CA2064803A1 - Cooling process and apparatus - Google Patents

Abstract

Material to be frozen is subjected to a cooling process which involves the efficient removal of latent heat of freezing. This can be achieved by subjecting the material being frozen to a greater rate of heat extraction when the latent heat is being given up than when the then solid material is being subsequently cooled further. Efficient removal of latent heat is also facilitated by inducing nucleation of the frozen liquid. Nucleation can be initiated acoustically and/or chemically. The invention, which has particular application in the frozen food industry and in the cryopreservation of biological material, allows shorter freezing times and/or improved quality or viability of the frozen product.

Description

WO 91/01635 PCI'/GB90/01231 ~.' ~.''1',~, 1 ''''~J`~ 6!~803 1 COOLING PROÇESS AND APPI~RATUS

3 This invention r~lates to a method o~ freezing a 4 material and to apparatus for use in such a method.

6 The invention has particular application in a number of 7 fields, as it can minimise the effects of undercooling 8 during freezing in order to alleviate or avoid damage 9 to the material being frozen. In particular, the 10 invention may be used in: .

12 ~A) the frozen food industry;

14 (B) the cryopreservation of human embryos and embryos of other animals;

17 (C) the freezing o~ human organs for transplantation;

19 (D) the freezing of small or large volumes of cell suspensions, such as blood, bone marrow and 21 microorgani~ms;

23 (E~ the freezing of other biological material, 24 particularly cellular (whether plant or animal) 25 material; and --27 (F) the freezing of other material, particularly where 28 freezing must take place in controlled conditions, 29 for examplQ, in freeze drying and/or in the production of hiyhly re~ular crystalline solids.

32 It is necessary to freeze or solidify many materials in 33 com~ercial and industrial processes. Freezing may be :

W091/01635 PCT/GBgO/01~31 2~ $ ~S~3 2 i part of a production process or be a means of enhancing 2 the storage characteristics of the material. The 3 storage of foodstuffs by freezing is a common method of 4 maintaining their viability for long periods of time.
Equally, in other technical fields, cryopreservation is 6 recognised as the principal method of preserving 7 biological material, particularly delicate and valuable 8 material~such as human or other animal embryos, u~til 9 required for use. It is anticipated that there are further possibilities for the application of 11 cryopreservation techniques to biological material:
12 there is a major shortage of human tissues and organs 13 for transplantation including corneas, pancreas, 14 kidney, liver and heart.
16 Although the freezing of foodstuf~s, the 17 cryopreservation of biological material and the 18 solidification of other materials may seem to be a l9 disparate collection of industrial and commercial processes, in fact they tend to share a common major 21 problem. During cooling of the "material" (which will 22 be used as a generic term), liquid in the material (for 23 example in medium surrounding cells in a biological 24 sample) tends to supercool to a point below its 2~ freezing or solidification point before nucleation of 26 the solid phase occurs. This is also known as 27 undercooling. Supercooling or undercooling can cause 28 damage to the material, and in the case for example of 29 embryos can even prevent their survival, because of the following e~fect. (Although the discussion that follows 31 relates to material comprising liquid water and the 32 formation o~ solid ice, the same principles would apply 33 to other liguid/solid systems.) .. . . .

WO91~01635 PCT~GB90/01231 .:, . . ~
rS~ 3 206~803 .
1 Conventionally, as an aq~eous material is cooled at a 2 steady rate, the temperature of the material will fall 3 with the surrounding falling temperature until the 4 nucleation point of the liquid is reached. Because of the tendency to supercool, this will be below the 6 melting point. At the nucleation point, water in the 7 materlal crystallises into ice, there~y liberating 8 latent heat of fusion. The temperature of the material 9 at this point rises fxom the nucleating point almost to the melting point. once the latent heat of fusion has ll been lost by the material and/or its associated water, 12 the temperature of the material again begins to fall.
13 However, because the surrounding temperature has by 14 this stage become cooler, there is a greater differential between the material temperature and the 16 surrounding temperature, so the material cools much 17 more quickly. This results in the relatively 18 uncontrolled formation of ice crystals, whose large l9 size can have a deleterious effect.
21 This leads to a real problem for the frozen food 22 industry. A convPntional technique employed by the 23 food industry to freeze ~ood is to use a blast or 24 tunnel freezer where the food is cooled by cold gas.
Inside the freezer these is a gradient o~ ~as 26 temperature, the temperature being warmest at the end 27 at which the food is introduced a~d gradually becoming 2~ lower as the fobd passes through the freezer.
29 Initially the sample cools in parallel with the gas temperature. However, a~ter nucleation the food 31 temperature rises to the latent heat plateau. Here, 32 the rate of loss of heat from the food to the 33 environment is proportional to the temperature - - ~ . . ~,............................................. .

. .

5 ~ PCr/GB90/Ot231 l difference which increases while the latent heat is 2 being given up. The food is therefore buffered at this 3 exotherm until the latent heat of fusion has been 4 dissipated, at which time the temperature of the sample will then rapidly equilibrate to the environment 6 temperature, resulting in a sharp drop in temperature.

8 In the frozen food industry, products such as some so~t 9 fruits (eg. peaches, plums, raspberries) and seafoods (eg. lobster, crab, prawn, finfish) are often of poor 11 quality when thawed. With other so~t ~ruits ~eg.
12 strawberries, kiwi fruit, mango), various vegetables 13 (such as new potatoes and asparagus) and some dairy 14 products (for example single cream) the problem is more extreme and thes~ products are not frozen on a 16 commercial basis. A major component of such 17 freeze-thaw injury is the loss of texture due to 18 mechanical damage caused ~y uncontrolled nucleation of 19 ice crystals and their subsequent growth associated with prolonged periods at the latent heat plateau.

22 The quality of product~ which are consumed in the 23 frozen state such as ice cream, sorbets and ices are 24 rPlated to the size and distribution of ice crystals, formation of which is often difficult to control.
26 Furthermore, in conventional freezing methods, water in 27 the sample nucleates on the outside and ice propagates 28 towards the centre. The evolution of latent heat at 29 the periphery of the sample results in the core being thermally bu~fered and "shell" freezing occurs.

32 With the cryopreservation of sensitive biological 33 cellular material, cellular material, there is an ''~''''''' ~ '.

, `,, : , ' ', ,, , i, ' . , . ,. ' .:: ~ .. ' : : ' ', . , ' . . ., . , . . , :

l additional harm~ul effect resultl?ng ~r8O~ 3supercooling 2 or undercooling. As ice forms in the medium the 3 concentration of any solutes in the remaining liquid 4 increases. By osmotic pressure, the cells will thus dehydrate, as a result of water moving to the more 6 concentrated medium If the cells have insufficient 7 time to dehydrate, then intracellular ice may form, 8 which is generally lethal to the cell.

In order to minimise the potential problems caused by ll supercooling, EP-A-0246824 teaches that a range of 12 solid materials can be used to cause water in an 13 aqueous medium to be nucleated at, or close to, the 14 free ing point of the medium. However, even with this considerable improvement over prior methods, caré still 16 needs to be taken in otherwise conventional cooling 17 methods that damage does not occur during the 18 relatively rapid cooling period after the temperature l9 plateau during which at least some of the latent heat of fusion of the medium is being lost.

22 The above discussion has centred on material comprising 23 (and in particular containing a significant amount of~
24 water. Water has a strDng tendency to cool below its 2S freezing point (the super~ooling or undercooling 26 effect) which introduces complications in cooling of 27 biological tissues which have many membrane bound 28 compartments which limit the propagation of ice. A
29 variety of met~ods have been described to initiate ice nucleation. A number of inorganic compounds, silver 3l iodide being a common example, and oryanic compounds 3Z (see EP-A-0246~24, discussed above) and "ice 33 nucleating" bacteria (merbers of the genera .~
' .1 .

. . .: . ., ~

. : .: : . ~ ~ ~

91/01 a6 4~ ~ PCT/G~90/0l231 .
1 Xanthomonas, Pseudomonas, and Erwinia) have been 2 demonstra~ed to have a crystal lattice structure which 3 are effectiv~ nucleators of ice in supercooled water.
4 Whilst these compounds have applications, for example in the seeding of rain clouds, biological 6 cryopreservation and snow formation respectively, they 7 cannot be readily applied to foodstuffs due to 8 toxicity, legislation or problems of application.

The problems of uncontrolled nucleation have been seen 11 effecti~ely to prevent the commercial freezing of 12 certain foodstuffs, as discussed above. Although 13 similar (or worse) problems have arisen in the somewhat 14 more specialist field of cryopreserving biological samples, some attempts have been made to initiate 16 nucleation in a relatively controlled manner, in 17 addition to the seeding process described in 18 EP-A-0246824. For example, ice nucleation has in the 19 past been initiated by either (a) mechanical shaking, (b) thermoelectric shock, (c) thermal shock or (d) 21 direct addition of ice crystals.

23 Mechanical shaking is an inefficient cumbersome process 24 that may damage the sample. Thermoelectric shock can be delivered by supplying a curren~ across the sample 26 in the case of a solid or container enclosing a liquid 27 sample. The technique uses the reverse of the Peltier 28 thermocouple effect. Thermal shock may be achieved by 29 contact of the sample with a much colder surface or the insertion of a precooled surface such as a metal wire 31 or glass rod. Perhaps the least inelegant of the ~2 presen~ processes is the direct addition of ice 33 crystals to a liquid sample or the surface o~ a solid.
: .

W O 91/01635 P ~ /GB90/01231 ~ 2 6 ~ 8 ~ 3 1 These last three invasive processes are unsuitable for 2 foodstuffs. There is therefore a need for an improved 3 non-invasive method of avoiding the serious 4 consequences of supercooling and subsequent nucleation.

6 The present invention addresses the problems discussed 7 above and provides a surprisingly simple and elegant 8 solution, which can be put into practice in a variety 9 of relatively straightforward ways.

11 At its broadest, the invention provides, in a first 12 aspect, a method of freezing material comprising a 13 liquid, the method comprising extraoting heat from the 14 material and varying the rate of heat extraction to compensate at least in part for latent heat being lost 16 during freezing.

18 More particularly, according to a second aspect of the 19 present invention, there is provided a method of freezing material comprising a liquid, the method 21 comprising extracting heat from the material at a first 22 rate while latent heat of fusion of the material is 23 being lost from the material and the temperature of the 24 material is not substantially falling and subsequently extracting heat from the material at a second rate when 26 the temperature of the material falls, the first rate 27 of heat extraction being greater than the second rate 28 of heat extraction.

The invention therefore seeks to minimise or at least 31 reduce the amount of time the sample spends at the 32 temperature "plateau" during which the latent heat of 33 fusion is being lost. In relation to the frsezing of . ~ .

~: , . . .
. ~ . .. . , : ,.

4~

l biological samples, there is evidence (Parkinson and 2 Whitfield, Therioqenology 27 ~5) 781-797, ~1987)) that 3 the survival of cryopreserved bull spermatozoa is 4 inversely related to the time at the latent heat plateau; however, Parkinson and Whitfield appear to 6 advocate a lower cooling rate between 5 and -lS~C than 7 between -15-C and -25C. The problem is however not 8 restricted to the viability of living systems: for 9 foodstuffs in particular, an excessively long time at the latent heat plateau leads to damage mediated ll mechanically by the effects of ice crystals and l2 chemically by unusual osmotic effects, for example, in 13 the semi-frozen state. It has been observed that 14 longer pariods of time at the latent heat plateau lead to the formation of longer ice crystals and to a 16 degeneration in quality of the subse~uently thawed 17 product.

l9 By means of the heat extraction regimen of the method of the present invention, the cooling rate ran be 21 controlled so that the material being frozen suffers 22 few or no deleterious effects. In particùlar, as at 23 least some of the latent heat of fusion is being given 24 up by the material, the heat extraction rate is greater. However, the temperature of the material will 26 not substantially decrease during the period when 27 significant quantities of the latent heat of fusion 28 being given up by the material. After at least some of 29 the latent heat has been given up, the lesser rate of heat extraction is necessary so as to prevent too great 31 a range of temperature drop. The first rate of heat 32 extraction may therefore take place when tha 33 temperature is increasing or constant or the rate of .

- - :

. .
~? ~

WO91/01635 PCT/GB9~/01231 ~ ,~,,. 9 2~6~.~'8`o3 l temperature drop of the material is not substantial 2 (for example, 1PSS than l-C/min or even 0~1C/min), and 3 the second rate may be applied when the rate of 4 temperature drop is at lea~;t O.l-C/min or even l-C/min.

6 The invention may also permit a shorter dwell time in a 7 freezing apparatus, before transfer of the material 8 being frozen to a cold storage environment, and this 9 may be of significant advantage.
11 It should be noted that the use of the term "rate" as 12 applied to heat extraction does not imply that either 13 the first or second rate of heat extraction is 14 constant. Either or both rate may vary, and in some instances a variable heat extraction rate may be 16 preferred, to achiave non-linear and/or interrupted 17 cooling. An "interrupted cooling" profile includes a 18 profile having an initial rate of cooling, followed by l9 an isothermal hold, which in turn is followed by a subsequent cooling rate (which may or may not be the 21 same as the initial cooling rate~. Non-linear and 22 interrupted cooling profiles have biological and 23 non-biological application. Overall, in this invention 24 the second heat extraction rate must be less tha~ the first.

27 It should also be notsd that the term "firstl', as 28 applied to heat extraction rate, does not preclude the 29 use of a different heat extraction rate prior to the latent heat temperature plateau being reached.

32 It will be understood that the word "frozen", as used 33 in this specification when applied to complex mixtures :. . .

`
.

~6~ lo ~

1 of solvent(s) and solute(s), such as biological 2 material and/or foodstuffs, does not necessarily imply 3 that all matter in the material is in the solid state.
4 For example, to take the case of a frozen ~oodstuff such as strawberries at -25C, about 10~ of the fruit 6 will be liquid at that temperature, yet the ; 7 strawberries ~ould in ordinary parlance be referred to 8 as "frozen": it is in this sense that the word 9 "frozen" is used, and cognate terms should be construed accordingly.
' 11 ~12 The second rate of heat extraction will determine the - 13 rate of cooling of the solidifying or solid material.
; 14 ~he rate of cooling selected should be such as not to 15 damage the material, for example by enabling 16 significant ice crystals to form in aqueous systems.

: . , .
18 The second rate of heat extraction will vary widely, 19 depending on the nature of the material. For mammalian embryos, for example, the second heat extraction rate 21 should be such that the cooling rate does not exceed 22 0. 5 C/min and should preferably be about 0.3 c/min at 23 least in the range of -5 to -30-C. However, for 24 reasons of expediency, within these limitations cooling should be as rapid as possible. Although these 26 criteria apply to mammalian embryos, other materials 27 may have their own criteria; for example, samples 28 containing hybridomas, lymphocytes, tissue culture 29 cells (eg mammalian) and various microorganisms may be cooled at a greater rate, for example from 0.5C/min to 31 1.5C/min, such as about 1C/min. For other material, 32 for example oyster embryos the cooling rate may be 3~ about 5C/min, and for red blood cells, the rate may be :. :
-, WO91/0163~ PCT/GB90/01231 '. '~ 11 ', 20~ 8~3 l several thousand C/min, for example up to about 3000~C/min.

4 In this invention, the first rate of heat extraction is applied while latent heat of fusion of the material i5 6 being lost. This should not be taken to mean that all 7 of the latent heat of fusion has to be lost during the 8 application of the first rate of heat extraction. In 9 any aqueous sample, for example, latent heat will be liberated from the temperature of nucleation down to ll the eutectic temperature or the glass transition.
12 However the ma~ority (for example at least 70% or 80%
13 or even at least 90%) is generally liberated at the 14 freezing point and a few (for example 5 or lO) degrees celcius below. The first rate of heat extraction is 16 for preference applied while a majority (for example at 17 least 80% or even at least 90%) of the water is 18 converted into ice, which is to say while a majority 19 (for example at least 80% or even at least 90%) of the total latent heat of fusion of the material is being 21 lost.

23 From phase diagrams of simple solutes such as sodium 24 chloride, the amount of unfrozen water in the system can be seen to decline exponenti lly with temperature.
26 At any sub-zero temperature, the proportion of unfrozen 27 water is directly related to the osmolarity of the 28 unfrozen solution. For solutions of interest to the 29 food industry (for example 0.5 and 0.25M sodium chloride solutions and their equivalents) 80% of the 31 ice will have formed by -lO~C.

-:
.
,~

. :. ~ , :::; ..
~: : : . ~ ,;,. , . : . . .
,::. . . .

WO91/0163~ PCT/GB90/01231 - ~b~4~ 12 ~

l The invention can therefore be seen to embody the 2 notion of efficient removal of latent heat during 3 freezing or, in preferred embodiments, during the 4 conversion of, say, 80~ of water into ice. In those systems where phase diagrams cannot be derived, then 6 the efficient removal of latent heat from the melting 7 point (ie the latent heat plateau) to 5C or lO~C below 8 the melting point. Although efficiency is to some g ex~ent a relative concept, in certain embodiments of the present invention latent heat removal (for example ll to the extent referred to above) may be considered 12 efficient if it is achieved in S0~ or less than 50% of 13 the time observed when following conventional blast 14 freeziny techniques at -30C.
16 The method is particularly applicable to the freezing 17 and cryopreservation of biological samples, which 18 thereby constitute preferred examples of material which l9 can be frozen by means of the invention. The tPrm "biological sample" includes cells (both eukaryotic and 21 prokaryotic), organs and tissues composed of cells, 22 embryos, viruses, all of which can be natural or 23 modified genetically or otherwise, and biologically 24 active molecules such as nucleic acids, proteins, glycoproteins, lipids and lipoproteins. The liquid 26 present in or constituting the material will generally 27 be water, but the invention is not limited to aqueous 28 materials.

The invention may be used in the cryopreservation of 31 animal cells, particularly gametes or fertilised 32 eggs/embryos. However, other animal cells and plant 33 cells can advantageously be frozen by means of this 34 invention.

:
, .. . . , . , .. . , . . . , ., .. , , . . ~ . ... ... . .. .

WOgl/01635 PCT/GB90/01231 ... . ~
: ~ I'! Ji 13 2 ~ 03 1 Another significant application for the invention is in 2 the frozen food industry, where it may be important for 3 reasons of preserving taste and/or texture or otherwise 4 to freeze food quickly and efficiently and without causing excessive damage to the biological or other 6 material which constitutes t:he food. For example, soft 7 fruit when frozen by conventional means loses much of 8 its taste and/or texture. The material is thus 9 preferably a foodstuff, such as vegetables, bread and other bakery products, meats, fish, sea food (eg.
11 lobster, crab, prawns, finfish) or fruit, in particular 12 soft fruit such as peaches, plums, raspberries, 13 strawberries, kiwi fruit and ~ango. Non-aqueous systems 14 and emulsions, such as chocolate ~whether plain, milk or white), ice cream, cream and mayonnaise, may also be 16 frozen by means of this invention, as may reconstituted 17 food products.
1~
19 The invention also has application to non-biological material which needs to be frozen in a controlled 21 fashion. This may be necessary or desirable for 22 certain foodstuffs and/or other material in which the 23 rate and nature of crystal formation is important.
24 Sorbets and ices may fall into this category.

26 The invention can also be applied to the 27 cryopreservation of organs Por transplantation and 28 large volumes of cell suspensions such as blood, bone 29 marrow and microorganisms.

31 The volume of the sample to be frozen is not 32 particularly critical, but when freeziny or 33 cryopreserving gametes or fertilised egg/embryos in the .

:

: - ,:
. ' ' ' ! ' ' ~ 14 I~ /CB9u/01231 . ; , .
~; 1 biological sciences, the sample volume will generally 2 be less than lml, typically less than 0.5ml and may 3 even be less than 0.2ml. Volumes of 0.5ml and 0.25ml ; 4 are common. For the frozen food industry, the volumes to be dealt with will of course be ~uch larger, often - 6 several dm3 or even m3.

- 8 Particularly in the case of cryopreserving biological 9 samples for scientific, clinical or commercial use, the material to be frozen may be in a container or on a 11 carrier. Suitable containers include ampoules, tubes, 12 straws and bags (particularly thin-sec~ioned bags, 13 which may be held between two heat conductive (eg 14 metal) plates). Appropriate polymers include plastics materials such as polypropylene or polyvinyl chloride.
- 16 Containers which are small in at least one dimension 17 are preferred, as temperature gradients may then be 18 ignored across the small dimension or dimensions.
19 Tubes, straws and thin-sectioned bags are particularly preferred for this reason.

22 In a ~urther important aspect, the invention involves 23 the use of acoustics, particularly acoustics of the 24 type generally known as high frequency sound or ; 25 ultrasound. The application of acoustics/ultrasound to 26 improve the crystalline structure of metal castings is 27 kn own as dynamic nuclea ti on. Wh il st 28 acoustics/ultrasound may induce nucleation in 29 supercooled metals, the predominant benefit is grain refinement. Irradiation with acoustics also improves 31 heat transfer at the boundary layer. Nucleation of ice 32 formation by acoustics has received scant attention in 33 the past. For example, Hobbs ("Ice Physics", Clarendon . .

. . .
.:

WOgl/01635 PCT/CB90/01231 ' : " .
~ ~ 6i-~ 03 - 1 Press, Oxford, 1874) which is regarded as a standard 2 work in the area, does not metnion the potential of 3 acoustics in ice formation. Two Russian patent 4 documents, with commercially impracticable teachings are however known.
7 In SU-A-0618098 food products were stated to be frozen 8 more rapidly and their quality improved by placing in a 9 coolant and simultaneously exposing to ultrasound at 18-66 kHz and 16-40 W. The treatment was stated to 11 incrPase heat exchange at the boundary layer and caused 12 ordered formation of finely-crystalline ice. The 13 document does not disclose ice nucleation, but, by 14 reference to and inference from the metallurgy industry, grain refinement i5 probably the result of ~ 16 ultrasonication.

- 18 SU-A-0395060 teaches a similar process where the 19 freezing process time was reduced from 5 min 10 sec to ~- 2Q 3 min 5 sec, clearly a manifestation of improved heat 21 transfer. Ultrasound was also stated to exert a 22 beneficial effect on crystalisation processes, but 23 again nucleation by the ultrasound W25 not stated.
24 Both these processes are, however, commercially unacceptable as disclosed for a number of reasons.

27 First, it has been found that when the process was 28 repeated with strawberries or strawberry slices ~4.5mm) 29 the thawed product was of unacceptable quality. There was no detectable improvement in the quality of the 31 fruit compared with material frozen in a conventional 32 (-30-C) blast freezer without the use of ultrasound.
33 Secondly, the processes described reguire ir~ersion of .

WO91/01635 4~ a PCT/CB90/01231 , '` '~ 16 ~, l the food in a bath of either ethylene glycol ~-22-C) or 2 freon 12 (-29.8 C). The possibility of contamination 3 of the food with,either of these substances would be an 4 unwelcome risk;~nder commercial circumstances, and the cost of thëse chemicals may in practice prove 6 prohibitive.

8 Thirdly, the power that is used (2 to 3 w/cm2) is very 9 high: this will not only have a severe warming e~fect on th~ food, it may also induce cellular damage to ll material b~ing frozen.

13 After nucleation of ice within à food the latent heat 14 of fusion should be removed as guickly as possible to minimise the effect of supercooling. It is known in 16 the food freezing industry that to achieve this the 17 samples may be immersed into cryogens, such as liquid 18 nitrogen (-196-C), liquid CO2 or freons, but this has l9 several associated problems.

21 First, with large biological samples (such as,above 5mm 22 diameter) ~Ishelll~ freezing will occur resulting in 23 fracture and cracking of the sample.

Secondly, in some fruits, such as strawberries, a 26 secondary type of tissue damage occurs if the fruit is 27 cooled below -lOO C. I~ is extremely difficult to 28 conduct a liquid nitrogen immersion process without 29 causing damage by exceediny the minimum storage temperature.

32 Thirdly, the immersion of samples into liquid nitrogen 33 is a costly process and therefore uneconomic and likely .
. .
~ ' , . I
:) ~ WO91/0163~ PCT/GB90/OIZ31 17 206~R3 . ' '' 1 to be unsustainable in the frozen food industry~

3 The teachings of SU-A-0618098 and SU-A-0395060 may be 4 unworkable on a practical basis if directly applied to freezing liquid-containing material such as biological 6 material and/or foodstuffs, and it appears that the 7 frozen food industry has largely ignored the 8 possibility of using acoustics in freezing processes.

It has now been discovered that the use of sound, 11 particularly high frequency sound, is highly benefical 12 when used in conjunction with or even independently of 13 a heat extraction method in accordance with the first 14 aspect of the invention. Preferably, therefore, the material being frozen is subjected to sound waves, 16 which may be high frequency sound waves.

18 The high frequency sound waves are preferably l9 ultrasound waves, generally at a frequency of at least 16 kHz, for example from 18-80 kHz. The frequency at 21 which acoustics is preferably applied ranges from 20 22 kHz to 50 kHz. Typically the applied frequency is from 23 20 kHz to 30 kHz; the optimal range for at least some 24 applicatons appears to be from 22.5 kHz to 25 kHz.

26 Supercooled material may be subjected to the sound 27 waves for from 0.1 to 1.0 seconds. Alternativelyl the 28 material may be pulsed or otherwise supplied with 29 acoustics throughout the freezing process. It is preferable for the acoustics to be applied as one or 31 more pulses. The pulse duration should on average 32 preferably be from 5% to 20% of the total time of 33 pulse-plus-interval; preferably the pulse lenth is from .

-W091/01635 PCT/CB9OtO1231 ` ~64~3 18 ~

l 0.5 to 5 seconds, with about 2 seconds being optimal.2 Pulses of about 2 seconds in 20 seconds have been ~ound 3 to be particularly effect:ive. ~he power and/or 4 frequency may be varied (either discreetly or continuously) during application. More than one 6 frequency may be used at the same time. It may be 7 particularly appropriate to apply acoustics when 8 certain material bein~ frozen is in the liquid phase;
g this may apply in particular to ice cream.
, , ll As far as the power at which the acoustics is applied, 12 there is clearly a conflict in requirements. On the 13 one hand the power should be high enough for the 14 acoustics to be effecti~e, and on the other hand the power should not be so hiqh as to cause unacceptable 16 heating of the material being frozen (as the energy 17 applied will be dissipated as heat). Power applied 18 between 0.05 and l.9 or 2.0 W/cm2 was found to be l9 acceptable, with a range of O.l to l.5 W/cm2 being preferred and about 0.2 to l W/cm2 being optimum.

22 This non-invasive technique of inducing ice nucleation 23 thus at least mitigat~s, or overcomes, problems 24 associated with prior art techniques.
26 The sound waves may be generated by sound wave 27 generators known in the art, such as ultrasonic baths, 28 piezoelectric transmitters and suitable transducers.
29 Thus the material may be in contact with the sound wave generator, for example inside a container such as a 31 mould in contact with a piezoelectric transmitter, or 32 ~n a conveyor belt in contact with a suitable 33 transducer. In this latter e=bodiment the materia1 may ~`
.

19 2 0 6 i 8 0 3 1 thus be moved within an environment having a 2 temperature gradient, such as a conventional blast or 3 tunnel freezer.

; 5 Four preferred methods of inducing ice nucleation using 6 high frequency sound waveC; are as follows.

- 8 1. The sample is immersed in an ultrasonic bath which g is preferably maintained at, or about, the freezing temperaturs of the material (eg. -20-C). Thus the 11 sound wave generator serves to both prsvide the high 12 frequency sound waves and also to cool the material.
13 The material will generally be immersed in a liquid, 14 preferably an aqueous liquid, such as water. However, the ~aterial, if desired, may be contained or enclosed 16 in a mould which is particularly suitable for the 17 freezing of ices.

19 2. The material may be placed in a contai~er, such as i` 20 a mould, which i5 cooled in a freezing bath. A
~ 21 piezoelectric transmitter is placed in contact with, or ;~ 22 built into, the mould to deli~er the high frequency 23 sound waves. This method is particularly suitable for ; 24 frozen sorbets, ices and ice creams.

26 3. The material may be placed on top of a conveyor 27 belt which is in contact with, or interrupted by, one 28 or more transducers. This method is particularly 29 suitable for thin layers of material, such as slices of foodstuffs such as so~t fruits. The contact between 31 the material and conveyor belt ensures that the sound 32 waves are transmitted efficiently to the whole o~ the 33 material. Cooling of the material can be achieved by i, .
~ .
...
.. .. - :
.

WO91/0163~ PCr/GB90/01231 l passing the conveyor belt through, ~or example, a 2 conventional blast freezer. It is preferred that a 3 short zone of acoustic transducers is placed at a 4 particular point along the conveyor belt to achieve maximum nucleation in the material.

7 4. For larger materials and those of non planar 8 geometry, such as spheres and cylinders, to achieve 9 more than a point contact with an ultrasonic source, it is preferable to immerse the sample either fully or ll partially in a liquid in a container. The high 12 frequency sound waves can then be applied via 13 transducers, but the material will be immersed in the 14 liquid for only a short period (for example less than one second). The temperature of the container is 16 preferably maintained so as to kèep the material at its - 17 freezing temperature, for example about -5C. The 18 liquid in the container is preferably kept below its l9 freezing point by the addition of non-toxic chemicals, for example food grade chemicals. This has the 21 advantage that the material may be simultaneously 22 coated with the food grade chemical. Preferred food 23 grade chemicals include sugars and glycerol, ~or 24 example to freeze the material and add a glaze. This ; 25 embodiment may be combined with a continuous process 26 such as the material being carried along a conveyor 27 belt as discussed above. For example, the conveyor 28 belt may dip into an ultrasonic bath, suitably for a 29 short period such as less than one second, when it is subjected to ultrasound.

32 The material is preferably precooled before subjection 33 to the high requency sound waves to induce ice .

:`
. ~ , . . : , . . , , :

; ~ 21 2 0'6'll'g ~'3 l nucleation. Suitably the material will be cooled so 2 that it is at the same temperature, namely of thermal 3 equilibrium, as the environment. This is since if a 4 large temperature differencé ~xists between the material and its environment then a temperature 6 gradient will be established across the material and 7 nucleation will occur on the outside and the ice front 8 will propagate towards the centre, resulting in 9 unwanted "shell" freezing. Thus, if the whole of the material is precooled to the temperature of the 11 environment, and in particular such that the inside of 12 the material is at the same temperature as the 13 environment, then on subjection to the high frequency 14 sound waves ice nucleation may be induced on the inside and preferably at the centre, of the material. Usually 16 the material will be thermally equilibrated with the 17 environment below its freezing point.

l9 The application of acoustics, as preferred for the present invention, as described above, itself forms an 21 independent aspect of the invention. It has been found 22 that if the immersion techniques suggested in the 23 Russian patent documents described above is avoided, it 24 is possible for acoustics to be beneficial and commercially feasible. According to a further aspect 26 of the invention, there is provided a method of 27 ~reezing material comprising a liquid, the ~ethod 28 comprising abstracting heat from the material and 29 applying sound waves to the material by means of a non-liquid conkact with the material. Generally, there 31 will in this aspect of the invention be solid or 32 mechanical contact between a source of high frequency 33 sound waves and the material to be frozen, but .,~,......................................................................... .
,. . ., ~
, ::, ~ o 3~ 22 ~

l gas-mediated contact may be adequate. The contact may 2 for example be achieved ~y the use of a source of high 3 frequency sound waves in the form of a probe, such as 4 the BRANSON LUCAS-DAWE probe, in direct contact with the material.- ~Alternat:ively or additionally, the 6 material could rest on a solid surface, to which was 7 mechanically connected, directly or indirectly, a 8 source of high frequency sound. It will be appreciated 9 that a layer of suitable material may be interposed between the material to be ~rozen and the solid ll surface, for example to prevent contamination and/or 12 undesirable sticking, but this is not to be regarded as 13 detracting from the mechanical connection, which is 14 just rendered somewhat more indirect. Further, it is to be understood that uniform contact betwe~n the 16 material and the surfac~ is not necessary: it is only 17 necessary for there to be sufficient contact for the 18 sound waves to ~e transmitted effectively.
lg A fluid-~illed (preferably liquid-filled) layer may be 2l interposed in the sound path between the source of high 22 frequency sound and the material to be frozen. This is 23 not to say that liquid is in contact with the material 24 to be frozen; on the contrary, the fluid layer simple aids transmission and/or distribution of the high 26 ~requency sound waves into the material. the fluid may 27 be any organic solvent, but is preferably freon, 28 glycol, ethanol or a food-compatible solvent such as 29 sold under the trade mark ISOPAR. The ISOPAR K product may be the most pre~erred.

32 It is to be understood that the "non-liquid contact" of 33 th~ =ateria1 to be frozen does not necessarily i=ply :

- ~ : ~ . . . . ...

23 ~

l complete dryness. For example, if cut fruit is being 2 frozen, a small amount of liquid may be released from ~ 3 the fruit itself. This is however to be contrasted - 4 with immersion within a sound-transmitting li~uid, which is not within this aspect of the invention.
: 6 ; 7 It has also been discovered that if the relatively high 8 power levels taught in the Russian patent documents g referred to above are avoided then, contary to expectations the results are better; further, a lower ll power level can be delivered by a more economical piece 12 of equipment. According to a further aspect o~ the 13 invention, there is therefore provided a method of -14 freezing material comprising a liquid, the method comprising a~stracting heat from the material and 16 applying sound waves to the materïal at a power level 17 of less than 2 ~/cm2. Preferred features o~ this 18 aspect of the invention are as described above.
';~ 19 Further, intermittent application of acoustics may 21 provide the basis for improved performance over the 22 disclo~ure of the Russian patent documents.
~r 23 .;24 Correspondingly, the invention relates in further aspects to an apparatus for freezing material 26 comprising a liquid, the apparatus comprising means for 27 abstracting heat from the liquid and means for applying `28 sound waves to the material, wherein (a) the sound 29 waves are applied to the material by means of a non-liquid contact with the material and/or (b) the 31 means for applying sound waves to the material is 32 adapted to deliver the sound waves at a power level of 33 less than 2 W/cm2 and/or (c) the means ~r applying :

WOgl/01635 ~3 24 PCT/GBU0/0l23l 1 sound waves to the material is adapted to deliver the 2 sound waves intermittently. Preferred features are as 3 described above.

Methods in accordance Wit}l the invention work well in 6 conjunction with the use of other means for inducing 7 ice to nucleate, such as by using chemical (for example 8 crystalline) ice nucleators, such as is disclosed in 9 EP-A-0246824. Such nucleators can be used to determine reasonably accurately when ice nucleates. The 11 nucleator may be coated on one or more walls of a 12 container for the material and/or on a carrier for the 13 material. As is disclosed in EP-A-0246824, cholesterol 14 is a preferred nucleator.

16 Heat extraction may be achieved by any convenient way.
17 In principle, it is possible for heat to be extracted 18 by an endothermic reaction taking place in the 19 material. However, it will usually be more convenient to provide a temperature gradient between the material 21 and at least part of the surrounding environment, which 22 should be cooler than the material. This embodiment of 23 the invention takes advantage of Newton's law of 24 cooling, which states that the heat loss will, for small temperature differences be proportional to the 26 temperature difference between the material and the 27 surroundings.

29 Heat extraction can therefore most easily be achieved in many applications of the present invention by 31 placing the material in a cold environment. It 32 therefore follows that, to achieve first and second 33 heat extraction rates where the first heat extraction , , .. , : : ... ~ .: .

:-~ . - . . : . -;: : . . .

: . ~ . '~ '.. : ~ .. :

' ,. ~i ,t; ,,~
~ ~ 25 ~6~3 l rate is greater tban and followed by the second, the 2 sample can be moved from a cold environment to a less 3 cold environment, for example by means of a conveyor 4 system. In practice in some applications, it may be easier to change the environment temperature rather 6 than to move the sample, in which case the environment 7 temperature is increased at the interface between the 8 first and second rates.

Suitable environment temperatures for the first and ll second heat extraction rates will be apparent to those 12 skilled in the art. For preference, the environment 13 temperature for the first heat extraction rate will be 14 at least 15~C, and preferably at least 25 C lower than the environment temperature for the second heat 16 extraction rate~ When the material to be frozen 17 comprises water, ~for example in the case of biological 18 material such as organs or, particularly, foodstuffs, l9 the environment temperature for the first heat extraction rate can be for example less than -50-C, or 21 even -80C or -lO0 C; the environment temperature for 22 the second heat extraction rate may be -20 C to -30 C.
23 For foodstuf~s, the environment temperature for the 24 second heat extraction rate may be the final desired ..
stor~ge temperature. For biological material that is i 26 to be cryopreserved, it may be desired to reduce the 27 environment temperature further, for example after the 28 second heat extraction rate.

The preferred minimum environment temperature for the 31 first heat extraction rate may in part be determined by 32 tolerance of the material being fro~en to temperature 33 gradients. For fruit ~t least, and possibly for other ~' . ~ ~, . ~, . ... . .. . .. .. .. .. . . .. ... .. .. .. . . . . . . ......... . ...... . .

.. ;, . . . .. ,, :- ,.: , . . ~,. .

0~ ~ 26 PCT/GB90/0123l : ' " ' : ,, 1 foodstuffs and biological material, placing material to 2 be ~rozen which has equilibrated with room temperature 3 in a~ environment temperature for the first heat 4 extraction rate of -100C or less appears to cause too large a temperature gradient to be acceptable in some ~ 6 circumstances. Strawberries, for example, suffer ;- 7 injury under such conditions, possibly caused by the - 8 non-uniform formation of glasses and eutectics.
:' . 9 As an alternative to altering the environment 11 temperature, different rates of heat extraction may be ! 12 achieved by altering the efficiency with which the 13 en~ironment extracts heat from the material: cold air 14 or other gas may be passed over the material at different rates for this purpose. A higher gas 16 velocity will achieve a higher heat extraction rate, as : 17 can be found with everyday experience of wind chill - 18 factors.
~`.' 19 0 It will be appreciated that the present invention can 21 be put into effect by making adjustments and 22 modifications to enable the appropriate heat extraction ~23 protocol to be carried out. As discussed above, this `~24 may be achieved by an appropriate protocol for changing the environment temperature. Such protocols can 26 readily be established for various foodstuffs and other 27 biological material by taking into consideration the 28 relevant parameters for each material, for example 29 including:

31 a) Size;
32 b) Geometry;

33 c) Water content;

. .
':
'' .
, .

., . . ~ . ,,j,. . ~ .

; ~ ~ : . . . . . . .

WO91/01635 PCT/GB90/0123l , , 27 20B~8 D3 l d) Freezing point (to a first approximation this 2 is dependent on solute concentration within ~ 3 the foodstuff or other material);
-~ 4 e) Thermophysical values of the material of th~
material, both before freezing and in the ; 6 frozen state; and 7 f) Container dimensions and other details.
~ 8 } 9 Because these parameters differ from material to material a computer can readily be used to derive 11 optimumi protocols.

13 The temperature history in a sample being cooled in a 14 controlled rate freezer (such ais the KRYO lO series Chamber Model 10-16 by Planar Biomed, Sunbury-on 16 Thames, England) can be calculated by solving 17 numerically the Fourier heat conduction equation in the 18 sample with convective or other boundary conditions as 19 appropriate. (The axpression KRYO 10 is a trade mark.) In general, the calculation method must allow for the 21 cooling of an aqueous solution or other material where - 22 c~mpositional as well as phase changes occur durin~
23 freezing. This requires the appropriate molarity-24 ~rePzing point depression data to be available, to provide the relationship between ice formation and - 26 melting temperature~ Supercooling of the sample may 27 also be suitably accounted for. In the ca~e of thin 28 slices the temperature gradients across the sample can 29 be assumed negligible and consequently the conduction equation reduces to a simple unsteady heat balance 31 between the time rate of change of enthalpy of the `. 32 sa~ple and the heat trans~er rate across its 33 boundaries. The validity o~ this si=plified :i :

W091/01635 PCT/GB9OtO1231 ~ 3 28 ~

l calculation has been compared against experimentally 2 derived data. The calculation method has been employed 3 to predict methods to reduce the latent heat plateau 4 within plum slices by manipulation of the environment temperature.
7 However for calculating the temperature history in 8 samples of finite thickness, where conduction within 9 the sample is important, it is necessary therefore to solve the full equation. Solving the full unsteady 11 equation with three space dimensions is computationally 12 very time consuming. However, in many cases the 13 temperature gradients in one direction are much greater 14 than in the other two and in these systems a reasona~le prediction for the temperature history can be obtained 16 from a one-dimensional model. This model could be 17 developed for l-d Cartesian, l-d spherical or l-d 18 cylindrical geometry.

In its broadest apparatus aspect, the invention 21 provides an apparatus for freezing material comprising 22 a liquid, the apparatus comprising means for extracting 23 heat from the material and control means for varying 24 the rate of heat extraction to compensate at least in part for latent heat being lost during freezing.
~6 27 According to a further aspect of the invention, there 28 is provided an apparatus for freezing a matexial 29 comprising a liquid, the apparatus comprising means for extracting heat from the material at a ~irst rate while 31 latent heat of fusion of the material is being lost 32 from the material and the temperature of the material 33 is not substantially ~alling and means for subsequently ... , . , . ~ .

- : .

WO91/Ot635 PCT/GB90/01231 i . .
" 2 0 6 ~1~ D 3 1 extracting heat ~rom the material at a second rate when 2 the temperature of the material falls, the first rate 3 of heat extraction being greater than the second rate 4 of heat extraction.

6 As-discussed above, the apparatus will preferably 7 comprise a (preferably high ~requency) sound generator.
8 The medium through which the sound is conducted from g the generator to the material may be gase~us, ~or example air, or solid.

12 Each heat extraction means can in gensral comprise a 13 refrigerated element, which may actively be cooled by 14 expansion of a gas. Conventional diffusion or compression/expansion refrigeration equipment may be 16 used in this emb3diment. However, this is not the only 17 form of heiat extraction means that can be used. For 18 example, a cold liquid or solid which is dissipated as 19 heat is extracted from the material can be used. An example of a cold liquid that can be used in this way 21 is liquid nitrogen, which will be the material of 22 choice for at least one of the heat extraction means 23 for cryopreservating biologi~al material, as biological 24 material is conveniently stored at the temperature of li~uid nitrogen. A cold solid which is similarly 26 dissipated is solid carbon dioxide (dry ice), although . .
27 the cooling effect of solid carbon dioxide will be less 28 than the cooling effect of liquid nitrogen, bscause the 29 sublimation point of the former is higher than the boiling point of the latter. A third possibility for a 31 heat extraction meians is to use a heat sink which warms 32 up to equilibrium with the material to be frozen, or as 33 nearly as any in~ervening (for exa~ple insulating) .
, ~
. . .

. ' . : ! : ' ' ' . , .' . .' , . .: , . . : ~
' ' ' . ; ", ~ ' ': ', ' ' .' .. .' , , "" ' . ' . . '" ' '" :' ' , '',' ~: , : ' " . . , ', ' WO91/01635 ~3 PCT/GB90/01231 ` 30 1 material allows in the time available. The heat sink 2 can therefore be a block of relatively cold material, 3 especially a material with high heat conductivity, for - 4 example a metal. To counter any adverse problems of - 5 condensation, the metal will preferably be non-6 corrosive, for example by being made of ~rass or 7 stainless steel. Howsver, any metal can be used if 8 appropriately protected, if necessary.

- lU Suitable insulating material may be polystyrene 11 (expanded or unexpanded) or another platics polymer 12 such as polytetrafluoroethylene or acetal but it will 13 be appreciated that any material with suitable 14 properties can be used.

16 An apparatus in accordance with the invention can 17 comprise a single heat extraction means, such as one of 18 those discussed above, and control means to control the 19 single heat extraction means to extract heat at the first and second rates. For example, a so called 21 "active" system in accordance with this embodiment of i-j 22 the invention could comprise a refrigerated element, 23 control means and temperature feedback means. The 24 control means could comprise a computer, microprocessor or other electronic means. The temperature feedback 26 means would continuously or continually monitor the 27 temperature of the material to be frozen and relay this 28 information to the control means, which could then 29 cause the refrigerated element to extract heat at the appropriate rate. Such an active system as this gives 31 considerable flexibility for a wide variety of material 32 to be frozen (particularly foodstuffs), but may involve 33 relatively high expense for small amounts o~ material.

;, ~, .

.
.

: , . . : ~ ~: . . , . ... ;. .
. ~ ; , : . :

WO91/01635 PCT/~B90/01231 ~ 31 2~6~803 2 A similar but simpler e~bodiment could comprise a 3 refrigerated element which is operable at two rates of 4 heat extraction. The element may be arranged to operate first at a higher heat extraction rate, and 6 then a timer may cause the element to switch to 7 operation at a low heat extraction rate. Such an 8 embodiment can be used when the characteristics of the 9 sample, or at least the environment surrounding the sample, are known, but this may be acceptable in many 11 circumstances, especially when various samples are 12 small compared to the apparatus of the invention, so 13 that any individual variation in characteristics will 14 be relatively small.
16 Other preferred embodiments of "active systems" are as 17 follows:

19 1) Batch systems. Mechanical freezers are generally cooled by the Joule-Thompson effect and operate at 21 temperatures down to -80-C; a minimum of -135-C is 22 possible. Material is placed into a closed chamber and 23 left until it has reached the desired temperature and 24 then removed for storage. The air in the chamber may be unstirred or fan driven to achieve forced 26 convection. Additionally, ~he material to be ~rozen 27 may be placed statically on shelves or rotated within 28 the freezer.

The desired thermal profile may be obtained in such a 31 closed system by direct control of the compressor 32 temperature by electxical or mechanical means. In some 33 cases this may be practically difficult as the response ~', .

Wos1/01635 ~ PCT/~B90/01231 ~ , 32 l time of such~' a control system may be too slow to 2 generate the desired profile. However, as the minimum 3 operating temperature will be required at the beginning 4 of the process the control of temperature may be achieved by ,m,ai'ntaining a constant compressor 6 temperature whilst varying the heat input into the 7 system from an independent heater which is programmed 8 electrically or mechanically to generate the desired 9 profile. In addition, a combination of direct control of compressor output together with an external heaker ll may be employed. The control of temperature may be 12 preprogrammed or alternatively may be actively 13 controlled from temperature sensors placed either in 14 the gas or in the samples to be frozen.
16 2) Continuous Systems. The material flows through 17 the freezer on a horizontal conveyor belt or spiral 18 system. Following a retention time within the fraezer, l9 the material emerges at a temperature suitable fsr storage. Gas circulation is usually fan driven; in 21 some cases the cold gas is forced upwards through a 22 perforated conveyor belt so that the samples are 23 suspended as in a fluidised bed. The temperature at 24 the point of entry is invariably warmer than at the point of exit. Cooling may be by mechanical means or 26 alternatiYely by vapour from a cold liquid such as 27 liquid nitrogen; in this case the minimum operating 23 temperature achievable (>-160-C) is lower than in 29 mechanical systems.
31 The desired thermal profile is to be achieved by the 32 manipulation of the temperature distribution of the gas 33 through the system. In contrast to the conventional , .: , ~: , ' , ' .:
-, . . . . . . . .
.. ..

WO91/01635 1'CT/CB90/01231 A, 2 0 6 q 8 0 3 l mode of operation the system will be at its minimum2 temperature at the point oE entry of the food and will 3 become warmer towards t:he point of exit. The 4 temperature gradient within the continuous system may be determined in several ways, including a system of 6 baffles to ensure the recirculation or removal of cold 7 gas, the introduction of warm gas or the positioning of 8 heaters. The velocity of ga~ flow will also modify the 9 heat transfer and will be selected to be at its maximum - lO at the point of entry, at later stages the flow may ll either be constant or reduced. In addition, the -; 12 temperature experienced by the sample may also be 13 modified by control of the speed of the conveyor belt.
14 By employing a series of conveyor belts running at different speeds, the retention times within different 16 areas of the freezer may also be manipulated. A
17 combination of several of these processes may also be 18 appropriate. The control of temperature may be ; 19 preprogrammed or alternatively be actively controlledfrom temperature sensors placed either in the gas or in 21 the samples to be frozen.

;:
23 3) Immersion in low temperature baths. This is a 24 process generally applied to ices, sorbets etc which are poured as liquids into moulds which are then 26 semi-immersed in a stirred low temperature bath, 27 typically at temperatures o~ -30 C. Such low 28 temperature baths are usually cooled by contact with a 2g heat exch~nger cooled by the Joule-Thompson effect.
Following freezing the sample is removed ~rom the mould 31 and placed into storage. The direct immersion of 32 non-moulded foods into liquid cryogens is generally not 33 considered good practice. ~owever, immersion into ' ' WO91/01635 ,~ PCT/GB90/01231 ~ 34 1 liquid CO~, which is considered to be non-toxic and 2 which evaporates at conventional storage temperatures, 3 may be safely employed for a variety of foodstuffs.
4 ::-The temperature profile achieved by immersion could be 6 modified by several potential methods. A series of - 7 baths, maintained at different sub-zero temperatures 8 could be employed, with the samples being immersed in g sequence through the various bathæ. Alternatively, the thermal gradient along a single bath may be manipulated ll to achieve the desired profile, the rate of passage -12 through such a gradient bath could also be modified in 13 a linear or non-linear manner to achieve the desired 14 profile. Again the control of temperature may be pre-programmed or alternatively may be actively 16 controlled from temperature sensors placed either in 17 the fluid or in the samples to be frozen.

19 In a quite different embodiment of the invention, apparatus in accordance with the invention can have 21 separate heat extraction means for providing the first 22 and second heat extraction rates, respectively.

24 What may be a preferred arrangement is again to have first and second extraction means, but to have the heat 26 eXtractiQn means so arranged that together they provide 27 the first heat extraction rate, whereas only one of 28 them (for example the first heat extraction means) 29 provides the second heat extraction rate.- This arrangement gives rise to a particularly effective 31 arrangement, particularly for the cryopraserYation of 32 biological material. The first heat extraction means 33 may bs a bath of liquid nitrogen or an environment of ..... .. . . . . ..... ... . . ...... .. . . .
. . . , , ~ , . , :, , i '-': . . '' : .:: : ': . , ', ; . , l cold nitrogen gas (eg above a bath o~ ~ d nitrogen), 2 which may be below -lO0 c. Biological or other 3 material to be frozen can be contained in a Dewar flask 4 also containing a cold (eg gaseous nitrogen) environment; the material can be appropriately 6 insulated to provide an acceptable second rate of heat 7 extraction. The cold gaseous nitrogen enviro~ment may 8 for preference be provid~d in a specialisied vessel g known as a "dry shipper" with which those skilled in the art will be familiar or, less preferably, above a ll liquid nitrogen bath. As a further possibility, 12 commercial deep freezes may provide an adequate cold 13 environment; they are frequently capable of achieving 14 and maintaining temperatures of from -80 C to -135-C.
More generally, mechanical co~mercial freezers can have 16 operating temperatures from -20 to -140-C, and 17 liquid/gas freezers based on cryogenic gases can 18 operate below thesa temperatures down to~ or at least 19 towards, absolute zero.
21 To augment the heat extracting effect of the nitrogen 22 or other primary coolant to a degree sufficient to 23 provide the greater first rate of heat extraction, a 24 second heat extraction means may be provided during the time at which the first rate of héat extraction occurs.
26 Appropriately, the second heat extraction means may be 27 a heat sink, for example, a block of cold brass or 28 another appropriate material, as discussed above. The 29 biological sample or other material to be frozen can again be suitably insulated from the heat sink so that 31 an appropri~te ~irst rate of cooling occuxs. -~

33 In a preferred embodiment, material to be frozen is .' - - ' ~ 5 . , ~ 3 36 ~

1 held within-a block of insulating material within the 2 Dewar flas~ àt! one or more points spaced between the 3 centre and the periphery of the block. The periphery 4 of the block will be continuously cooled by a cold environment. The centre of the block can receive the 6 brass or other heat sink, which provides the additional 7 rate of cooling necessary for the first rate of 8 cooling.
g The way in which the heat extraction means can be 11 constituted is not limited to any of the embodiments 12 discussed above, and may for example be a combination 13 of the particular embodiments described or indeed any 14 other suitable arrangement.

16 From the above discussion of a preferred embodiment of 17 a passive arrangement, it will be appreciated that the 18 invention also provides means which can be used in 19 conjunction with a dry shipper, liquid nitrogen bath, freezer or any other cold environment, including those 21 discussed above.

23 According to another aspect of the invention thPre is 24 provided a device for use in freezing material comprising a liquid, the device comprising a heat sink, 26 insulating means at least partially surrounding the 27 ~eat sink and means for holding, within the insulating 28 means, material to be frozen, the device being adapted 29 to withstand a temperature at which the material is frozen.

32 The heat sink may, as before, comprise a block of heat 33 conductive raterial such as a =etal, for example brass.

'~
, , ' ~
. ` . ... . . .
, , : ''::; , ' r~.,~ 37 20~180~ ,.

: .
1 It may be formed as a core, for example a generally 2 cylindrical core, around which the insulating means may 3 be placed. The core is preferably detachable from the 4 insulating means; the reason for this preference will be discussed below.

7 The insulating means may be any suitable gaseous, 8 liquid or, preferably, solid insulator. Polystyrene, . g polytetrafluoroethylene (ptfe) and acetal are -~ lO acceptable. It will be appreciated that the insulator 11 should have low, but not zero, heat conductivity and~or 12 diffusivity. Polystyrene (unexpanded), for example has ' 13 a thermal conductivity of 0.04 W.m l.X 1 and a thermal 14 diffusivity of 2.9 x 10~8m2. 5-l, The figures for ptfe and acetal are as follows:
; 16 17 PTFE Acetal 19 thermal conductivity 0.24 0.22-0.24 - -W.m l.K 1 @ 23-C
21 thermal diffusivity 0.74 0.30 22 m2 -1 24 The holding means may be any appropriate shape or con~iguration for holding the material to be frozen.
26 Since at least part of the material will be li~uid, the 27 holding means may be adapted to receive a container, 28 for example a straw, ampoule or bag, as discussed 29 above, for the material. Ampoules may be made of glass, plastics or any other suitable material;
31 suitable plastics ampoules include those æold under the 32 trade mark CRYOTUBES. For the case of straws or 33 ampoules to be held in a solid insulating block, the .:. .
,, .' -.
.
. .
. .
'' ' , .

' ' . : ' , ., I ~ : . .' ' . ':

~3 38 ~

l holding means may simply comprise holes drilled or2 otherwise formed in the block. Several containers may 3 be received in the same hole. It may be that the 4 insulating block has more than one components, which can is used in a single operation of the device: the 6 components may be stacked, one upon the other, with the 7 cylindrical core being extended appropriately such that 8 it accommodates the entire depth of the stacked -~ g insulator blook components.

11 In use, the heat sink (in the preferred embodiment, the 12 brass core) will fixst be cooled, for example by 13 placing it in a cold environment. The insulating means 14 and the material to be frozen can then be positioned around the heat sink, so that the cold environment at - 16 least partially surrounds the insulating means. The - 17 material to be frozen will therefore be cooled at the 18 first heat extraction rate by the combined effects of 19 the heat sink and the cold environment until the temperature of the ~eat sink equilibrates the 21 temperature of the adjacent portion of the insulating 22 means; thereafter, the material to be frozen will be 23 cooled at the second heat extraction rate solely by the 24 effect oP the cold environment, the temperature at any time being dependent upon the properties of the cold 26 environment and the thermal properties and dimensions 27 of the insulating means and the heat sink. (The 28 temperature profile is predictable using the computer 29 simulations involved in the design of this piece of equipment, and can be adjusted to suit a required 31 application by varying the parameters considered 32 above.~

. .
.:

'~ ~

WO91/0163~ PCT/~B90/01231 39 20~ 8 ~3`' 1 The thermal characteristics of the heat sink and the 2 insulating means, the position of the holding means 3 within the insulating means and the nature of the cold 4 environment will ba chosen so that heat is extracted from the material to be frozen at the first extraction 6 rate for the appropriate length of time, ie while 7 latent heat is being extracted from the material and 8 the temperature of the material is not substantially 9 falling.
11 According to a further aspect of the invention, there 12 is provided a method of freezing material comprising a 13 liquid, the method comprising providing material to be 14 frozen within insulating means, at least partially surrounding a cold heat sink with the insulating means, 16 and providing a cold environment at least partially 17 surrounding the insulating means.

l9 The cold environment may be defined by a container which may be well insulated (ie having lower heat 21 conductivity than the insulating means) for exa~ple 22 provided by vacuum insulation. The environment may 23 therefore be defined by a Dewar flask or a dry shipper.

A ~urther application of nucleation of aqueous 26 solutions by acoustics would be the controlled, 27 simultaneous nucleation of multiple samples during the 28 cooling phase of ~re~ze-drying. A possible scenario is 29 the freeze-drying of vaccines, where several thousand 30 small glass vials would be cooled, ~rozen and dried in 31 the freeze-drying apparatus in a single run.
32 Undercooling of the samples during the cooling phase o~
33 freeze-drying is, to some extent, inevitable and ,, , . . . ' . ~ ; ' ' : , ' WO91/0163~s S~3 PCT/GB90/01231 l without any attempt at synchronised nucleation the ice 2 formation points of individual vials (or other sample 3 container) will vary by several degrees. This will 4 lead to varià~ions in processing time, sample quality as drying begins and inconsistencies in the quality of 6 the completed, dried batch of samples. Th~ problem 7 could be solved if a source of acoutstics was 8 appropriately configured and placed within the g freeze-dryer to be used to brin~ about controlled nucleati~n and ensure that it coccurred at a required 11 temperature, and uniformly between the samples.

13 In the foregoing discussion, reference has primarily 14 been made to systems in which liquid water is frozan to ice. However, it will be appreciated that the 16 invention is not limited to water based systems.

18 Other preferred features of each of the aspects of the l9 present invention are as for the other aspects mutatis mutandis.

` 22 , WO91/01635 PCT/CB90/01231 ~ 41 ~6'180~

l For a better understandi~g of the invention, and to 2 show how it may be put into effect, preferred 3 embodiment of t~e invention will now be described by 4 reference to the accompanying drawings, in which:

6 FIGURE 1 is a graph showing how the temperature of 7 a biological sample Yaries against time as it is 8 cooled through its ~reezing point;

FIGURE 2a shows a vertical sectional view through ll a device which is a "passive freezer" embodiment 12 of the invention;

14 FIGURE 2b shows an exploded perspective view of a further passive freezer embodiment;

17 FIGURE 2c shows an exploded perspective view of a 18 still further passive freezer embodiment;

FIGURE 3 shows five temperature cooling curves for 21 material cooled in accordance with the invention;

23 FIGURE 4 shows a temperature cooling curve for 24 plum slices frozen in accordance with Example l of the invention and a comparative temperature 26 cooling curve for plum slices frozen by a 27 conventional blast ~reezing apparatus; and 29 FIGURE 5 shows a temperature cooling curve for strawberry halves frozen in accordance with 3l Example 2 of the invention and a comparatiYe 32 temperature cooling curve for matched strawberxy 33 halves ~rozen by a conventional blast freezing 34 apparatus.

.

,. ' .

` 4~ ~

Referring now to the drawings, Figure 1 illustrates a 2 general problem which is solved by means of the 3 invention. Figure 1 is a graph of time agai~st 4 temperature for a bovine embryo being cooled through its freezing point towards its cryopreservation 6 temperature in liquid nitrogen. ~he embryo is kept in 7 bovine embryo culture medi~m plus 10% v/v glycerol as a 8 cryoprotectant, as is conventional, in an 0.25 ml 9 plastic embryo cryopreservation straw. Line A shows the temperature of the cooling environment surrounding 11 the embryo and Line B shows the temperature of the 12 cryporotectant contained in the straw and immediately 13 surrounding the embryo itsel~. Over time, the 14 environment temperature falls steadily. For the cryoprotectant medium, however, (and, it can be 16 assumed, for the embryo itself, as the temperatures of 17 the cryoprotectant and the embryo will ~ot be expected 18 to be significantly different) the temperature starts 19 to fall steadily, towards and below the melting point (Tm) of the medium containing the embryo. The 21 biological material then supercools until the 22 nucleation point (Tn) is reached. At this point, the 23 water in the material begins to crystallise, and the 24 latent heat of fusion of the water in the sample is released. The temperature of the embryo sample thus 26 increases from Tn to Tm. After the latent heat of 27 fusion has been relea~ed, the sample continues to cool, 28 but by this stage the temperature differential between 29 the sample and the surroundings is greater than it previously was. The rate of temperature drop for the 31 sample therefore increases, because of the operation of 32 Newton's law of cooling. The slope of curve B becomes 33 unacceptably steep, which is reflected in damage :

. ~ .
.
. j .
.~ ~

.. . .

~ 43 20~4803 .~ ................................................................. .
.
l occurring to the embryo. In this context, 2 "unacceptablel' means the recorded rate of cooling 3 differs (by being more rapid) from the rate recommended 4 or used in conventional practice to achieve successful 5 cryopreservation; an unnacepta~le rate is that which 6 could be expected to contribute to serious injury in 7 the frozen sample. This general principle would hold 8 whenever the cooling rate recommended in a published 9 procedure differs signi~icantly from the rate recorded during operation of the protocol: hence the requirement ll to control cooling rate.

13 Such difficulties can be a~oided by means of the 14 present invention, part of one embodiment oP which is shown in Figure 2a, which shows a device 1 which is in 16 accordance with the third aspect~of the invention and 17 which is adapted to be placed in a cold environment 18 such as in a Dewar flask or dry shipper containing 19 liquid nitrogen.

21 The device l comprises a vertically arranged, circular-22 sectioned cylindrical brass core 3, which is 140mm long 23 and 27mm in diameter. The core 3 is provided at its 24 bottom end with a spigot 5 for location in a corresponding socket in a bevelled, centrally located 26 ~oss 7 integral with a base plate 9. The base plate 9 27 and boss 7 are constructed from laminated polystyrene.
28 The base plate 9 is in the form of a disc 200mm in 29 diameter and 20mm thick. The boss 7 has a minimum diameter of 27mm, to correspond with the brass core 3, 31 a height of 20mm, and is ~evelled outwardly towards the 32 base plate 9 at 45. In use, the brass core 3 is 33 ~irmly attached to the boss 7 and base plate 9.

' - ' ~

. ~ , ~, .

WO91/0~635 PCT/GB90/01231 ,,,,',~-^ ~ 44 2 0 ~; ~8 ~
1 An insulating block 11, generally in the form of a 2 hollow circular-sectioned cylinder is configured to 3 slide and fit over the brass core 3 and to seat snugly 4 in the boss 7 an~d,~ase plate 9. The insulating block 11 is also constructed from laminated polystyrene and 6 it has a maximum height of 180mm and a diameter of 7 200mm. Its hollow has a diameter of 2.7cm to 8 correspond with the brass core.

A first series of twelve holes 13 are formied in the 11 insulating block 11. They extend vertically 12 downwardly r parallel to the axis of the brass core 3 13 and are symmetrically arranged about the core's axis.
14 Each hole 13 in the first series is 3mm in diameter and extends down from the uppermost surface of the 16 cylindrical blocX 11 to a depth of 140mm. The axis of 17 each oP the holes 13 'lies 35mM from the axis of the 18 brass core 3 or 21.5mm from the periphery of the brass 19 core 3.

21 Second, third and fourth series of twelve holes lie, in 22 register, radially outwardly from the first series;
23 representative holes are indicated by reference 24 numerals 15, 17 and l9, respectively. The axis of the holes of the second series 15 lie 50mm radially 26 outwardly from the axis of the brass core 3, and the 27 corresponding distances for the third and fourth series 28 17 and 19 are 65mm and 80mm; otherwise the holes of the 29 second, third and fourth series 15, 17 and 19 are as for the first series 13.

32 The purpose of each series of holes 13, 15, 17 and 19 33 is to hold plastios straws (not shown) convention~lly ~ .

: ~ ~5 2~6Q8~3 1 used for the cryopreservation of mammalian.embryos and 2 gametes. Such straws are available ~rom IMV, L'Aigle, 3 France, and are internally coated with cholesterol, as 4 taught in EP-A-0246824. Instead of coating straws (or any other container) with cholesterol, crystals of an 6 appropriate nucleator, including cholesterol, can be 7 added to the contents. Appropriate nucleators are 8 available from Cell Systems Limited under the trade g marks CRYOSEED or XYGON.

ll On top of the insulating blocX ll, and covering the top 12 of the brass core 3 and the first to fourth series of 13 holes is an insulating cover plate 21 in the form of a 14 disc o~ 200mm diameter to correspond to the insulating block ll. The cover plate 21 i5 constructed of 16 laminated polystyrene and is 20mm thick.

18 In use, the brass core 3 and base plate 9 are first l9 placed in a cold environment, for example in a dry shipper. ~A dry shipper is a well insulated container 21 resembling a large Dewar flask lined with absorbent 22 material containing liquid nitrogen; because the 23 nitrogen is absorbed, there is little or no free liquid 24 in the shipper.) The brass core 3 is allowed to equilibrate with the cold environment, whereafter the 26 insulating block ll, containing twelve straws in the 27 first series of holes 13, each containing a bovine 28 embryo, is positioned round the brass core 3 to seat on 29 the base plate 9. The cover plate 21 is then placed on the insulating block l9, and the device l is left to 3l cool.

... ~.. ~... ... ...... . . . ... . .. . .
- , : , .
. -: . : . . : . , ~
. . :: . : . . , . :

:. . - . . : . . , :. :

.: : ~ . .. . . ,:

, . . .

~ WO91/01635 PCT/GB90/01231 3 ~ 3 ~6 ~

.. ~ . . :~ , l Initially, the straws are cool~d both by the influence 2 of the brass core. 3 and by the cold environment. This 3 combined action provides a relatively high rate of heat 4 extraction from ths embryos. The cooling curves of five samples of cooling medium for bovine embryos in 6 the first series of holes 13 are shown in Figure 3.
7 (The embryos are in cryopreservation straws containing 8 bovine embryo culture medium plus lO % v/v glycerol as 9 a cryoprotectant.) The first heat extraction rate is applied whilP the water is supercooling, shown at 11 region C of the curve. The temperature of the sample 12 drops below the melting point (Tm) and supercooled 13 slightly to the nucleating point (Tn). The nucleating 14 temperature is not far below the melting point, because of the presence of the cholesterol ice nucleator within 16 tha straws. However, when the temperature reaches the 17 nucleating point (Tn) the sample temperature rises as 18 shown at D to the melting point (Tm). By the time the 19 temperature of the embryos begins significantly to drop again, the brass core 3 has substantially equilibrated 21 with the embryos and the intervening material of the 22 insulating block ll~ Therefore, the continued heat 23 extraction is solely towards the periphery of the 24 insulating block ll, and so the rate of heat extraction from the samples is lower. The slope of the graph at E
26 is therefore acceptably smooth and no too steep and no 27 damage results to the embryos, which can then safely be 28 allowed to cool to the temperature of the cold 29 environment (-80 C). In the temperature range -25 to -30-C, the average rate of cooling was found to be 31 0.32C~min with this configuration.

' - .

WO91/01635 . PCr/GB90/0l231 ,~ ~J 47 2~

1 Figure 2b shows a further embodiment of a passive 2 ~reezer, broadly similar to that shown in Figure 2a, 3 but including a handle as.sembly 101 and locating lugs 4 103 on an insulating block 105 adapted to extend through a cover plate 107 and to engage apertures in a 6 locating disc 109 of the handle assembly 101. A
7 locating lug 111 on the cover plate 107 locates in a 8 spigot 113 of the handle assembly 101. The insulating 9 block 105 is made of acetal and has sample placement holes 106 adapted to receive 2.5ml ampoules for 11 cryopreservation of, for example, mammalian cell lines.
12 The insulating block 105 is seated on a bevelled boss 13 115 on a base 117 and surrounds a brass core 119. All 14 components other than the brass core are made of acetal. Salient dimensions of the device of Figure 2b 16 are as follows:

:

.. . ,, . : : :.
~i .: - ~: . : . . .~. , , . i . , 2 0 6 4 a ~ 48 PCl/GB90/0l23l ~: 1 ACETAL CONSTRUCTION
cryo-ampoules [c2.5ml]

. 5 DIMENSION [mm]
; 6 Component Diameter:Depth/height 7 - - ~
:~ 8 1 Lid 107 200 : 40 2 Block 105 200 :140 9 3 Locating lugs 103 [2] 15 : 52 4 Brass rod 119 57 :140 . 5 Base 117 200 : 20 -- _ . _ , .- 12 Machined holes ; 6 Sample placement holes 106 13 : 50 15 7 Countersink for boss 115 ` 5 -i. 16 8 Centre of sample placement , hole 106 to perimeter of 17 block 105 44 lR 9 Centre of locating lug 103 ;. to perimeter of block 105 22.5 .- 19 10 Hole for brass rod 119 . 57 : 140 . 20 ~
-~ 21 Note 1: the height of the locating lugs 103 does not - 22 include threaded portion inserted into block -23 dimensions not critical : 24 Note 2: the height of the brass rod 119 does not ~ 26 include locating lug on base - dimensions not critical .~ 27 : 28 Note 3: the base 117 has three cmall acetal feet 29 mounted, equally spaced, at the periphery. Feet 5mm high x 5mm diam. Size of boss to locate brass rod and 31 block not critical.
:~ 32 . 33 ,' ' : ' .:

., : .
., ~.

WO91/0163~ PCT/CB90/01231 ~ 49 20~8~ `
.' l This construction, when used in conjunction with a ; 2 liquid nitrogen-containing dry shipper, allows a 3 cooling rate of -1C/min.

A different embodiment, essentially similar in 6 construction to that shown in Figure 2b but for use in .
7 connection with cryopreservation straws (eg for bovine 8 embryos), has the acetal component parts replaced with 9 P~FE parts. The salient dimensions are as follows:

13 plastic straws 14 [0.25/0.5ml]
- ___ 16 DI~ENSION [mm]
l7 Component Diameter:Depth/height 18 l Lid 107 200 : 20 19 2 Block 105 200 : 160 20 3 Locating lugs 103 ~2] 35 : l0 4 Brass rod ll9 22 : 160 21 5 Ba~e 117 200 : 20 22 -~
23 Machined holes 24 - ~ - .
25 6 Sample placement holes l0S 3 : 133 26 7 Countersink for boss 115 5 8 Centre of sample placPment 27 hole 106 to perimeter of 28 block 105 63 9 Centre of locating lug 103 29 to perimeter of block 105 30 l0 Hole for brass rod ll922 : 160 ,.
:
: , . . .

.,' ... . .

WO91/0163~ PCT/GB90/01231 ' 2~ ~8'~3 '' ' ' `' ~ ~

1 Note 1: the height of the locating lugs 103 does not 2 include threaded por~ion inserted into block -3 dimensions not critical Note 2: ~he height of the brass rod 119 does not 6 include locating lug on base - dimensions not critical 8 Note 3: the base 117 has three small acetal feet 9 mounted, equally spaced, at the periphery. Feet 5mm high x 5mm diam. Size of boss to locate brass rod and 11 block not critical.

13 This construction, when used in conjunction with a 14 liquid nitrogen-containing dry shipper, again allows a cooling rate of -0.3C/min.

17 Figure 2c shows a still further embodiment of a passive 18 freezer. ~he construction is a modification of that 19 shown in Figure 2b, and like components have been given the same reference numerals. The principal difference 21 is that in the Figure 2c construction the insulating 22 bloc~ 105 has been replaced with two half height blocks 23 105a and 105b; this allows for more of ampoules to be 24 present (up to 15). Salient dimensions of the device of Figure 2c are as follows:

, . .

; ,, ., .
., W09t/01635 PCr/GB90/0123l ` ~ 51 206~
.; . . ..
.~ 1 2 ACETAL CONS~RUCTION
3 cryo-ampoules [c2.5ml]
4 ----~
DIMENSION [mm]
6 Component Diameter:Depth/height 7 ------- _ 8 1 Lid 107 200 : 40 2 Block 105a 200 : 70 - 9 3 Block 105b 200 : 70 4 Locating rods 103 [2] 15 : 123 5 Brass rod 119 57 : 120 ; 11 6 Base 117 200 : 20 12 Note 1: the height of the locating lugs 103 does not 13 include threaded portion inserted into block -14 dimensions not critical ~-; 16 Note 2: the height of the brass rod ll9 does not 17 include locating lug on base - dimensions not critical 19 Note 3: the base 117 has three small acetal feet mounted, equally spared, at the periphery. Feet 5mm 21 high x 5mm diam. Size of boss to locate brass rod and 22 block not critical.

26 Machined holes 28 7 Sample placement holes 106 13 : 50 8 Countersink for boss 115 5 29 9 Centre of sample hole 106 to perimeter of block 105 44 10 Centre of locating lug 103 31 to perimeter of block 105 22.5 32 ll Hole for brass rod 119 57 : 120 .

:

, ..~

. ~ .

~ , - : . .
. i . :~ ~ . - . - . : .

.:

~,~6~3`' 52 ~
"~ 1 2 It must be noted that the configuration described here i 3 in detail aie only a few of a great number of possible 4 configurations, depending upon the cooling rate : 5 required and the type of sample holder (for example 6 straw or ampoule) to be cooled.
:~ 7 . 8 The variables can be:
. g ~ lO i the diameter of the insulator (although in ~ ll practice it may be convenient to use a 12 standard diameter for a range vf products for . 13 manufacturing and marketing reasons);

ii the depth of the insulating block;

. 17 iii the diameter of th~ metal core;

l9 iv the number, size and placement of the holes for the samples; and 22 v the materials of the insulating block and 23 metal core.
; 24 `~ 25 The invention will now be illustrated by the following 26 examples, which relate to active, as well as passive, 27 systems. Unless otherwise stated, all examples of ~ 28 active systems in accordance with the invention (ie ; 29 those active examples other than comparative examples) ; 30 were carried out in a PLANAR KRYO lO/16 controlled rate ~ 31 freezing machine. (The expression PLANAR KRY0 lO/16 is `
32 a trade mark). Temperature was measured with T type 33 thermocouples connected to a SQUIRREL data logger (1200 ~' , ' '' '., ` . '`
- -.

W091/0163~ PCT/GB90/01231 ; 53 20 6~8 03 1 series). (The word SQUIRREL is a trade mark.) Data were -~ 2 transferred to an IBM-compatible computer for storage 3 and analysis. In order to compare different treatments, ~; 4 the time the sample is at the latent heat plateau, defined here as the exotherm time (ET), is used; this 6 is further defined by the final temperature eg ET 5 or 7 ET-10 being the time from the exother~ to -5C or -lOC
8 respectively. Application of acoustics was either from 9 a Branson model 250 sonicator operating at 20kHz, a Branson ~odel 2200 ultrasonic cleaner, a Lucas-Dawe - ll series 6266 immersible transducer, a Telesonics tube 12 resonator type TR connected to a ultrasonic generator 13 type USR-20 (20kHz) or a HILSONICS acoustic driver, 14 model IMG 400 (Hilsonic Ltd, Merseyside, England).
l6 Example_l 18 This example shows that plums freeze better when using 19 an efficient latent heat removal protocol of the invention, even in the absence of acoustics, as 21 compared to conventional methods. Korean dark skinned 22 plums (Tesco foodstores) were sliced into 4.5mm slices - 23 and were frozen by a method in accordance with the 24 present invention. For comparison purposes, plum slices were also frozen by conventional methods. The methods 26 used are as follows.

28 l. Slices were frozen by a method in accordance :l 29 with the inventionO The initial environment temperature was -75'C, which was held for 2 3l minutes. The environment temperature was then 32 warmed to -30-C at lO-C/min. The temperature 33 reduction in the plum slice was significantly ~, .
. . ..

, WO91/01635 pcT/Gs9o/ol23l ~ ~?,~3 '` ;`'1``,''-:` ' 5"

1 faster than in the blast freezer treatment (2, 2 below), with a measured exotherm (ET-10) of 80 3 seconds (Figure 4).

2. (This is a comparison method.) Slices were 6 placed in a commercial blast freezer operating at 7 -40C; the measured exotherm (ET 10) was 554 8 seconds tFigure 4~. They were then transferred to 9 a commercial deep freeze operating at -20-C.

11 3. (This is a comparison method.) Material was ; 12 immersed directly into liquid nitrogen and 13 transferred to a commercial deep freeze. The 14 sample cooled quickly through its exotherm;
however the final temperatur~ attained was below 16 -100C.

18 Sensory evaluation of frozen/thawed material was made 19 against fresh plum slicPs. Frozen plums were removed from the freezer 45 minutes before evaluation and laid 21 on a plate with cling film to cover them. The plums 22 were placed on paper plates before panellists singly, 23 on demand, according to a statistically randomised 24 design. The panellists were instructed to assess the flesh only and to discard the skin of the fruit.
26 Malvern water was used as a mouth wash betwe~n samples.
27 24 replicate tastings of each sample were carried out.
28 The assessment took place under purple lighting to 29 disguise any colour differences.

31 Results 33 Adjusted mean scores for the whole trial are shown Wog1/0l635 pcr/GB9o/o123]
2 ~ 6 1 below; the scores are on a scale of 1-10.
2 Texture: 1 2 3 4 4 Firmness 5.46 3.46 6.08 7.83 Wetness 6.46 7.75 5.67 2.92 ` 6 Crispness 5.42 4.00 6.33 6.79 7 Fibrous/Chewiness 6.25 5.29 6.71 7.42 8 Particulateness 5.25 4.71 5.75 6.88 9 Juiciness 6.92 7.46 6.08 3.79 11 Flavour:

13 Overall strength 6.33 6.88 6.04 3.75 14 Sweetness 4.79 4.88 4.38 3.63 Sharp/Acidic 4.79 4.71 5.00 2.96 16 Bitterness 2.83 2.96 2.88 2.25 18 Key: 1 = Present invention; 2 = Blast frozen; 3 =
19 Liquid nitrogen; 4 = Fresh 21 Discussio 23 Present invention vs. Fresh.
i 24 The fresh sample is significantly firmer, drier, more 26 fibrous/chewy than the sample frozen by the invention.
27 In flavour terms the fresh sample is lower in flavour 28 overall, less sweet and less sharp/acidic than the 29 plums frozen by the invention.
t, 30 Present invention vs. blast freezing.

32 The plums frozen by the present invention are 33 significantly firmer and more fibrous/chewy than the ., ., ..,, . .. , ,,.,. ~ ~. .. ., - , ~.
- ;,: . . : :
~:. , .. ,., , : . :

.
. .
?
. . . ..
. . ~ , .

WO91/01635 PCr/GB90/0123 1 blast frozen plums. The remaining parameters show no 2 significant differences.
3 Present invention vs. liquid nitrogen freezing.

There were no significant differences for any 6 parameters.

8 Example 2a This example shows that strawberries freeze better when 11 using an efficient latent heat removal protocol of the 12 invention, even in the absence of acoustics, as 13 compared to conventional methods. Spanish class 1 14 strawberries (Sainsburys Foodstores) were halved and frozen by the following methods:
16 ` ~ -17 1) Simulation of blast freezing in a Planar 18 controlled rate freezer, with a rate of cooling of 19 the gas temperature of 1C/min. The measured I 20 exotherm was 6~0 seconds (Figura 5). ,~

22 2) Frozen by a method in accordance with the 23 invention. The initial environment temperature was 24 -50 C for 7 minute with rewarming at lO~C/minute to -30-C. The measure exotherm in the matched 26 strawberry half to treatment 1 was 280 seconds 27 (Figure 5).
2~
29 3) Strawberries were frozen by immersion into liquid nitrogen.

33 , WO91/0163$ PCT/GB90/01231 57 20~ ~803 l Results.
3 Following ~reezing in li~uicl nitrogen many strawberries 4 fractured. Strawberries blast frozen and immersed in liquid nitrogen displayed significant leakage of 6 cellular contents. For those frozen by the present 7 invention leakage was less pronounced and the 8 straw~erries were significantly firmer. The exudate g was less pigmented than following blast freezing or liquid nitrogen freezing, clearly demonstrating that ll less intracellular damage occurred following the l~ current method.

14 Sensory evaluation of the frozen/thawed material was made against fresh strawberries. Frozen strawberries 16 were removed form the freezer 45 minutes before 17 evaluation. 25 independent replicate tastings of each 18 sample were carried out.

Texture:
2l Treatment 23 Rating l 2 3 ~4 Excellent - -26 Very Good 0 3 27 Good 2 6 2 28 Fairly Good 3 7 lO
29 Moderate 12 6 9 Poox 7 3 2 3l Very Poor 1 0 33 Key: 1 = Blast Freezing; 2 = Invention; 3 = liquid N2 ., .

. . .

- -- - . . . . . .. .

, wo 91/01635 03 P~r/GB9o/ol23l ?~&~ sa 1 Flavoyr:
.. 2 Treatment 4 Rating 1 2 3 . 5 6 Excellent - - -7 Very Good - 1 3 . 8 Good 3 7 5 . g Fairly Good 4 5 3 Moderate 8 8 4 . 11 Poor 5 2 8 lZ Very Poor 5 3 2 ., 13 14 Xey: 1 = Blast Freezing; 2 = Invention; 3 = liquid N2 ,~ 15 16 There appeared to be little ef~ect of storage time, 17 within the range of from 1 to 30 days, on the quality 18 of the material frozen by the method in accordance with 19 the present invention.
21 Both the type of strawberry and the degree of ripeness 22 also determined the quality on thawing; the 23 obserYations here are not intended to be exclusive but 24 rather to be a ~uide to the trends observed. The best results were obtained with slightly under-ripe class 1 2 6 Spanish strawberries. Poorer results were obtained with : 27 riper class 1 strawberries of the same type. Good 28 results were achiev d with slightly under-ripe class 2 29 Carmel strawberries (from Israel). With ripe class 1 30 Carmel strawberries and class 1 Kenyan strawberries 31 (Sainsburys Foodstores) poorer results were obtained~
~i 32 It must be emphasised that with such riper starting : 31 =aterial the results ~ollowing the method in accordance -,i ", , ; , ,, ",~ "; "" ~ " ,, WO91/01635 ~ PCT/~B90/01231 59 2~6 ~8 0 ~

1 with the present invention outlined above was always 2 superior to blast freezing or liquid nitrogen freezing 3 of the same material.
~i3~E~
7 This example shows that even better results are 8 obtained when strawberries are frozen using an 9 efficient latent heat removal protocol, with the application of acoustics. Strawberries (Californian 11 guadalupe) were obtained in bulk from a retail outlet 12 and sorted to discard all over- or under-ripe material.
13 The selected strawberries were washed and then halved.
14 The separated halves of each fruit were collected together to provide two populations of 280, essentially 16 matched strawberry halves.

18 The strawberries were frozen in batches of 70 halves.

A 12"x12" (30.5cm x 30.5cm) acoustic plate (22.5 k~z, 21 220V, Hilsonic ~td, Birkenhead, UX) was precooled to 22 -70-C in a CryoMed 2700 freezer and the strawberry 23 halves loaded on to it, which resulted in a temperature 24 rise to -50C. The material was cooled according to the following protocol: (1) providing an initial 26 environment temperature at ~58C for one minute; (2) 27 warming at 10C/minute to -48C.

29 Sample temperature was monitored using type T
thermocouples embedded in the mid~point of 31 representative strawberry halves, connected to a 32 microprocessor data-logger (Grant Instruments, 33 Ca~bridge, UR). When the samples reached -20'C they .

-: , - , . . .. .
::.. ; ' .

WO91/01635 . PCT/CBg0/01231 ~6~3 i ~ 60 ~

- 1 were transferred ~o storage at -30C for 5 days.
2 Samples were thawed by exposu;re to room temperature for 3 90 minutes before sensory evaluation.
When an acoustic treatment was applied a pulse of 2 sec . ~ i .
6 every 30 sec was used throughout the entire cooling 7 cycle.
,~ 8 9 Subsequently thawed strawberries were subjected to a ~ 10 sensory evaluation panel, with the following results:
:'' 11 13 Characteristic - acoustics + acoustics sig. dif. in mean 14 scores due to acoustic treatment . _ . . . ~ .

17 Berry colour 5.~ 6.2 nsd 1=dull red 18 9=bright red 19 Free liquid on plate 4.3 3.4 0.01 20 1=small amount 21 9=large amount 22 Firmness 3.2 4.5 0.01 l=so~t 23 9=firm Mushiness 6.2 4.9 0.01 25 l=not mushy 26 9=very mushy 27 Overall appearance 5.4 6.4 0.05 28 l=dislike extremely 29 9=like extremely 30 Overall Texture 4.2 5.5 0.01 31 l=disliXe extremely 9=extremely 3 Overall flavour 5.0 6.0 nsd 3 1=dislike extremely 34 9=like extremely 35 Overall opinion 4.6 5.8 0.l0 36 1=dislike extremely 37 9=like extremely ,, .

~ wosl/ol63s - PCT/GB90/0123l ,." ~ 20l~48~''3 ''-`

l Example 3a 3 This example shows that a blanched vegetable, celery, 4 freezes better when using an efficient latent heat removal protocol of the invention, as compared to 6 conventional methods, and that even better results are 7 obtained in the additional presence of acoustics.
9 Celery was obtained from a retail outlet. Celery samples were cut into 0.6cm (~ inch) pieces, and 250g 11 were blanched per run at 90~C (l90~F) for 2 minutes.
12 There was a loss of 10% material on blanching. The 13 samples were rinsed with cold water to bring them to 14 room temperature (20 C). The celery samples were then frozen in accordance with the invention using the 16 following protocol:

18 (1) The initial environment temperature was 19 maintained at -75~C for 2 minutes;
21 (2) The environment temperature was then warmed 2Z to -30-C at 10C per minute. This protocol was 23 followed with and without the application of acoustics.
24 When acoustics was applied, an ultrasound frequency of 22.5kHæ was used, and the power lev~l was 220 watts, 26 applied over an area of 929cm2 (144 square inches), 27 resulting in a power level of 0.24W/cm2. The 28 ultrasound was not applied continuously, but rather was 29 applied for 3 seconds every 30 seconds.
31 As a control, the blanched celery was also blast frozen 32 at an environment temperature of -40 C. The samples 33 were reroved when they reached -30'C. After treat=ent, ., , 6 ~ ~ "` ` 62 ~J

1 som~ of the frozen celery samples were stored at -30OC
2 and some were subjected to a standard temperature abuse 3 protocol.
~ 4 '~ 5 The resulting samples were evaluated in a balanced, -~ 6 sequential order by a tasting panel consisting of 42 7 panelists, who had been pre--screened to have a positive 8 attitude towards evaluating frozen celery slices that 9 had been thawed. A serving consisted of 6 slices of celery that had undergone a given treatment. The 11 celery had heen thawed at ambient temperature for 60 12 minutes prior to serving; this was sufficient to 13 eliminate any ice crystals, yet still to be slightly 14 chilled. The panelists were instructed to evaluate all slices having undergone a given treatment before rating 16 the attributes, so that the rating would reflect the 17 majority of slices.

19 The results showed that the efficient latent heat removing protocol in accordance with the ivention Zl resulted in better firmness, less mushiness and a 22 better overall impression of freshness of flavour than 23 the control, blast-frozen samples. Further, when 24 acoustics was also applied, it was not only found that the samples offered textural advantages over the 26 control samples, but it was also found that they held 27 up better under temperature abuse than the control 28 samples. An additional advantage of the invention - 29 displayed was the reduction in the time taken for the sample temperature to be reduced from ambient to the 31 storage temperature (-30C). Using prior art blast 32 freezing techniques, the time taken to reach -30C is 33 in the order of ~0 minutes. Using an effioien~ latent .

- . : . . , i . - . .
- . . , . - . - ,. . ~: . ~ .
. , ... . - . . .. . .

WO9l/01635 PCT/GB90/01231 63 2 0~ ~80 3 ~
", ~
1 heat removal protocol in accordance with the invention, 2 this time is reduced to about 8.2 minutes. A further 3 improvement to about 5.2 minutes, is seen with the 4 additional application of acoustics.

6 Example 3b 8 Celery sticks were purchased from a local supermarket 9 (Tesco foodstores), washed and cut into lcm sections.
They were blanched for 3 minutes at 80C, then ~lushed 11 with cold water. Samples were frozen according to 12 three methods:

14 (1) Simulated blast freezing ~Planar Kryo 10 set at -40~C);

17 (2) According to the invention, using an initial 18 environment temperature of -50 C, with a hold time of 19 8 minutas, and then warming to -20C at a rate of 10C/min.

22 (3) As in (2) with the addition of acoustics 23 supplied from a 20cm x 20cm plate equilibrated at -50C
24 (25kHz, 260W power, 2 seconds per 30 seconds pulse time).

27 On thawing, texturs of the three samples was assessed 28 according to a subjective assay, the results of which 29 were as follows:

31 Scored 0-5 (0=poor, 5=excellent) `' :

.

~: ., . . : . ... ,,,, ... ,.. ,, . .... -,, , ~ , ... .

: . :. . .: . ., ~ ;:, ~. : ,; : ; :, . : : ,::; , . . :,: , :

2~4~Q3 64 ~
. . . . .
1 The average taste panel scores for each treatment were:
; 3 Treatment (1) - 2.5 4 Treatment (2) - 3.0 Treatment (3) - 4.0 7 Ex~ample 4a 9 Small new potatoes of less than 4 cm in diameter (Sainsbury's Foodstores) were frozen by a number of 11 treatments, as described below, and evaluated on 12 thawing. Potatoes were neither cooked nor blanched 13 before freezing.

1) The potatoes were 'blast frozen' as for 16 strawberries in Example 2a above; on thawing the 17 potatoes were ~ery soft, leaked cell water and 18 were considered unacceptable after cooking.

2) The potatoes were frozen by liquid nitrogen 21 immersion; they invariably fractured during 22 freezing.
! 23 24 3) The potatoes were frozen by a method in accordance with the present invention by (13 26 providing an initial environment temperature of 27 -80 C for 1 minute, (2) warming at 10-C/minute to 28 -20-C. On thawing, the potatoes were intact and 29 retained their original texture with no leakage.
On boiling, the potatoes were acceptab~e.

.

, . ,.. . . .- . . - . . , .... - . . . ~ ~ . , , - .... - . . . . .... .. -WO91/0l635 PCT/GB90/01231 ` 65 2~ ~/18Q3 1 Example 4b 3 Small new po~atoes (3-5cm length, var. M.Bard, Tesco ~ foodstores), were cooXed in boiling water for 15 minutes, then flushed with c:old water until cool. 200g 6 batches were frozen to -30 C by the following methods;

8 (1) Simulated blast freezing (-40 C) in a Planar 9 Kryo 10 fre~zer.
11 (2) According to the invention, using a Planar 12 Kryo 10 freezer. The initial temperature was -50~C, 13 which was held for 6.5 minutes; the temperature was 14 then allowed to rise a a rate of lO C per minute until -20-C was reached.

17 (3) As (2) with the addition of ultrasound, 18 supplied over 20cm x 2cm at 360W, 25kHz, and various 19 pulsing lengths, as described below.
21 The lengths of latent heat plateaus in the various 22 treatment were measured. Following thawing, batches 23 were assessed by a taste panel, and ~uantitative drip 24 loss by halving tubers, wrapping in gauze in a funnel, and placing a 31b (1.36kg) weight on the sample for 20 26 minutes. Smears o~ sample material were mounted on a 27 microscope slide, and observed using light microscopy.

29 The results are given b~low.
31 (l) Lengths of latent heat plateaus (LHP's) in various 32 cooling treatments,- were as follows:

-- :

I .

., . . . ;. . ,~.,. , . .. : .. ~ .

WO91/0163s PCT/C~90/01231 3~,; 66 ~

1 LHP length (minutes) 2 Treatment 1 8 3 Treatment 2 6.5 4 Treatment 3,.: 2s in 15s 7.0 .:2s in lOs 5.0 ::
6 2s in 5s 4.0 8 According to sensory evaluation, the treatments were 9 ranXed for texture in the following order;

11 Treatment 3 2s in Ss > Treatment 3 2s in lOs >
12 Treatment 3 2s in 15s > Treatment 2 > Treatment 1.

14 (2) Fluid extrusion.
16 TreatmentFluid Extruded 18 1 ll 3 2s in 40s 7 22 (3) Microscopy 24 Cells from Treatments 1 and 3 were compared. Blast frozen cells showed a loss of organized cell structure 26 and contents, with extensive folding of the cell 27 membrane. By contrast, cells frozen by Tr~atment 3 28 (acoustics), showed good retention of cellular 29 integrity,`and less folding of the cell membrane.

.~ ,.
:
. . .

., .. .. ~.,, . .. , , ".. . . .

, . ~ . . . . : . . . . . . . ` .

;, . ! , ~ , . , . I, ' ., , ' . . ,;' .

~ 67 20B'`~8`~3;,`;
. . .
1 Example 5 3 Two types of asparagus obtained from Sainsbury's 4 Foodstores, which were Peruvian and Thai in origin respectively, were frozen by a number of methods as 6 described below and evaluated following steaming of the 7 thawed product.

9 1) Both types of asparagus were blast frozen as described in Example 2a. The subsequently thawed 11 product had poor taste and texture and scored 12 4/20.

14 2) Both types of asparagus were frozen in liquid nitrogen. The spears fractured and, on thawing, 16 had very poor taste and texture; they scored 2/20.

18 3) ~oth types of asparagus were frozen by a method 19 accordance with the present invention by ~1) providing an initial environment temperature of 21 -80 C for 1 minute and ~2) rewarming to -20C at 22 15~C/minute. On thawing, the taste of the spears 23 was improved, as was their texture on cooking;
24 they scored 10/20.

26 Example 5b 28 Raw asparagus spears (produce of Thailand, purchased at 29 Sansibury's foodstore) werie trimmed to 6 inch (15cm) lengths, and frozen by:

32 (1) Simulation of blast freezing in a Planar 33 controllecl-rate freezer, set at -40 C.

, 1/01635 PCT~GB90/0123l ~ `; 68 1 (2) Frozen in a KRYO lO series chamber Model 2 10-16 controlled rate freezer by Planar Biomed, Sunbury 3 on Thames, England, in accordance with the invention 4 optimised by - computer modelling. The initial environment`temperature was -50C, which was held 12 6 minutes, and the temperature was then increased at a 7 rate of 10C per minute until -20 C was reached.

9 (3) ~rozen as in (2) with addition o~ acoustics (22.5kHz, 360W power, 2 seconds per 20 seconds).
ll Acousrtics was supplied by a HILSONIC acoustic driver 12 model IMG 400 (Hilsonic Ltd, Merseyside, England) 13 coupled through an ISOPAR M liquid filled chamber to an 14 8" x 8" (20cm x 20cm) plate forming the floor of the freezer chamber. Following freezing, the samples were 16 thawed to ambient temperature over 6 hours. The spears 17 werethen cooked for 4 minutes in boiling water, and the 18 three frozen treatments compared with an unfrozen l9 sample using a taste panPl.

21 The panel recorded average scores (O - 5, O=poor, 22 5=excellent):

24 Unfrozen - 5 Method (l~ - l.5 26 Method (2~ - 2.5 27 Method (3) - 3 2~
29 Examplio 6a 31 Single cream is an example of a oil in water emulsion.

32 Single pasteurised cream was obtained from Sainsbury's 33 Foodstores. Following freezin~ and thawing of this .. ..

.~' ' -, ~, . . : , . . : . , .. . : . . " ,. "
i. ; ., , . ,, , :: ,. : ~ ,. ~ . ~ ;

WO91/0l635 PCT/CB90/01231 ~3 69 2a ~ ~g 03' .: 1 product, separation of the cream solids ~rom the 2 liquids occurs. Freezing damage may be assessed by the .. 3 loss of liquid through a small mesh filter. 10 ml 4 aliquots were placed in glass universals and frozen by a variety o~ methods, as described below:
: 6 7 1) Blast freezing, as described in Example 2: on 8 thawing the cream is discoloured yellow, curdled.
9 The liquid loss is 34~;
`, 1 0 . 11 2) Liquid nitrogen immersion; as described in 12 Example 2a; on thawing the cream does not 13 visually separate but becomes very viscous. The ; 14 liquid loss is 12%; and '' 15 16 3) Freezing by a method of the present invention, 17 with an initial environment temperature of -80C
18 for 1 minute, followed by warming at 15C/minute 19 to -20C. On thawing the cream does not visually separate; there is an increase in viscosity but . 21 not as pronounced as with liquid nitrogen 22 freezing. The liquid loss is 10%.

24 4) Freezing as for method (3) except that ultrasound was applied for 0.1 seconds for every .;` 26 1C cooling of the cream from O to -20C. This .` 27 combination of acoustic nucleation and efficient 28 removal of latent heat consistently, in five 29 independent trials, further reduced the drip loss ` 30 by 10-16% o~ that observed in method (3).
31 :~:
: 32 It can be seen, therefore, that the present invention 33 gives results which are appreciably better than blast :.

., . .

. ` ' ` . .'' ;' '. '" ' '~ ,.'.. .`, ' `' ' ,'''. '' , '' '':',.' ' '; '' ' W091/01635 PCT/GB90~01231 2a,~ ,o3, ., : 1 freezing and which are also better than the more 2 expensive and relatively inconvenient process of 3 freezing by liq~id nitrogen i~mersion.
4 ..
Example 6b 7 Single cream (Tesco foodstores) was divided into lOOml : 8 batches, either in freezer bags supported by metal g frames or in metal moulds.

11 The cream was frozen according to the following 12 methods:

14 (1) Simulated blast freezing (-40C) using a Planar Kryo 10.

17 (2) According to the invention, involving rapid 18 freezing by immersing samples in a Planar Kryo 10 19 controlled rate free2er initially at -80~C (hold 10 minutes), then warmed to -20C at lO~C per minute, with 21 the addition of acoustics throughout the cycle (300W
22 over 20cm x 20cm, 22kHz, 2 seconds every 60 seconds 23 pulsing).

(3) According to the invention, using a Planar 26 Xryo 10 freezer at -507C, holding 15 minutes, with the 27 addition of acoustics throughout the cycle as in (2).

29 Sensory analysis of the three tratments post-thaw, indicated as follows:

32 (1) Separation of the cream had occurred, 33 resulting in liquid 10s5, very grainy, and buttery : 34 tasting.

.~;~ , . . .
. , . ~:" : , ::, ,, : .

,:. : - ,i :
,:

W091/0163~ PCT/GB90/01231 ~ 71 2 0 6 4 8 ~ 3 1 (2) Very good texture, no fluid loss.
.~ 2 3 (3) No fluid loss, but texture not as good as in 4 (2).
. 5 6 Example 7 : 7 8 Mayonnaise is an example of a water in oil emulsion.
9 Commercial mayonnaise, such as Hellman's, appears to be stable following a wide range of freezing methods. This 11 probably reflects the degree of physico-chemical 12 stabilisation of the product. Home-prepared mayonnaise 13 and non-stabilised commercial mayonnaise such as Kite 14 wholefood mayonnaise separate following freezing and th~wing. Such mayonnaises were frozen in 10 ml aliquots . 16 in glass universals by the following methods:
; 17 18 1) Blast freezing, as in Example 2a; total . l9 separation of the oil occurred on thawing;

21 2) Liquid nitrogen immersion, as in Example 2a;
- 22 total separation of oil occurred on thawing; and ~ 23 :~ 24 3) Freezing by a method in accordance with the present invention, in which the mayonnaise was 26 cooled at 20 C/minute from O-C to -50-C, held at ; 27 -50-C for 2 minutes, warmed at 15-C/minute to 28 -20 C. On thawing, there was good retention of 29 texture with littl~ or no separation of constituents.

: ,' .~

~ W O 91J01635 PC~r/GB90/01231 ~ 6~3 i 72 l Example 8 3 Prepared prawn and mayonnaise sandwiches were obtained 4 from Tesco and Sainsbury's Foodstores and singly frozen by a variety of methods, as follows:

7 1) Blast freezing as described in Example 2a; on 8 thawing there was a total separation of the 9 mayonnaise: the oil component seeped through the lower slice of bread and the product was totally ll unacceptable;

13 2) Lîquid nitrogen immersion as described in 14 Example 2a; fracturing of the sandwich occurred and on thawing there was total separation of 16 mayonnaise as in ~l) above;
~7 18 3) Freezing by a method in accordance with the 19 present invention, in which each sandwich is cooled at 20C/minute to -50C, held isothermally 21 at that temperature for 30 minutes and then warmed 22 at 10UC/minute to -20'C. on thawing the product 23 was acceptable. There was little or no separation 24 of the mayonnaise, good retention of prawn quality and no fracturing of the bread.

27 Exam~le 9 29 Fill~ts of fresh Scottish smoked salmon (Sainsbury's foodstore) were frozen according to two methods:

32 (l) Simulation of blast freezing in a Planar Kryo 33 10 controlled-rate ~reezer st at -~O'C.

.

, . .

" , ' . . ' . , ~ ', , ~ , ' . ' ' , ' ' '; , ~ " '' ' '' . . :
'' ~ ' ~ . . .
. .

WO91/01635 ~CT/GB90/01231 :: 2 ~ 3 3 (2) In accordance with the ivnention, using 4 thermal modelling and ultrasonics application. The initial environment temperature was -50 C, which was 6 held for 4 minutes, and the temperature was increased 7 at a rate of lO-C per minute until -20-C was reached.
8 Ultrasonic acoustics was supplied at 360W over 20cm x 9 20cm, 22.5kHz and 2 seconds per 40 seconds pulsing.

ll Following thawing, samples were tested by a panel for 12 texture and taste. The panel recorded average scores 13 of:

Unfrozen : 5 l6 Method (l~ : l 17 Method (2) : 3 19 (0-5, O=poor, 5sexcellent).
21 Example lO

23 25ml ice pops (similar to sorbets) were obtained from a 24 local supermarket (Tesco Foodstores), and frozen according to two methods:

27 (l) By processing according to the invention by 28 holding first at -50'C for 5 minutes and then 29 increasing the temeprature at lO-C/~in until -20C was rached in the sample, as detected by a thermocouple .

32 (2) As (l), with the addition of ultrasound 33 delivered from a 20cm x 20cm plate equilibrated at .~
.

~ ~ , `' ' " . `' . ' " ., `., ' ''`. ' '' ''' . ~ ': '' ; ~ ? ~ 3 50C, powered by a 260W, 22.5kHz generator, 2 seconds 2 per 40 seconds pulsing. There results were as follows:
3 Cooling profiles in the two treatments varied, with 4 acoustic treatment considerably reducing latent heat plateaus, and freezing time to -20 C. An assessment of 6 crystal size by~eye indicated smaller ice crystals were 7 present in the sample frozen with acoustics compared to 8 the sample frozen without. In addition, the ice pops g frozen with acoustics were harder to the bite and crispier in texture than those without acoustics.

12 Example 11 14 Cream cheese (Rraft General Foods) was sliced into ~ inch (1.3cm) cubes, and samples frozen according to 16 the following methods:

18 (1) Simulated blast feezing in a Planar Kryo 10 19 controlled rate freezing apparatus held at -40'C;

21 (2) According to the invention, again using a 22 Planar Kryo 10 apparatus but using a hold time at -50 C
23 for 5 minutes then warming at lO C/min to a temperature 24 of -20 C.

26 (3) As (2), with the addition of ultrasound, 27 supplied at 360W over 20cm x 20cm, 25kHz, 2 seconds per 28 30 seconds pulsing.

When thawed, the samples were analysed by a taste panel 31 on a 0-5 ranking (o=poor, 5=excellent). The average 32 scores were:

~ . ' .- -... .
:
. .
, . ~ , . ~

~ ' ' ' ' ' ' ' ' ' ~ ' ' , , .~ WO91/01635 PCT/GB90/01231 ,,, ~ 206~8Q3 ~. 1 Unfrozen : 5 -~ 2 Method (1) : 3 ; 3 Method (2) : 3.5 r 4 Method (3) : 4.0 :. 5 :- 6 Example 12 -~ 8 Lean beef was obtained from a local butcher and sliced ~ 9 into approximately 1" (2.5cm) cubes. Four samples of - 10 375g each were frozen according to the following 11 methods: ~
12 .
: 13 - (l) Using a -20~C chest freezer ~: 14 15 (2) Simulation of blast freezing (-40-C, Planar :~
16 Kryo 10).
: 17 18 (3) According to the invention, in a Planar Kryo 19 10 controlled rate freezer kept initially at -50C for 15 minutes and then warmed at a rate of lO-C/min until 21 the temeprature reiached -20 C. Acoustics (360W over : 22 20cm x 20cm, 25kHz, 2 seconds per 30 seconds pulsing) 23 was supplied.

Following incubation at -20~C overnight, samples were : 26 thawed, and ~luid loss from the samples assayed over 6 27 hours.

29 (l) 14ml ~.
(2) 3ml . 31 (3) 2.5ml 32 ;

.~ '',''''' .'' . .
.:
' :
.
: i . .

WO9l/01635 PCT/CB90/01231 ~ 2~6~3 76 1 Example 13 - 3 This example demonstrates that acoustics imporve an 4 otherwise conventional blast free~ing process.
-` 6 Belgian strawberries were purchased from a local - 7 supermarket (Tesco Foodstores), washed, halved and 8 divided into lOOg batches.

Batches were frozen according to the following methods:

12 (1) Simulation of blast freezing in a Planar Kryo 13 10 controlled-rate freezer, set at -40C.
~4 (2) As (1), with the additionof a 20cm x 20cm ~16 ultrasonics plate equilibrated at -40~C, supplied by an - 17 external generator with 360W, 25kHz, with pulsing of 2 - 18 seconds every 30 seconds, 2 seconds every 60 seconds 19 and 2 seconds every 120 seconds.

21 (3) As (2) with 260W power.

; 23 Following freezing, samples were assayed for drip loss 24 over a 6 hour period.

26 The results obtained were as follows:

;` 28 ' :.
., .

.. ..
.- . - .

.. . :
,~ .

., . .. . ,,,.~

'~ 77 2b6~803 . .. .
~` 1 Freezing Method ¦ Drip loss (ml) 2 ~
,: 3 1 260W power 1 360W power .~- 4 (1) 1 ~L2 1 14 -~ 5 (2) 2s in 30s 1 13 1 18 ., 6 2s in 60s 1 10 1 15 ~:
: 7 2s in 120s 1 12 ¦ 12 These results indicate that improved freezing can be 11 obtained when blast freezing/acoustics are combined, 12 providing pulsin~ intervals are optimized.

:- 14 Examvle l~a " 15 -; 16 This example demonstrates that àcoustics improves an 17 otherwise conventional chest freezing process.
~:. 18 19 ~oneydew melons wee frozen to -20C according to two . 20 methods:
. 21 . 22 (1) In a chest freezer set at -20-C.
23 -: ~:
- 24 (2) on a 20cm x 20cm ultrasonics plate .:. 25 equilibrated at -20C powered by a generator providing --26 22.5kHz frequency, 260W power, at on/off intervals of 2 ;~ seconds every 40 seconds. .
' 28 :~-. 29 (3) As (2) with a fluid-filled plate, incorporating a glycol-filled layer.
`'~ 31 :. 32 Upon thawing, the treatments were assayed by a taste - 33 panel, which scored for texture on a range of 0 (poor) 34 - 10 (excellant).

.

- : .
: .

35 PCT/GBgO/0l231 : ~ 96`~~ ; 78 1 Treatment (1) 2 2 Treatment (2) 4.5 3 Treatment (3) 3.5 Example 14b .~s 7 Honeydew melons (Tesco Foodstores) were halved and, 8 using a 3cm diameter scoop, samples were removed, mixed 9 and 200g portions frozen by the following methods:

11 (1) Simulation of blast freezing in a Planar Kryo 12 10 controlled-rate freezer, set at -40C.

14 (2) Frozen in accordance with theinvention. The 15 environment temperature was initially -50 C, with a . 16 holding time of 16 minutes, and the temperature was 17 raised at a rate of lO C per minute to -20-C.

19 (3) Frozen as in (2), with the addition of :~: 20 acoustics (22.5kHz, 2~0W over 20cm x 20cm, 2 seconds 21 per 30 seconds).
22 Following freezing, the samples were maintained at .; 23 -20'C overnight, then thawed for 6 hours. The fluid : 24 lost from each sample was recorded:
~, 25 26 ~1) 31mls 27 S2) 15mls 28 3) 13mls .-~
. 30 Example 15 #/ 31 32 A typical ice cream mix without preservatives was - 33 frozen in a chest ~reezer zt -50'C with and without the ~ . -;' ::, . ; : ':; ' ' .. :. . , . :~ ' :
..

?

~ W091/0163~ PCT/GB90/01231 79 2 ~ 6 ~

1 application of acoustics. 13 samples (25 to 27ml) were 2 placed in stainless steel cylindrical moulds (length 3 12cm, mean diameter 2.2cm) and immersed in a 30% w/v 4 solution of calcium chloride in a Branson (Shelton, Connecticut, USA) Model 2200 ultrasonic cleaner. The 6 ultrasonic cleaning bath was placed in the chest 7 freezer and the bath solution was maintained at -40 C.
8 For the samples under test, acoustics was applied at 70 9 to 80% of the maximum power level (120W) at a frequency of 47kHz. The frequency was pulsed for 45 seconds 11 every 30 seconds. The samples were removed when a 12 temperature of -30OC was reached. The control and 13 experimental samples of the frozen ice cream mix were 14 divided into halves, with one part being stored at -30 C and the other being subiected to accelerated 16 thermal abuse.

18 A significant improvement in quality was observed in a 19 blind taste test for the ice cream that had been subjected to acoustics during the freezing process.
21 Additionally, the time taken to reach -30-C was 22 significantly less, when acoustics was applied.
23 Freezing could therefore be a~hieved more rapidly with 24 the application of acoustics.
26 Example 16 28 This example demonstra~es that the acoustics aspect of 29 this invetion has application during the cooling phase of a freeze-drying (lyophylisation) operation.

32 0.5ml of distilled water was placed in each o~ 20 33 conventional glass freeze-drying vials and cooled to ~ ' , ':
--. .

2~ 03 80 1 -4C without freezing. The vials were placed on a 2 precooled (-5C) 20cm x 20cm acoustic plate (Hilsonic 3 Ltd) and immedi~ateiy subjected to 2 seconds of 25kHz 4 acoustics at~320W. The contents of each of the vials nucleated instantly, demonstrating the feasibility of 6 nucleating undercooled aqueous or other solutions in 7 glass vials, using an acoustic source that was 8 configured such that it could also be used as the shelf 9 upon which the vials were standing.
ll ExamDle 17 - Bacterial Cells 13 Bacteria were harvested from culture slopes in lOml of 14 nutrient broth + 10% v/v glycerol and the resulting suspended bacterial population measured into lml 16 aliquots in polypropylene CRYOTUBES [2ml]. CryoSeedsTM
17 cholesterol crystals tCell Systems, Camhridge] were 18 added to each tube to ensure reproducible ice 19 nucleation.
21 The tubes were transferred either to a Planar Kryo 10 22 conventional programmable freezer rPlanar Products, 23 Sunbury on Thames, Middx] or to a passive freezing 24 device as described above in relation to Figure 2b and configured to be cooled at l-C per minute. The tubes 26 were cooled to -70C, when they were removed and 27 plunged into liquid nitrogen. Samples temperatures 28 were monitored using a Type T thermocouple/~lectronic 29 thermometer combination with the probe immersed in one of the samples.

32 The tubes were thawed by immersion in water at 25-C and 33 the samples spirally-plated onto nutrient broth to 34 provide a viable cell count.

, '~`

' ` . ' . . . ~ . , ' :
. ' , ,' ~ ~ ', ' ' ' ! . . . .
' ', ' ' .
"' , `' " ' ~. "' ` . ~ '' ' ' . '. ' , ' , ` ' ~ ' ' I
''., ~ , ' ' ,' , ' . ~ :, , , ~ ' ' ' . ' . . . .

81 20~803 1 Bacterium ~ ble cells rmeans 2 of duplicate cultures]
3 Planar freezer Passive freezer Escherichia coli82.45 82.70 6 Staphylococcus aureus 80.70 81.45 7 Neisseria meninqitidis 63.85 59.45 8 Haemophilus influenzae 5g.50 70.65 g Vibrio cholerae 75.70 72.45 11 The results show that the passive freezer of this 12 invention enables good results to be obtained even with 13 a small and portable piece of equipment.

Example ~8 -Bovine embryos 17 Bovine embryos at the 4-cell stage o~ development were 18 incubated in ovum culture mediu~ + 10% v/v glycerol and 19 then loaded individually into 0.25ml plastic straws.
XYGONTM cholesterol was incorporated into 5 straws 21 which were cooled in the passive freezer as described 22 in relation to Figure 2, configured to provide a 23 -0.3C/min cooling rate, before plunging into liquid 24 nitrogen. The remaining 5 straws were cooled in a Planar R2~6 controlled rate freezer and seeded manually 26 at -6-C.

28 The cooling profile for this machine was:
2g cool Q 5.0C per min from 20 to -5 C
31 cool @ 0.2 -5 -6 C
32 seed during the second step 33 cool @ 0.5 C per min from -6 to -32 C
4 plunge into li~uid nitrogen ~,o~4~3 .
1 Embryo~ were thawed by immersion of the straws in water 2 at 30 c, rinsed in several washes of culture medium 3 with decreasing concentrations of cryoprotectant and 4 incubated in culture medium overnight.

6 Of the five embryos frozen :in the passive freezer, four 7 were in excellent condition after culture and the ~ifth 8 was still of an acceptable quality for transplanting.
g The embryos cooled in the Planar freezer were scored a~
(three) excellent and (two) still viable but not 11 acceptable for transplanting.

13 Example 19 - ~ammalian Cell Lines A range of cultured mammalian cells were suspended in 16 91% FBS culture medium with 10% v/v D~S0, placed in 17 2.Sml plastic ampoules and then frozen in the passive 18 freezer described above in relation to Figure 2b and 19 configured to cool at 1.0C per min. The cells were removed from the freezer when the samples had reached 21 -18-C and were plunged directly into li~uid nitrogen 22 for a minimum storeage period o~ 24h.

24 Recovered cells were cultured n vitro and viable cell counts taken, based on the mean of two ampoules.

.

., :-Wo 91/01635 PCr/GB90/01231 83 2,0 ~ U 3 Cell Line S~ Viability 4 Rat fibroblast : 5 7 Monkey ~cidney cells : :l 8 9 3T3-Li 95 10 ~ouse fibroblast ;~ 12 . 13 :

: 15 ; 17 : 19 . 22 : 23 , 24 .... .

~ .
, . .

. . .
-;, , '-~ -. ~.

Claims (28)

1. A method of freezing material comprising a liquid, the method comprising extracting heat from the material and varying the rate of heat extraction to compensate at least in part for latent heat being lost during freezing.

2. A method of freezing material comprising a liquid, the method comprising extracting heat from the material at a first rate while latent heat of fusion of the material is being lost from the material and the temperature of the material is not substantially falling and subsequently extracting heat from the material at a second rate when the temperature of the material falls, the first rate of heat extraction being greater than the second rate of heat extraction.

3. A method as claimed in claim 1 or 2, wherein the liquid is aqueous.

4. A method as claimed in claim 3, wherein latent heat removal is achieved in at most 50% of the time observed when following conventional blast freezing techniques at -30°C.

5. A method as claimed in any one of claims 1 to 4, wherein the material to be frozen comprises cells of biological origin.

6. A method as claimed in claim 5, wherein the cells are animal gametes or embryos.

7. A method as claimed in any one of claims 1 to 5, wherein the material to be frozen comprises a foodstuff.

8. A method as claimed in claim 8, wherein the foodstuff is for human consumption.

9. A method as claimed in claim 7 or 8, wherein the foodstuff comprises a vegetable, bread or another bakery product, meat, fish, sea food or fruit.

10. A method as claimed in claim 9, wherein the fruit is soft fruit.

11. A method as claimed in claim 7 or 8, wherein the foodstruff comprises ice cream and/or chocolate.

12. A method as claimed in any one of claims 1 to 11, which comprises initiating nucleation of solidifiable liquid.

13. A method as claimed in any one of claims 1 to 12, wherein the material being frozen is subjected to sound waves.

14. A method of freezing material comprising a liquid, the method comprising abstracting heat from the material and applying sound waves to the material by means of a non-liquid contact with the material.

15. A method of freezing material comprising a liquid, the method comprising abstracting heat from the material and applying sound waves to the material at a power level of less than 2 W/cm2.

16. A method of freezing material comprising a liquid, the method comprising abstracting heat from the material and intermittently applying sound waves to the material.

17. A method as claimed in any one of claims 13 to 16, wherein the sound waves are at a frequency of at least 16 kHz.

18. A method as claimed in any one of claims 13 to 17, wherein the sound waves are pulsed.

19. A method as claimed in any one of claims 13 to 18, wherein the sound waves are applied at a power level of less than 2 W/cm2.

20. A method as claimed in claim 12, wherein nucleation is achieved at least partly by use of a chemical nucleator.

21. A method as claimed in any one of claims 1 to 20, wherein the material is being freeze-dried.

22. An apparatus for freezing material comprising a liquid, the apparatus comprising means for extracting heat from the material and control means for varying the rate of heat extraction to compensate at least in part for latent heat being lost during freezing.

23. An apparatus for freezing a material comprising a liquid, the apparatus comprising means for extracting heat from the material at a first rate while latent heat of fusion of the material is being lost from the material and the temperature of the material is not substantially falling and means for subsequently extracting heat from the material at a second rate when the temperature of the material falls, the first rate of heat extraction being greater than the second rate of heat extraction.

24. A device for use in freezing material comprising a liquid, the device comprising a heat sink, insulating means at least partially surrounding the heat sink and means for holding, within the insulating means, material to be frozen, the device being adapted to withstand a temperature at which the material is frozen.

25. A device as claimed in claim 24, wherein the heat sink comprises metal.

26. A device as claimed in claim 24 or 25, wherein the insulating means comprises plastics material.

27. A method of freezing material comprising a liquid, the method comprising providing material to be frozen within insulating means, at least partially surrounding a cold heat sink with the insulating means, and providing a cold environment at least partially surrounding the insulating means.

28. An apparatus for freezing material comprising a liquid, the apparatus comprising means for abstracting heat from the liquid and means for applying sound waves to the material, wherein (a) the sound waves are applied to the material by means of a non-liquid contact with the material and/or (b) the means for applying sound waves to the material is adapted to deliver the sound waves at a power level of less than 2 W/cm2 and/or (c) the means for applying sound waves to the material is adapted to deliver the sound waves intermittently.