hard plate 124, may selected as mentioned above in connection with the apparatus of Fig. 1; and the 45 ejecting and the recovering of frozen particles may be conducted in the manner shown in Fig. 9 and 10.

Example

Explanation will next be made to a process for producing cells-containing frozen particles 50 according to the present invention with reference to the following Examples, but the invention is no way restricted only thereto.

Example 1

Commercially available bread-making living yeast manufactured by Toyo Jozo Co., was sus- 55 pended in water to prepare a yeast suspension of about 0.1 9/mi.

Then, experiment was conducted by use of the apparatus shown in Fig. 1 and described above. A tank which comprises a square body portion having 400 mmx400 mm cross section and 900 mm height and an inverted pyramidal bottom portion having 300 mm height, was formed as a liquid coolant storage tank 1 of the apparatus. Then, liquid nitrogen as the liquid 60 coolant 2 was charged to the height of 500 mm from the bottom of the tank as shown in Fig.

2. In other words, the distance from the ceiling of the tank to the liquid surface L of the liquid coolant was set to 700 mm.

Further, a bubbling pipe 3 shaped like a square loop (350 mmx350 mm) was disposed horizontally at a position 50 mm below the liquid surface. To the pipe 3, nitrogen gas from the 65 9 GB2196018A 9 gas region in a liquid nitrogen source 8 was supplied at a flow rate of 300 liters/m2min. and sparged from the openings 3d inwardly of the tank 1 to generate ripples with an average wave height of 8 mm on the outer surface of the liquid nitrogen.

The yeast suspension as a liquid to be frozen and nitrogen gas at 25'C stored in a high pressure tank 10 as gas to be mixed, were introduced into a spraying means 4 at flow rates of 5 0.2 liter/min and 1 liter/min, respectively, so as to mix with each other in the spraying means 4. The spraying means 4 was disposed about 700 mm above the liquid surface L of the liquid nitrogen and had a nozzle aperture of 0.7 mm 0. Then, the gas-liquid mixture was sprayed to the liquid surface under spraying pressure of 4 kg/cm2. The highest temperature of the upper space in the tank 1 was -20'C. 10 On the other hand, in order to maintain the liquid surface of the liquid nitrogen 2 in the tank 1 at a constant level, liquid nitrogen was supplied from the pipe 11 to the inside of the tanke at a flow rate of 2-0 liters/hr. The flow rate of the nitrogen gas discharged from the tank 1 was set to 20xO.65 NM3/hr in order to pre-cool the yeast suspension and the nitrogen gas to be mixed by the cooling means 7. 15 When the mixture of the yeast suspension and the nitrogen gas was continuously sprayed from the spraying means 4 for about 10 minutes under the foregoing conditions, about 1000 mi of flouring yeast cells-containing frozen particles were obtained in the form of ice spheres. The average particle size of the spheres was from 50 to 100 pm.

The ice spheres were stored by way of a lifter 60 and a conveyor 70 in a stocker to which, 20 nitrogen gas under pressure of 2 kg/CM2 and at temperature of 70'C was supplied for fluidizing the ice spheres from the bottom, and for keeping the temperature of the ice spheres at -700C.

Then, they were ejected from an ejector 91 having a nozzle of 8.3 mm diameter. Drive nitrogen gas supplied from the central inlet of the ejector 91 was at an acceleration pressure of 43-5 kg/CM2 and at temperature of -70'C, while the temperature of ice spheres upon ejecting from 25 the nozzle was -70'C. The ice spheres were ejected under the foregoing conditions while setting the eject distance D as 20 mm, and 50 mm, and then respective yeast cells-containing destroyed particles were recovered.

The yeast suspension, the yeast cells-containing ice spheres and the yeast cells-containing destroyed particles were respectively suspended in wast as specimens. And then, they were 30 centrifugally separated to obtain a yeast cell fraction and a supernatant fraction thereof for each of the specimens.

For the supernatant fractions, quantitative protein determination was carried out by Lowry method, and enzyme activity was determined by means of alcohol dehydrogenase activity. The results are shown in Table 1. 35 (Test Method) The alcohol dehydrogenase activity was measured by an absorptiometric method, using 1.00 mi of a liquid reaction mixture consisting of 0.63 mi of 0.05 M barbital buffer solution (pH 8.6), 0.10 mI of an aqueous solution of 0.25 % nitrotetrazolium blue (NTS) manufacutred by Dojin 40 Kagaku Institute, 0.02 mi of 10 % albumin (bovine serum), i.e., Fraction V Powder manufaured by Armour Pharmaceutical Co., 0.10 mi of 10 mM NAD, i.e., flNAD+ manufactured by Oriental Kobo Co., 0.05 mi of 100 U/m] of Diaphorase (D1) manufactured by Toyo Jozo Co. and 0.10 m] of 1 M ethanol. - On one hand, after charging 1.00 mI of the liquid reaction mixture in a glass cell and 45 preliminarily heating to 37'C for 5 minutes, 10 pi each of the specimens as described above was added to the liquid reaction mixture and left at 37'C for 10 minutes. Then, 2 mi of 0AN hydrochloric acid was added to terminate the reaction and the absorbance As of the specimen at a wavelength of 550 urn was determined.

On the other hand, after charging 1 mi of the liquid reaction mixture into a glass cell and 50 leaving at 37'C for 15 minutes, 2 mi of 0AN HCI and 10 p] of the specimen were added to the liquid reaction mixture. Then, the absorbance Ab at a wavelength of 550 urn was determined as a blank.

In view of the absorbance As of the specimen and the absorbance Ab of the blank, the alcohol dehydrogenase activity of the specimen was determined by the following equation: 55 GB2196018A 10 (As-Ab)/10 3.01 (activity) X 16.4 0.01 5 where 16.4 molecular extinction coefficient of reduction type NTB (CM2/MrlnC)I) reaction time (min) 3.01 final dissolve amount (mi) 0.01 enzyme solution amount (mi) 10 Quantitative protein determination by the Lowry method was conducted as described below.

After mixing 0.5 mi of the specimen thoroughly together with 5 m] of a reagent A prepared by the method described later in a glass cell and leaving the mixture at room temperature for 10 minutes, 0.5 m] of Folin reagent prepared by diluting a commercially available product (manufactured by Wako Seiyaku Co.) by two times with water was rapidly added to the mixture, then it was left for 30 minutes. Then, the absorbance was measured at 750 pm by an absorptiometric method and quantitatively determined based on the calibration curve of bovine serum albumin.

The reagent A is an alkaline reagent prepared by mixing a reagent (1) obtained by dissolving NaCO, into 1N NaOH to 2 % concentration and a reagent (2) obtained by dissolving CuSO,.5H,0 20 into 1 % sodium citrate to 0.5 % concentration just before the use.

Example 2

Using the same yeast suspension as in Example 1, frozen yeast cellscontaining particles were prepared under the same conditions as in Example 1 except for setting the flow rate of the 25 yeast suspension to 0.2 1/min, the flow rate of the nitrogen gas to 1 1/min, the nozzle diameter to 0.5 mm and the spraying pressure to 2-2.5 kg /CM2.

As a result, yeast cells-containing ice spheres having diameter from 200 to 300 urn were obtained.

In the same manner as in Example, the ice spheres were ejected, and then yeast cell- 30 containing destroyed particles were recovered. Thereafter, analysis was conducted in the same manner as in Example 1.

The result is shown in Table 2.

Example 3 35

Experiment was conducted in the same manner as in Example 1 except for using a yeast suspension containing 0.0583 g/mi of yeast cells, The experiment was conducted with the eject distance D only of 20 mm.

The result is shown in Table 3.

40 Comparative Example Yeast cell bodies were pulverized by use of a commercially available cell body pulverizer, i.e., Dyno-Mill KIDL type manufactured by Willy A Bachofen Maschinefabrik. A grinding container of 0.6 liter volume for use in Dyno-Mill charged with glass beads of the type of MK-2G having particle grain size 0.25-0.5 mm 0, was rotated at 2000 rpm. To the container, yeast cells- 45 containing suspension which has been prepared by suspending 0.23 g/m] of bread-making living yeast manufactured by Toyo Jozo Co. in wate, was continuously supplied at a flow rate of 100 mi/min to conduct the destruction of the yeast cells.

The result is shown in Table 4.

50 Example 4

Experiment was conducted in the apparatus shown in Fig. 12 by use of 1/2 and 1/4 solutions of the 0.1 g/m] yeast suspension. A tank comprising the square body portion which has 400 mmx400 mm cross section and 1400 mm height and is provided in the lower portion thereof a screen-like member 115 made of SUS 303 metal gage of 150 mesh at an angle 0 of 60', was 55 formed as a tank for making frozen particles in the apparatus. And liquid nitrogen was charged as a liquid coolant to the height of about 100 mm from the bottom of the tank. The distance between the highest portion of the screen-like member and the surface of the liquid nitrogen was about 300 mm and the distance between the highest portion of the mesh- like member and the nozzle was about 1000 mm. 60 Further, ascending vaporized gas was generated from the coolant at 40 NM3/h to maintain the temperature inside the tank for making frozen particles to about -80'C.

Then, using the yeast suspension as a liquid to be frozen and the nitrogen gas at 2WC as a mixing gas, they were supplied to a spraying means 118 which was situated about 1000 mm above the heighest portion of the mesh-like member and had a nozzle aperture of 0.5 mm 0, at 65 GB2196018A 11 a flow rate of 0.2 1/min for the yeast suspension and at a flow rate of 5 1/min for the nitrogen gas. They were mixed in the spraying means 118 and then the mixture was sprayed down wardly under spraying pressure after the mixing of 3.5 kg/cm2.

After the mixture of the yeast suspension and the nitrogen gas was continuously sprayed for about 10 min under the foregoing conditions, about 1000 mi of highly flouring yeast cells- 5 containing ice spheres were obtained. The average particle size of the spheres was about 100 Am.

- The ice spheres thus obtained were further ejected continuously from the ejector 125 having a nozzle of 8.3 mm diameter while maintaining the temperature of the product at -70'C. In this case, the drive nitrogen gas supplied from the central inlet of the ejector 125 was at an 10 acceleration pressure of 4 kg/CM2 and at a temperature of -70'C. The temperature of the ice spheres upon ejecting from the nozzle was -70'C. Further, the distance from the nozzle to the hard plate 128 upon ejecting was 20 mm.

After ejecting, destroyed particles of the yeast cells-containing ice spheres were recovered and applied to the same analyses as in Example 1. The result as shown in Table 5 was obtained. 15 Example 5

The experiment was conducted under the same conditions as in Example 4 except for using only the 0.05 g/mi yeast suspension, setting the flow rate of the yeast suspension to 0.2 1/min, the flow rate of the nitrogen gas to 1 Ilmin and the spraying pressure after the mixing of the 20 spraying means 118 to 25 kg/cm2. As a result, yeast-containing ice spheres having diameter of about 200 urn were obtained in the unit for making frozen particles.

After ejecting, the yeast-containing destroyed particles were recovered and applied to the same analyses as in Example 1. The result is shown in Table 6.

12 GB2196018A 12 Table 1

Alcohol dehydro- Protein genase activity: content: 5 supernatant supernatant fraction fraction (U/9) (mg/g) 10 Yeast suspension 0 3 (0.1 g/ml) Yeast cells-containing 101 108 15 ice spheres Yeast cells-containing 99 158 destroyed particles I (eject distance 20 mm) 20 Yeast cells-containing 108 105 destroyed particles II (eject distance 50 mm) 25 g indicates the weight of the yeast cell 30 Table 2

Alcohol dehydro- Protein 35 genase activity: content:

supernatant supernatant fraction fraction (U/g) (mg/g) 40 Yeast suspension 0 3 (0.1 g/ml) Yeast cells-containing 55 69 45 ice spheres Yeast cells-containing 65 107 destroyed particles 1 50 (eject distance 20 mm) Yeast cells-containing 73 121 destroyed particles 11 55 (eject distance 50 mm) 13 GB2196018A 13 Table 3

Alcohol dehydro- Protein genase activity: content: 5 supernatant supernatant fraction fraction (U/9) (mg/g) 10 Yeast suspension 0 3 (0.0583.g/ml) Yeast cells-containing 115 155 15 ice spheres Yeast cells-containing 359 244 destroyed particles (eject distance 20 mm) 20 25 Table 4

Alcohol dehydro- Protein 30 genase activity: content:

supernatant supernatant fraction fraction (U/g) (mg/g) 35 Yeast suspension 0 3 (0.23 g/ml) 40 Dyno-Mill treated 245 165 solution 14 GB2196018A 14 Table 5

Alcohol dehydro- Protein genase activity: content: 5 supernatant supernatant fraction fraction (U/g) (mg/g) 10 Yeast suspension 1 5 (0.025 g/ml) Yeast cells-containing 189 87 15 ice spheres Yeast cells-containing 314 121 destroyed particles (eject distance 20 mm) 20 - - - - - - - - - - - - - - - - - - 25 Yeast suspension 2 6 (0.05 g/ml) Yeast cells-containing 217 102 30 ice spheres Yeast cells-containing 469 175 destroyed particles (eject distance 20 mm) 35 Table 6 40

Alcohol dehydro- Protein genase activity: content: 45 supernatant supernatant fraction fraction (U/9) (mg/g) 50 Yeast suspension 2 6 (0.05 g/ml) Yeast cells-containing 197 96 55 ice spheres Yeast cells-containing 416 177 destroyed particles (eject distance 20 mm) 60 GB2196018A 15

Claims (17)

1. A process for treating cells to facilitate the extraction of useful substances therefrom which comprises imparting impact energy to frozen particles containing cells therein so as to disrupt the walls of the cells.

2. A process according to claim 1 wherein the frozen particles are caused to collide with 5 each other.

3. A process according to claim 1 wherein the frozen particles are caused to collide with an impact point or surface.

4. A process according to any one of the preceding claims wherein the frozen particles have diameter of 500 microns or less. 10

5. A process according to any one of the preceding claims wherein the frozen particles are ejected from an ejecting device.

6. A process according to claim 1 which comprises producing liquid microfine droplets having diameter of 500 microns or less from a cells-containing suspension, contacting the liquid micro fine droplets which a coolant so as to produce frozen particles having diameter of 500 microns 15 or less, and then ejecting the frozen particles to impart an impact energy thereto.

7. A process according to claim 6 wherein the frozen particles are produced by dropping the liquid microfine droplets into a coolant of liquefied gas so that the liquid microgine droplets are frozen by liquid-liquid contact with the coolant.

8. A process according to claim 7 wherein the liquid microfine droplets are dropped into a 20 coolant on the surface of which ripples are generated.

9. A process according to claim 6 wherein the frozen particles are produced by dropping the liquid microfine droplets into a cool gas region in which vaporized coolant is ascending, so that the liquid microfine droplets are frozen by gas-liquid contact with the coolant.

10. A process according to any one of claims 6 to 9 wherein the frozen particles are caused 25 to collide with each other.

11. A process according to any one of claims 6 to 9 wherein the frozen particles are caused to collide with an impact point or surface.

12. A process according to any one of claims 6 to 11 wherein the suspension contains 0.005 g/ml to 0.5 g/ml of cells. 30

13. A process according to any one of claims 6 to 12 wherein the liquid microfine droplets are produced by use of a sprayer.

14. A process according to any one of claims 6 to 13 wherein the frozen particles are ejected from an ejecting device.

15. A process according to claim 1 substantially as described with reference to any one of 35 Examples 1 to 5.

16. A process according to claim 1 substantially as described by reference to the accom- panying drawings.

17. A process which comprises the additional step of recovering cell contents from cells treated in accordance with the process claimed in any one of the preceding claims. 40 Published 1988 at The Patent Office, State House, 66/71 High Holborn, London WC1R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Burgess & Son (Abingdon) Ltd. Con. 1/87.