CA2068993A1

CA2068993A1 - Cooling process and apparatus

Info

Publication number: CA2068993A1
Application number: CA 2068993
Authority: CA
Inventors: George John Morris
Original assignee: Individual
Current assignee: CELL SYSTEMS Ltd
Priority date: 1989-11-20
Filing date: 1990-11-20
Publication date: 1991-05-21
Also published as: EP0502033A1; AU659076B2; GB2255164B; GB9210005D0; GB2255164A; JPH05504052A; AU6757390A

Abstract

Material to be frozen is subjected to a cooling process which involves the application of sound waves, such as ultrasound. The invention has particular application in the frozen food products as margarine, chocolate and ice cream but may also be applied to freeze drying and cryopreservation processes. The material being frozen may also be subjected to a cooling regimen which efficiently removes the latent heat of freezing, for example by increasing the heat extraction rate when the latent heat of freezing is being given up. The invention may permit shorter freezing times and/or improved viability of the frozen or solid product.

Description

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3 This invention relates to a method of freezing a I 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 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 of human organs for transplantation;

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

3 (E) the freezing of other biological material, 24 particularly cellular (whether plant or animal) ~5 material; and 27 (F) the freezing of other material, particularly where 28 freezing must take place in controlled conditions, 29 for example, in freeze drying and/or in the production of highly regular crystalline solids.
;1 ' . -: ' 32 It is necessary to freeze or solidify many materials in 33 commercial and industrial processes. Freezing may be . .

wo91/0708~ PCT/GB90/01783

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1 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 maintaining their viability for long periods of time.
S 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, until 9 required for use. It is anticipated that there are lo 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 foodstuffs, the 17 cryopreservation of biological material and the 18 solidification of other materials may seem to be a 19 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 2~ sample) tends to supercool to a point below its 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 effect. (Although the discussion that follows 31 relates to material comprising liquid water and the .2 formation of solid ice, the same principles would apply 33 to other liquid/solid systems.) ..... ~ .. .. .. - - . - .. ... . - ~ . . . . . . .
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3 23~8~3 1 Conventionally, as an aqueous material is cooled at a 2 steady rate, the temperature of the material will fall with the surrounding falling temperature until the 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 material crystallises into ice, thereby liberating ~ latent heat of fusion. The temperature of the material g at this point rises from the nucleating point almost to the melting point. Once the latent heat of fusion has 11 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 .his stage become cooler, there is a greater differential between the material temperature and the 16 -urrounding temperature, so the material cools much 17 more quickly. This results in the relatively 18 uncontrolled formation of ice crystals, whose large 19 size can have a deleterious effect.
21 ~his ieads to a real problem for the frozen food 22 lndustry. A conventional technigue employed by the 3 ^ood industry to freeze food is to use a blast or 24 ~unnel freezer where the food is cooled by cold gas.
Tnside the freezer these is a gradient of gas 26 .emperature, the temperature being warmest at the end 27 at which the food is introduced and gradually ~ecoming ?8 lower as the food passes through the freezer.
29 Initially the sample cools in parallel with the gas ~emperature. However, after 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 ~ ... , .. . " . .. . ~ . . .

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1 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 soft 9 fruits (eg. peaches, plums, raspberries) and sea~oods (eg. lobster, crab, prawn, finfish) are often of poor 11 quality when thawed. With other soft fruits (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 these 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 by uncontrolled nucleation of 19 ice crystals and their subsequent growth associated ~-with prolonged periods at the latent heat plateau.

22 The quality of products which are consumed in the 23 frozen state such as ice cream, sorbets and ices are -24 related 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 buffered and "shell" freezing occurs.

32 With the cryopreservation of sensitive biological 33 cellular material, cellular material, there is an . , ' . '. .' , ~ , ~,.. ,-, .. ....... .
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WO 91/û7085 pcr~GB9o/o1783 ~ 2 additicnal harmful effect resulting from supercooling 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

5 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.

10 In order to minimise the potential problems caused by 11 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 freezing point of the medium. ~owever, even with this 15 considerable improvement over prior methods, care 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 19 plateau during which at least some of the latent heat 20 of fusion of the medium is being lost.
~1 22 ~he above discussion has centred on material comprising ~3 (and in particular containing a significant amount of) 2~ .~7ater. Water has a strong tendency to cool below its 5 freezing point (the supercooling 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 methods have been described to initiate ice '0 nucleation. A number of inorganic compounds, silver '1 lodide being a common example, and organic compounds ~2 (see EP-A-0246824, discussed above) and "ice 33 nucleating" bacteria (members of the genera ... . .. .. . . .. ..

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WO9l/07085 PCT/GB90/01~83 ~ 3~ ~ ` 6 1 Xanthomonas, Pseudomonas, and Erwinia) have been 2 demonstrated to have a crystal lattice structure which 3 are effective nucleators of ice in supercooled water.
Whilst these compounds have applications, for example 5 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.

9 :
The problems of uncontrolled nucleation have been seen 11 effectively 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 cryoprecerving 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 current 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 32 present processes is the direct addition of ice 33 crystals to a liquid sample or the surface of a solid.

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t 2~68993 l 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 consequences of supercooling and subsequent nucleation.

6 The present invention addresses the problems discussed 7 above and provides a surprisingly simple and elegant ~ 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 extracting 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 ?resent invention, there is provided a method of _reezing material comprising a liquid, the method 21 ^omprising extracting heat from the material at a first 22 ra~e while latent heat of fusion of the material is 23 being lost ,rom the material and the temperature of the ~4 ~aterial is not substantially falling and subse~uently extracting heat from the material at a second rate when 26 _he temperature of the material falls, the first rate 27 of heat extraction being greater than the second rate 28 of heat extraction.

'0 The invention therefore seeks to minimise or at least '1 -educe the amount of time the sample spends at the 32 .empera~ure "plateau" during which the latent heat of 33 fusion is being lost. In relation to the freezing of - - . . . .. . .. . ~ .

WO 91/07085 PCl tG890/Ot783 1 biological samples, there is evidence (Parkinson and 2 Whitfield, Theriogenoloq~ 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 -15'C than 7 between -15-C and -25-C. 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 11 mechanically by the effects of ice crystals and 12 chemically by unusual osmotic effects, for example, in 13 the semi-frozen state. It has been observed that 14 longer periods of time at the latent heat plateau lead to the formation of longer ice crystals and to a 16 degeneration in quality of the subsequently thawed 17 product.

19 By means of the heat extraction regimen of the method of the present invention, the cooling rate can be 21 controlled so that the material being frozen suffers 22 few or no deleterious effects. In particular, 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 ~uantities 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 the 33 temperature is increasing or constant or the rate of WO9l/07085 PCT/GB90/01783 9 2Q~993 1 temperature drop of the material is not substantial 2 (for example, less than l-C/min or even 0.1-C/min), and 3 the second rate may be applied when the rate of 4 temperature drop is at least 0.1-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 achieve non-linear and/or interrupted 17 cooling. An "interrupted cooling" profile includes a 18 profile having an initial rate of cooling, followed by 19 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 than the first.

27 It should also be noted that the term "first", 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 ~ . . . ~ . . . . . . ` . .

WO 91/0708~ PCl /G1~90~01783 ?,~6~93 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 foodstuff such as strawberries at -25 C, about 10% of the fruit 6 will be liquid at that temperature, yet the 7 strawberries would in ordinary parlance be referred to 8 as "frozen": it is in this sense that the word g "frozen" is used, and cognate terms should be construed accordingly.

12 The second rate of heat extraction will determine the 13 rate of cooling of the solidifying or solid material.
14 The rate of cooling selected should be such as not to 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. ~owever, 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 0.5-C/min to 31 1.5-C/min, such as about l C/min. For other material, 32 for example oyster embryos the cooling rate may be 33 about 5-C/min, and for red blood cells, the rate may be ,, , . . ~ ., ........... : . - : : . . . . .

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W O 91/07085 PC~rlG B90/01783 ~' 11 20~89n3 `' 1 several thousand C/min, for example up to about 2 3000'C/min.
In this invention, the first rate of heat extraction is 5 applied while latent heat of fusion of the material is 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 11 the eutectic temperature or the glass transition.
12 However the majority (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 10) 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 ~otal 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 ae_line exponentially with temperature.
26 .~t any sub-zero temperature, the proportion of unfrozen 27 water is directly related to the osmolarity of the 2~ unfrozen solution. For solutions of interest to the 29 food industry (for example 0.5 and 0.25M sodium cAloride solutions and their equivalents) 80% of the 31 ice will have formed by -lO-C.

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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 9 extent a relative concept, in certain embodiments of the present invention latent heat removal (for example 11 to the extent referred to above) may be considered 12 efficient if it is achieved in 509~ or less than 50% of 13 the time observed when following conventional blast 14 freezing techniques at -30-C.
16 The method is particularly applica~le to the freezing 17 and cryopreservation of biological samples, which 18 thereby constitute preferred examples of material which 19 can be frozen by means of the invention. The term "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.

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~ 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 to freeze food quickly and efficiently and without causing excessive damage to the biological or other 6 material which constitutes the 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 s~rawberries, kiwi fruit and mango. Non-aqueous systems -14 and emulsions, such as chocolate (whether plain, milk 15 or white), ice cream, cream and mayonnaise, may also be 16 'rozen by means of this invention, as may reconstituted 17 food products.
19 The invention also has application to non-biological 20 material which needs to be frozen in a controlled 21 .ashion. 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 4 27 cryopreservation of organs for transplantation and 28 iarge 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 freezing or ~3 cryopreserving gametes or fertilised egg/embryos in the - r `. ': ' ,' ,,,~ . - ' . , '., ~ . :
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: : .' ': ' :: .. : , WO9l/07085 PCT/GB90/0l783 ~ 14 r 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 O.Sml and 0.25ml 4 are common. For the frozen food industry, the volumes to be dealt with will of course be much larger, often 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-sectioned 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 further important aspect, the invention involves 23 the use of acoustics, particularly acoustics of the 24 type generally known as high frequency sound or ultrasound. The application of acoustics/ultrasound to 26 improve the crystalline structure of metal castings is 27 known as dynamic nu cleat ion. Whil 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 ... : - . - : ....................... : , ,, , ,~
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wo 91/07085 PCr/GB90/01783 ~ 15 2~ 93 Press, Oxford, 1874) which is regarded as a standard 2 work in the area, does not mention the potential of 3 acoustics in ice formation. Two Russian patent documents, with commercially impracticable teachings, 5 are however known.
7 In SU-A-0618098 food products were stated to be frozen 8 more rapidly and their quality improved by pl cing in a 9 coolant and simultaneously exposing to ultrasound at 18-66 kHz and 16-40 W. The treatment was stated to 11 increase 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 '5 industry, grain refinement is probably the result of 16 ultrasonication.

18 SU-A-0395060 teaches a similar process where the 19 rreezing process time was reduced from 5 min 10 sec to ~ min 5 sec, clearly a manifestation of improved heat 21 rransfer. Ultrasound was also stated to exert a 22 beneficial effe~t on crystalisation processes, but -;
23 again nucleation by the ultrasound was not stated.
24 ~oth 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) ~9 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.
'3 Secondly, the processes described require immersion of 1 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 under commercial circumstances, and the cost of these 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 effect on the food, it may also induce cellular damage to ll material being frozen.

13 After nucleation of ice within a food the latent heat 14 of fusion should be removed as quickly 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 19 several associated problems.
21 First, with large biological samples (such as above 5mm 22 diameter) "shell" 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. It is extremely difficult to 28 conduct a liquid nitrogen immersion process without 29 causing damage by exceeding the minimum storage temperature.

32 Thirdly, the immersion of samples into liquid nitrogen 33 is a costly process and therefore uneconomic and likely WO 91/07085 PCl/GB90/01783 , 17 2 ~ 9 3 i to be unsustainable in the frozen food industry.
3 The teachings of SU-A-0618098 and SU-A-0395060 may be ~ unworkable on a practical basis if directly applied to freezing liquid-containing material such as biological 6 material andjor 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 3 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 Possibly the application of acoustics reduces the size 19 of the ice crystals being formed, thereby resulting in less cellular disruption. This may be particularly 21 useful when freezing vegetables, such as potatoes, as a 22 lesser auantity of intracellular oxidative or other ,3 degradative enzymes (which are believed to be the cause 24 of tissue degradation or discolouration such as browning) may be released; acoustics may therefore 26 reduce or even obviate the need for blanching the 27 vegetables, which is conventionally used to reduce or 28 ~revent biological activities associated with 29 degradation, by heat-inactivation of the enzymes.
Acoustics may also have a beneficial effect when 31 applied during the freezing of material (for example, 32 rood) which may be subjected to (at least partial) ~3 freeze-thaw cycling. Partial thawing of frozen food .. , ,., ., . .,. .. . , ,. ................. ., . .- , . .
.:: .: . . . . - - . , , , - :~ ,. : . : . -. ~ , . .:, WO91/0~085 PCT/GB90/01783 1 can be a problem in the frozen food industry because or 2 temperature fluctuations during the distribution 3 process from manufacturer to retailer to consumer.
4 Food subjected to acoustics when originally frozen may suffer less oxidative damage than food not so treated.
7 The high frequency sound waves are preferably 8 ultrasound waves, generally at a frequency of at least 9 16 kHz, for example,from 18-80 kHz. The frequency at which acoustics is preferably applied ranges from 20 11 kHz to 50 kHz. Typically the applied fre~uency is from 12 20 kHz to 30 kHz; the optimal range for at least some 13 applica~ons appears to be from 22.5 kHz to 25 kHz.

Supercooled material may be subjected to the sound 16 waves for from 0.1 to 1.0 seconds. Alternatively, the 17 material may be pulsed or otherwise supplied with 18 acoustics throughout the freezing process. It is 19 preferable for the acoustics to be applied as o~e or more pulses. The pulse duration should on average 21 preferably be from 5% to 20% of the total time of 22 pulse-plus-interval; preferably the pulse lenth is from 23 0.5 to 5 seconds, with about 2 seconds being optimal.
24 Pulses of about 2 seconds in 2~ seconds have been found to be particularly effective. The power and/or 26 frequency may be varied (either discreetly or 27 continuously) during application. More than one 28 frequency may be used at the same time. It may be 29 particularly appropriate to apply acoustics when certain material being frozen is in the liquid phase;
31 this may apply in particular to ice cream.

33 As far as the power at which the acoustics is applied, . . . . ~

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W091/0708~ PCT/GB90/017~3 "_ ~ ~` 19 20~9~3 1 there is clearly a conflict in requirements. On the 2 one hand the power should be high enough for the 3 acoustics to be effective, and on the other hand the 4 power should not be so high as to cause unacceptable heating of the material being frozen (as the energy 6 applied will be dissipated as heat). Power applied 7 between 0.05 and 1.9 or 2.0 W/cm2 was found to be 8 acceptable, with a range of 0.1 to 1.5 W~cm2 being 9 preferred and about 0.2 to 1 W/cm2 being optimum.

11 This non-invasive technique of inducing ice nucleation 12 thus at least mitigates, or overcomes, problems 13 associated with prior art techniques.

The sound waves may be generated by sound wave 16 generators known in the art, such as ultrasonic baths, 17 piezoelectric transmitters and suitable transducers.
18 Thus the material may be in contact with the sound wave 19 generator, for example inside a container such as a 2~ mould in contact with a piezoelectric transmitter,-or 21 on a conveyor belt in contact with a suitable 22 transducer. In this latter embodiment the material may 23 ,hus be moved within an environment having a 24 temperature gradient, such as a conventional blast or _unnel freezer.

27 ~our preferred methods of inducing ice nucleation using 28 high fre~uency sound waves are as follows.

1. The sample is immersed in an ultrasonic bath which 31 is preferably maintained at, or about, the freezing 32 temperature of the material (eg. -20-C). Thus the 33 sound wave generator serves to both provide the high . . . , ~ ~. ~ . . . - -:, ~ . - . . : , . : . - ~ - .,.

1 frequency sound waves and also to cool the material.
2 The material will generally be immersed in a liquid, 3 preferably an aqueous liquid, such as water. However, 4 the material, if desired, may be contained or enclosed in a mould which is particularly suitable for the 6 freezing of ices.

8 2. The material may be placed in a container, such as 9 a mould, which is cooled in a freezing bath. A
piezoelectric transmitter is placed in contact with, or 11 built into, the mould to deliver the high frequency 12 sound waves. This method is particularly suitable for 13 frozen sorbets, ices and ice creams.

3. The material may be placed on top of a conveyor 16 belt which is in contact with, or interrupted by, one 17 or more transducers. This method is particularly 18 suitable for thin layers of material, such as slices of 19 foodstuffs such as soft fruits. The contact between the material and conveyor belt ensures that the sound 21 waves are transmitted efficiently to the whole of the 22 material. Cooling of the material can be achieved by 23 passing the conveyor belt through, for example, a 24 conventional blast freezer. It is preferred that a short zone of acoustic transducers is placed at a 26 particular point along the conveyor belt to achieve 27 maximum nucleation in the material.

29 4. For larger materials and those of non-planar geometry, such as spheres and cylinders, to achieve 31 more than a point contact with an ultrasonic source, it 32 is preferable to immerse the sample either fully or 33 partially in a liquid in a container. The high : ' .,, ! : . : ' ~ ' ' . - , ' ' ' ~; ' ' , ' :, . ~ , . ' . . . , ' . : ' -: :

WO9l/07085 PCT/GB9OtO1783 f;; A 21 8 .9 ~ 3 1 frequency sound waves can then be applied via 2 transducers, but the material will be immersed in the 3 liquid for only a short period (for example less than 4 one second). The temperature of the container is preferably maintained so as to keep the material at its 6 freezing temperature, for example about -5-C. The 7 liquid in the container is preferably kept below its 8 freezing point by the addition of non-toxic chemicals, 9 for example food grade chemicals. This has the advantage that the material may be simultaneously 11 coated with the food grade chemical. Preferred food 12 grade chemicals include sugars and glycero~, for 13 example to freeze the material and add a glaze. This 14 embodiment may be combined with a continuous process such as the material being carried along a conveyor 16 belt as discussed above. For example, the conveyor 17 belt may dip into an ultrasonic bath, suitably for a 18 short period such as less than one second, when it is 19 subjected to ultrasound.
21 The material is preferably precooled before subjection 22 to the high frequency sound waves to induce ice 23 nucleation. Suitably the material will be cooled so 24 that it is at the same temperature, namely of thermal equilibrium, as the environment. This is since if a 26 large temperature difference exists between the 27 material and its envir~nment then a temperature 28 gradient will be established across the material and 29 nucleation will occur on the outside and the ice front will propagate towards the centre, resulting in 31 unwanted "shell" freezing. Thus, if the whole of the 32 material is precooled to the temperature of the 33 environment, and in particular such that the inside of W09l/0708s ~9 3 2~ PCT/GB90/~1783 1 the material is at the same temperature as the 2 environment, then on subjection to the high frequency 3 sound waves ice nucleation may be induced on the inside 4 and preferably at the centre, of the material. Usually the material will be thermally equilibrated with the 6 environment below its freezing point.

8 The application of acoustics, as preferred for the g present invention, as described above, itself forms an independent aspect of the invention. It has been found 11 that if the immersion technigues suggested in the 12 Russian patent documents described above is avoided, it 13 is possible for acoustics to be beneficial and 14 commercially feasible. According to a further aspect of the invention, there is provided a method of 16 freezing material comprising a liquid, the method 17 comprising abstracting heat from the material and 18 applying sound waves to the material by means of a 19 ~on-liquid contact with the material. Generally, there will in this aspect of the invention be solid or 21 mechanical contact between a source of high frequency 22 sound waves and the material to be frozen, but 23 gas-mediated contact may be adequate. The contact may 24 for example be achieved by the use of a source of high frequency sound waves in the form of a probe, such as 26 the BRANSON LUCAS-DAWE probe, in direct contact with 27 the material. Alternatively or additionally, the 28 material could rest on a solid surface, to which was 29 mechanically connected, directly or indirectly, a source of high frequency sound. It will be appreciated 31 that a layer of suitable material may be interposed 32 between the material to be frozen and the solid 33 surface, for example to prevent contamination and/or . ., , - .

~.~ .............................................. ;
23 2Bg:3~3 !

1 undesirable sticking, but this is not to be regarded as 2 detracting from the mechanical connection, which is 3 ~ust rendered somewhat more indirect. Further, it is 4 to be understood that uniform contact between the material and the surface is not necessary: it is only 6 necessary for there to be sufficient contact for the 7 sound waves to be transmitted effectively.

9 A fluid-filled (preferably liquid-filled) layer may be interposed in the sound path between the source of high 11 frequency sound and the material to be frozen. ~his is ~2 not to say that liquid is in contact with the material 13 to be frozen; on the contrary, the fluid layer simple 14 aids transmission and/or distribution of the high lS frequency sound waves into the material. the fluid may 16 be any organic solvent, but is preferably freon, 17 glycol, ethanol or a food-compatible solvent such as 18 sold under the trade mark ISOPAR. The ISOPAR K product 19 may be the most preferred 21 It is to be understood that the "non-liquid contact" of 22 the material to be frozen does not necessarily imply 23 complete dryness. For example, if cut fruit is being 24 frozen, a small amount of liquid may be released from the fruit itself. This is however to be contrasted 26 with immersion within a sound-transmitting liquid, 27 which is not within this aspect of the invention.

29 It has also been discovered that if the relatively high power levels taught in the Russian patent documents 31 referred to above are avoided then, contary to 32 expectations the results are better; further, a lower 33 power level can be delivered by a more economical piece ":

" ' : ' . ' ' " ~ '' . ' . ~ ' ; ' WO9l/07~85 PCT/GB90/01783 ~,~5~993 1 of equipment. According to a further aspect of the 2 invention, there is therefore provided a method of ~ freezing material comprising a liquid, the method 4 comprising abstracting heat from the material and applying sound waves to the material at a power level 6 of less than 2 W/cm2. Preferred features of this 7 aspect of the invention are as described above.

9 Acoustics, either in conjunction with other aspects of the invention or on its own, has particular application 11 in the preparation of certain foodstuffs, notably ice 12 cream, chocolate and margarine, as well as in freeze 13 drying.

Ice cream is conventionally made by a process, 16 illustrated in Figure 6 (which will be described in 17 detail below), which involves the following steps:

19 (a) mixlng the solid and liquid components;
(b) pasteurisation;
21 (c) homogenisation;
22 (d) cooling (generally to 4 C or thereabouts);
23 (e) ageing/maturation;
24 (f) freezing (g) packaging;
26 (h) hardening (often to -40-C); and 27 (i) storage.

29 Acoustics can usefully be applied during ice cream manufacture, generally when solid material (whether 31 a~ueous or non-aqueous) is crystallising or about to 32 crystallise, particularly during one or more of the 33 steps of (e) ageing/maturation; (f) freezing and (h) ..... ... . . .

W091/0708~ PCT/GB90/01783 2~5~993 1 hardening or during any other step involving the 2 solidification of aqueous or fatty material.
3 Solidification of fat takes place to a significant 4 extent during the ageing or maturation step (e).
Crystallisation of a significant proportion (for 6 example about 50%) of the water present takes place 7 during freezing (step (f)), which may involve reducing 8 the temperature of the material to within a range of 9 -5 C to -30 C, typically about -20-C; air or another suitable gas may be introduced at this stage, depending 11 on the desired characteristics of the final product.
12 At present, scrape freezers are used for this step; the 13 application of acoustics and/or a suitable cooling 14 profile may improve it.
16 It is within the ageing/maturation step (e) and the , 17 freezing step (f) that it is preferred that acoustics 18 be applied. Application may be by means of a probe at 19 least partly immersed in the product, by means of transponders in operative contact with the vessel or 21 other equipment used in the step or in any other 22 convenient way. If a stirrer is used in the ageing 23 step, it may also be used to mediate the acoustics.
24 Similarly, an air or gas nozzle or a scraper used in ~
25 the freezing step may also be used to mediate the ~ -26 acoustics. It is also possible that acoustics and/or 27 an improved cooling profile may be used in the 28 hardening step (h3, to improve the crystal structure of 29 the product and/or to decrease the process time;
30 acoustics may be mediated as described above. ~

32 Chocolate manufacture is a well established process.
33 one of the critical steps is tempering, which involves :'.`': ~ ' . -. ' -', ' '' ' ' , :
, ' ' ', . ', . ~ , ~ . ' - . ' `"' - : , .' ' , : -: :.

WO 91/n708s PCr/GB90/U1783 2~ 26 1 the production of seed crystals from liquid fat wh1cn 2 grow during subsequent cooling. Cocoa butter is 3 polymorphic, and the particular form of crystals formed 4 in the solidifying chocolate determine the quality of the product in terms of its gloss, hardness, bloom 6 resistance, viscosity and contraction properties.
7 According to Willie & Lutton, the various forms of 8 cocoa butter have the following melting points:
Form Melting_Point/C Crystal Structure 12 I 17.3 gamma 13 II 23.3 14 III 25.5 B~' IV 27.5 B~
16 V 33.8 B
17 VI 36.4 B

19 There is some discrepancy in the industry about the precise melting point values, but the above figures can 21 be .aken as exemplary.

23 During the tempering process, molten chocolate (for 24 example at 50 C or so) containing cocoa butter in the liquid state is allowed to cool. Cooling is controlled 26 ~o allow formation of form IV crystals (B'), which may 27 then metamorphose to form V (B) crystals. it is 28 important to avoid the formation of form III (B") 29 crystals, as these crystals (and forms II (~) and I
(gamma), to which they can metamorphose, are unstable 31 as far as their growth is concerned. An excess of 32 forms I, II and/or III lead to undesirable 33 characteristics in the chocolate whereas form V is ~ ' - . , ~ -WO 91tO708~ PCr/GB90/01783 A
27 ~ 3 1 predominant in well tempered fat. It is desirable to 2 form a large number of crystals, evenly distributed 3 throughout the product.
Acoustics can be used to promote and control the 6 formation of the desirable form(s) of cocoa butter 7 crystals. In particular, acoustics may be applied to 8 promote the formation of form IV crystals at a 9 temperature af the order of the form IV melting point (and generally above that of the form III melting 11 point). Acoustics may therefore be applied at about 12 27.57C or 28c to 32C or 33 C.

14 Many forms of chocolate-containing confectionery involve enrobing chocolate wholly or partially around a 16 core or centre. Conventionally, in the enrobing 17 process, the temperature of the chocolate is dropped to 18 about 28 C to give good seeding but is then raised to 19 32-C because it is too viscous if kept at the lower temperature. Acoustics, appropriately applied, may 21 shorten or dispense altogether with the need for the 22 temperature drop to 28'C. Acoustics may additionally 23 or alternatively help break up any large crystals that 24 form and/or promote even crystal distribution throughout the product.

27 The application of acoustics to chocolate undergoing 28 temperlng may be by any appropriate method. A tank, 29 reservoir or hopper may be supplied with, or operatively connected to, one or more ultrasonic 31 transducers or probes. A probe or transducer may be 32 operatively connected to a stirrer. Further, a probe 33 or transducer may be connected to a nozzle or other : ;; : . :, . . . . . . ..

W O 91/0~085 PC~r/GB90/01783 ~ 993 28 l dispensing device, for example during enrobing.

3 Margarine is an emulsion which includes oils and/or 4 fats, generally but not necessarily of vegetable origin, and water. In manufacture, margarine is 6 prepared as a liquid emulsion, which is allowed to cool 7 and set. As for chocolate, acoustics may play an 8 important part in fat crystal formation and its 9 control. Evidence indicates that acoustics may enhance the stability of margarine.

12 Acoustics may be applied via a probe at least partially 13 immersed in margarine or by a transducer operatively 14 connected to a vessel containing the margarine.
Alternatively, a stirrer or other device in contact 16 with the margarine may be operatively connected to the 17 transducer.

19 Lyophilisation or freeze drying is in common use in industry today. Foods and pharmaceuticals are two of 21 the areas in which it is widely (but not exclusively) 22 used. The process generally involves two or three 23 stages, as follows:

(1) Freezing;

27 (2) Primary drying, often achieved by partial 28 evacuation for 24 hours or more and resulting 29 typically in a moisture content of 5 to 10%
w/w; and '1 . :
32 (3) optionally, secondary drying, which may be 33 achieved chemically.

: - ~ .-. . -, -; :- :: -W09l/07085 PCT/GB90/01783 ~ 29 2~6~993 `

2 ~rotocols for the efficient removal of latent heat and 3 the application of acoustics, in accordance with the invention can help realise several advantages, as follows.
7 First, the primary drying time may be reduced. Roy and 8 Pikal (J. Parenteral Sci. & Tech. 43(2) 60-66 (1989)) g show that l-C undercooling tends to lead to a 1%
increase in drying time. The problem of undercooling 11 is greater for small quantities of liquid and for clean 12 vessels an~ so becomes particularly acute in the 13 pharmaceutical industry where many products are 14 routinely lyophilised in small, extremely clean containers. Antibiotics and monoclonal antibodies, for 16 example, are routinely packaged in lml sterile vials;
17 5ml vials are also common in the industry. Under these 18 conditions, the degree of undercooling may be up to 19 20-: this can represent an increase in drying time of a matter of hours, thereby adding considerably to 21 processing costs.

23 Secondly, the residual moisture content after primary 24 drying may be (a) lower and (b) less variable.
26 The residual moisture content may be lower because of 27 'he crystal configuration formed. In a non-supercooled 28 vial, for example, nucleation will generally take place 29 at the vial bottom (which will usually be next to a cold surface) and will spread upwards. This allows the ~1 formation of "sublimation chimneys", which are 32 essentially low energy exit paths for ice in the 33 sublimation process. In a supercooled vial, nucleation .Y.... - .. ... ~ .. . , - - , . ............... , . :

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

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

WO91/07085 PCT/G~90/01783 $
~9 ~3 :` ` 30 1 generally takes place throughout the volume of liquid;
2 sublimation chimneys are not formed, and ice subliming 3 off the preparation has a more tortuous path to follow.
The residual moisture content may be less variable, 6 because normally there would be a spread of nucleation 7 points, ranging from the theoretical freezing point to 8 20-C below that temperature; acoustics in particular 9 may lower the statistical spread of these nucleation temperatures.

12 ~ ~hird advantage resulting from the use of the 13 invention is that, when biological material is being 14 lyophilised, higher viability and/or biological activity may result.

17 When applying the various features of the invention to 18 lyophilisation, the latent heat energy may be removed 19 by applying the cooling protocols described earlier in this specification. Similarly, acoustics can be 21 applied in accordance with the general principles 22 taught earlier, but it will be appreciated that 23 ultrasonics transducers may for example be attached or 24 otherwise operatively connected to supports (for example shelves) for containers whose contents are 26 being lyophilised and/or to the walls or other 27 components of the lyophilisation chamber itself.

29 Further, intermittent application of acoustics may provide the basis for improved performance over the 31 disclosure of the Russian patent documents.
.2 33 Correspondingly, the invention relates in further :. :

WO 91tO7085 PCr~GB90/01783 f~i' '' ` ' 31 2~993 1 aspects to an apparatus for freezing material 2 comprising a liquid, the apparatus comprising means for 3 abstracting heat from the liquid and means for applying 4 sound waves to the material, wherein (a) the sound waves are applied to the material by means of a 6 non-liquid contact with the material and/or (b) the 7 means for applying sound waves to the material is 8 adapted to deliver the sound waves at a power level of g less than 2 W/cm2 and/or (c) the means for applying sound waves to the material is adapted to deliver the 11 sound waves intermittently. Preferred features are as 12 described above.

14 Methods in accordance with the invention work well in conjunction with the use of other means for inducing 16 ice to nucleate, such as by using chemical (for example 17 crystalline) ice nucleators, such as is disclosed in 18 EP-A-0246824. Such nucleators can be used to determine 19 reasonably accurately when ice nucleates. The nucleator may be coated on one or more walls of a 21 container for the material and/or on a carrier for the 22 material. As is disclosed in EP-A-0246824, cholesterol 23 is a preferred nucleator.

Heat extraction may be achieved by any convenient way.
26 In principle, it is possible for heat to be extracted 27 by an endothermic reaction taking place in the 28 material. However, it will usually be more convenient 29 to provide a temperature gradient between the material and at least part of the surrounding environment, which 31 should be cooler than the material. This embodiment of 32 the invention takes advantage of Newton's law of 33 cooling, which states that the heat loss will, for ,.. . .. .

- . .
: .: . . . . . .

WO 91/07085 PCr/GB90/01783 32 ~i l small temperature differences be proportional to the 2 temperature difference between the material and the 3 surroundings.
Heat extraction can therefore most easily be achieved 6 in many appIications of the present invention by 7 placing the material in a cold environment. It ~ therefore follows that, to achieve first and second 9 heat extraction rates where the first heat extraction rate is greater than and followed by the second, the 11 sample can be moved from a cold environment to a less 12 cold environment, for example by means of a conveyor 13 system. In practice in some applications, it may be 14 easier to change the environment temperature rather than to move the sample, in which case the environment 16 temperature is increased at the interface between the 17 first and second rates.

19 Suitable environment temperatures for the first and second heat extraction rates will be apparent to those 21 skilled in the art. For preference, the environment 22 temperature for the first heat extraction rate will be 23 at least 15'C, and preferably at least 25C lower than 24 'he environment temperature for the second heat extraction rate. When the material to be frozen 26 comprises water, for example in the case of biological 27 material such as organs or, particularly, foodstuffs, 28 the environment temperature for the first heat 29 extraction rate can be for example less than -50 C, or even -80-C or -lOO-C; the environment temperature for 31 the second heat extraction rate may be -20 C to -30-C.
32 For foodstuffs, the environment temperature for the 33 second heat extraction rate may be the final desired W09l/0708~ PCT/GB90/01783 f-` 33 2Q5~993 1 storage temperature. For biological material that is 2 to be cryopreserved, it may be desired to reduce the 3 environment temperature further, for example after the 4 second heat extraction rate.

7 The preferred minimum environment temperature for the 8 first heat extraction rate may in part be determined by g tolerance of the material being frozen to temperature gradients. For fruit at least, and possibly for other 11 foodstuffs and biological material, placing material to 12 be frozen which has e~uilibrated with room temperature 13 in an environment temperature for the first heat 14 extraction rate of -lOO C or less appears to cause too large a temperature gradient to be acceptable in some 16 circumstances. Strawberries, for example, suffer 17 injury under such conditions, possibly caused by the 18 non-uniform formation of glasses and eutectics.

As an alternative to altering the environment 21 temperature, different rates of heat extraction may be 22 achieved by altering the efficiency with which the 23 environment extracts heat from the material: cold air 24 or other gas may be passed over the material at different rates for this purpose. A higher gas 26 velocity will achieve a higher heat extraction rate, as 27 can be found with everyday experience of wind chill 28 factors.

It will be appreciated that the present_ invention can 31 be put into effect by making adjustments and 32 modifications to enable the appropriate heat extraction 33 protocol to be carried out. As discussed above, this W O 91/07085 PC~r/GB90/01783 6~93 1 ~ay be achieved by an appropriate protocol for changing 2 the environment temperature. such protocols can 3 readily be established for various foodstuffs and other biological material by taking into consideration the relevant parameters for each material, for example 6 including:
7 a) Size;
b) Geometry;
g c) Water content;
d) Freezing point (to a first approximation this 11 is dependent on solute concentration within 12 the foodstuff or other material);
13 e) Thermophysical values of the material of the 14 material, both before freezing and in the frozen state; and 16 f) Container dimensions and other details.

18 Because these parameters differ from material to 19 material a computer can readily be used to derive optimum protocols.
22 The temperature history in a sample being cooled in a 23 -ontrolled rate freezer (such as the KRYO 10 series 24 ~hamber ~odel 10 16 by Planar Biomed, Sunbury-on Thames, England) can be calculated by solving 26 numerically the Fourier heat conduction equation in the 27 sample with convective or other boundary conditions as 28 appropriate. (The expression KRYO 10 is a trade mark.) 29 In general, the calculation method must allow for the cooling of an aqueous solution or other material where 31 ~ompositional as well as pnase changes occur during 32 freezing. This requires the appropriate molarity-33 freezing point depression data to be available, to , .. . . . . .

~. .. .
2~993 1 provide the relationship between ice formation and 2 melting temperature. Supercooling of the sample may 3 also be suitably accounted for. In the case of thin 4 slices the temperature gradients across the sample can be assumed negligible and consequently the conduction 6 equation reduces to a simple unsteady heat balance 7 between the time rate of change of enthalpy of the 8 sample and the heat transfer rate across its 9 boundaries. The validity of this simplified calculation has been compared against experimentally 11 derived data. The calculation method has been employed 12 to predict methods to reduce the latent heat plateau 13 within plum slices by manipulation of the environment 14 temperature.
16 However for calculating the temperature history in 17 samples of finite thickness, where conduction within 18 the sample is important, it is necessary therefore to 19 solve the full equation. Solving the full unsteady equation with three space dimensions is computationally 21 very time consuming. However, in many cases the 22 temperature gradients in one direction are much greater 23 than in the other two and in these systems a reasonable 24 prediction for the temperature history can be obtained from a one-dimensional model. This model could be 26 developed for l-d Cartesian, 1-d spherical or l-d 27 cylindrical geometry.

29 In its broadest apparatus aspect, the invention provides an apparatus for freezing material comprising 31 a liquid, the apparatus comprising means for extracting 32 heat from the material and control means for varying 33 the rate of heat extraction to compensate at least in .: , . , ~ : - . -.

~ Q,9~3 1 part for latent heat being lost during freezing.
3 According to a further aspect of the invention, there 4 is provided an apparatus for freezing a material comprising a liquid, the apparatus comprising means for 6 extracting heat from the material at a first rate while 7 latent heat of fusion of the material is being lost 8 from the material and the temperature of the material 9 is not substantially falling and means for subsequently extracting heat from the material at a second rate when 11 the temperature of the material falls, the first rate 12 of heat extraction being greater than the second rate 13 of heat extraction.

As discussed above, the apparatus will preferably 16 comprise a (preferably high frequency) sound generator.
17 The medium through which the sound is conducted from 18 the generator to the material may be gaseous, for 19 example air, or solid.
21 ~ach heat extraction means can in general comprise a 22 refrigerated element, which may actively be cooled by 23 expansion of a gas. Conventional diffusion or 24 compression/expansion refrigeration equipment may be used in this embodiment. However, this is not the only 26 form of heât extraction means that can be used. For 27 example, a cold liquid or solid which is dissipated as 28 heat is extracted from the material can be used. An 29 example of a cold liquid that can be used in this way is liquid nitrogen, which will be the material of 31 cholce for at least one of the heat extraction means 32 for cryopreservating biological material, as biological 33 material is conveniently stored at the temperature of . . ,. ` ' ,', ;.-.
.~ ... ., . . , . , ~
.: . :: :
.... . . . . . ..

W~9l/07085 PCT/GB90/017~3 ,.~ 2~689~3 1 liquid nitrogen. A cold solid which is s1mIlar1y 2 dissipated is solid carbon dioxide (dry ice), although 3 the cooling effect of solid carbon dioxide will be less 4 than the cooling effect of liquid nitrogen, because the S sublimation point of the former is higher than the 6 boiling point of the latter. A third possibility for a 7 heat extraction means is to use a heat sink which warms 8 up to equilibrium with the material to be frozen, or as 9 nearly as any intervening (for example insulating) material allows in the time available. The heat sink 11 can therefore be a block of relatively cold material, 12 especially a material with high heat conductivity, for 13 example a metal. To counter any adverse problems of 14 condensation, the metal will preferably be non-corrosive, for example by being made of brass or 16 stainless steel. However, any metal can be used if 17 appropriately protected, if necessary.

19 Suitable insulating material may be polystyrene (expanded or unexpanded) or another plastics polymer 21 such as polytetrafluoroethylene or acetal but it will 22 be appreciated that any material with suitable 23 properties can be used.

An apparatus in accordance with the invention can 26 comprise a single heat extraction means, such as one of 27 those discussed above, and control means to control the 28 single heat extraction means to extract heat at the 29 first and second rates. For example, a so called "active" system in accordance with this embodiment of 31 the invention could comprise a refrigerated element, 32 control means and temperature feedback means. The 33 control means could comprise a computer, microprocessor ~-. ~. . , . . . . -, . ~ - ............................... .
.~ , ~,5~93 3 8 l or other electronic means. The temperature feedback 2 means would continuously or continually monitor the 3 temperature of the material to be frozen and relay this 4 information to the control means, which could then cause the refrigerated element to extract heat at the 6 appropriate rate. Such an active system as this gives 7 considerable flexibility for a wide variety of material 8 to be frozen (particularly foodstuffs), but may involve g relatively high expense for small amounts of material.
11 A similar but simpler embodiment could comprise a 12 refrigerated element which is operable at two rates of 13 heat extraction. The element may be arranged to 14 operate first at a higher heat extraction rate, and then a timer may cause the element to switch to 16 operation at a low heat extraction rate. Such an 17 embodiment can be used when the characteristics of the 18 sample, or at least the environment surrounding the 19 sample, are known, but this may be acceptable in many circumstances, especially when various samples are 21 small compared to the apparatus of the invention, so 22 that any individual variation in characteristics will 23 be relatively small.

Other preferred embodiments of "active systems" are as 26 follows:

28 1) Batch systems. Mechanical freezers are generally 29 cooled by the Joule-Thompson effect and operate at temperatures down to -80-C; a minimum of -135-C is 31 possible. Material is placed into a closed chamber and 32 left until it has reached the desired temperature and 33 then removed for storage. The air in the chamber may . . ' ~ ` . ' '~

, WO9l/07085 PCT/GB90/01783 , -39 2~689~3 1 be unstirred or fan driven to achieve forced 2 convection. Additionally, the material to be frozen 3 may be placed statically on shelves or rotated within 4 the freezer.
6 The desired thermal profile may be obtained in such a 7 closed system by direct control of the compressor 8 temperature by electrical or mechanical means. In some g cases this may be practically difficult as the response time of such a control system may be too slow to 11 generate the desired profile. ~owever, as the minimum 12 operating temperature will be required at the beginning 13 of the process the control of temperature may be 14 achieved by maintaining a constant compressor temperature ~hilst varying the heat input into the 16 system from an independent heater which is programmed 17 electrically or mechanically to generate the desired 18 profile. In addition, a combination of direct control 19 of compressor output together with an external heater may be employed. The control of temperature may be 21 - preprogrammed or alternatively may be actively 22 controlled from temperature sensors placed either in 23 the gas or in the samples to be frozen.

2) Continuous Systems. The material flows through 26 the freezer on a horizontal conveyor belt or spiral 27 system. Following a retention time within the freezer, 28 the material emerges at a temperature suitable for 29 storage. Gas circulation is usually fan driven; in some cases the cold gas is forced upwards through a 31 perforated conveyor belt so that the samples are 32 suspended as in a fluidised bed. The temperature at 33 the point of entry is invariably warmer than at the .. . : . .. .. . .

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

091/07085 PCT~GB9OtO1783 ~ ~ 40 1 point of exit. Cooling may be by mechanical means or 2 alternatively by vapour from a cold liquid such as 3 liquid nitrogen; in this case the minimum operating 4 temperature achievable (>-160-C) is lower than in mechanical systems.
7 The desired thermal profile is to be achieved by the 8 manipulation of the temperature distribution of the gas 9 through the system. In contrast to the conventional mode of operation the system will be at its minimum 11 temperature at the point of entry of the food and will 12 become warmer towards the point of exit. The 13 temperature gradient within the continuous system may 14 be determined in several ways, including a system of baffles to ensure the recirculation or removal of cold 16 gas, the introduction of warm gas or the positioning of 17 heaters. The velocity of gas flow will also modify the 18 heat transfer and will be selected to be at its maximum 19 at the point of entry, at later stages the flow may either be constant or reduced. In addition, the 21 temperature experienced by the sample may also be 22 modified by control of the speed of the conveyor belt.
23 By employing a series of conveyor belts running at 24 different speeds, the retention times within different areas of the freezer may also be manipulated. A
26 combination of several of these processes may also be 27 appropriate. The control of temperature may be 28 preprogrammed or alternatively be actively controlled 29 from temperature sensors placed either in the gas or in the samples to be frozen.

32 3) Immersion in low temperature baths. This is a 33 process generally applied to ices, sorbets etc which ... . . . . . .

W O 91/07085 PC~r/GB90/01~83 ~ ,.,, ~,. . .
41 2a~8~3 1 are poured as liquids into moulds which are then 2 semi-immersed in a stirred low temperature bath, 3 typically at temperatures of -30 C. Such low 4 temperature baths are usually cooled by contact with a heat exchanger cooled by the Joule-Thompson effect.
6 Following freezing the sample is removed from the mould 7 and placed into storage. The direct immersion of 8 non-moulded foods into liquid cryogens is generally not g considered good practice. However, immersion into liquid C02, which is considered to be non-toxic and 11 which evaporates at conventional storage temperat~res, 12 may be safely employed for a variety of foodstuffs.

14 The temperature profile achieved by immersion could be modified by several potential methods. A series of 16 baths, maintained at different sub-zero temperatures 17 could be employed, with the samples being immersed in 18 sequence through the various baths. Alternatively, the 19 thermal gradient along a single bath may be manipulated to achieve the desired profile, the rate of passage 21 through such a gradient bath could also be modified in 22 a linear or non-linear manner to achieve the desired 23 profile. Again the control of temperature may be 24 pre-programmed or alternatively may be actively controlled from temperature sensors placed either in 26 the fluid or in the samples to be frozen.

28 In a quite different embodiment of the invention, 29 apparatus in accordance with the invention can have separate heat extraction means for providing the first 31 and second heat extraction rates, respectively.

.

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

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

WO 91/07085 PCr/GB90/Ot783 .
99 42 ~; .

1 What may be a preferred arrangement is again to have 2 first and second extraction means, but to have the heat 3 extraction means so arranged that together they provide 4 the first heat extraction rate, whereas only one of them (for example the first heat extraction means) 6 provides the second heat extraction rate. This 7 arrangement gives rise to a particularly effective 8 arrangement, particularly for the cryopreservation of 9 biological material. The first heat extraction means may be a bath of liquid nitrogen or an environment of 11 cold nitrogen gas (eg above a bath of liquid nitrogen), 12 which may be below -lOO C. Biological or other 13 material to be frozen can be contained in a Dewar flask 14 also containing a cold (eg gaseous nitrogen) environment; the material can be appropriately 16 insulated to provide an acceptable second rate of heat 17 extraction. The cold gaseous nitrogen environment may 18 for preference be provided in a specialised vessel 19 known as a "dry shipper" with which those skilled in the art will be familiar or, less preferably, above a 21 liquid nitrogen bath. As a further possibility, 22 commercial deep freezes may provide an adequate cold 23 environment; they are frequently capable of achieving 24 and maintaining temperatures of from -80-C to -135 C.
More generally, mechanical commercial freezers can have 26 operating temperatures from -20 to -140-C, and 27 liquid/gas freezers based on cryogenic gases can 28 operate below these temperatures down to, or at least 29 towards, absolute zero.
31 To augment the heat extracting effect of the nitrogen 32 or other primary coolant to a degree sufficient to 33 provide the greater first rate of heat extraction, a ..
. ~ .. . ~ ... .
, ~, , . . : , . . ~. ..
.

WO9lt07085 PCT/GB90/0178~

43 20~ 3 1 second heat extraction means may ~e provided during the 2 time at which the first rate of heat extraction occurs.
3 Appropriately, the second heat extraction means may be 4 a heat sink, for example, a block of cold brass or another appropriate material, as discussed above. The 6 biological sample or other material to be frozen can 7 again be suitably insulated from the heat sink so that 8 an appropriate first rate of cooling occurs.
In a preferred embodiment, material to be frozen is 11 held within a block of insulating material within the 12 Dewar flask at one or more points spaced between the 13 centre and the periphery of the block. The periphery 14 of the block will be continuously cooled by a cold environment. The centre of the bloc~ can receive the 16 brass or other heat sink, which provides the additional 17 rate of cooling necessary for the first rate of 18 cooling.

The way in which the heat extraction means can be 21 constituted is not limited to any of the embodiments 22 discussed above, and may for example be a combination 23 of the particular embodiments described or indeed any 24 other suitable arrangement.
26 ~rom the above discussion of a preferred embodiment of 27 a passive arrangement, it will be appreciated that the 28 invention also provides means which can be used in 29 conjunction with a dry shipper, li~uid nitrogen bath, freezer or any other cold environment, including those 31 discussed above.

33 According to another aspect of the invention there is 34 provided a device for use in freezing material .. . . .

: . : .

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

W O 91/0708~ PC~r/GB90/01783 ~ 9~3 44 ~

1 comprising a liquid, the device comprising a heat sin~, 2 insulating means at least partially surrounding the 3 heat sink and means for holding, within the insulating 4 means, material to be frozen, the device being adapted to withstand a temperature at which the material is 6 frozen. ~ ;
7 ~ .
8 The heat sink may, as before, comprise a block of heat 9 conductive material such as a metal, for example brass.
It may bé formed as a core, for example a generally 11 cylindrical core, around which the insulating means may 12 be placed. The core is preferably detachable from the 13 insulating means; the reason for this preference will 14 be discussed below.
16 The insulating means may be any suitable gaseous, 17 liguid or, preferably, solid insulator. Polystyrene, 18 polytetrafluoroethylene (ptfe) and acetal are 19 acceptable. It will be appreciated that the insulator should have low, but not zero, heat conductivity and/or 21 diffusivity. Polystyrene (unexpanded), for example has 22 a thermal conductivity of 0.04 W.m l.K 1 and a thermal 23 diffusivity of 2.9 x 10 3m2.S 1. The figures for ptfe 24 and acetal are as follows:
26 PTFE Acetal 28 thermal conductivity 0.24 0.22-0.24 29 W.m l.K 1 @ 23-C
thermal diffusivity 0.74 0.30 31 m2 5-l 33 The holding means may be any appropriate shape or - .; . , - ,: . ~ , .. .

WO91J07085 PCTtGB90/01783 2 ~
1 configuration for holding the material to be frozen.
2 Since at least part of the material will be liquid, the 3 holding means may be adapted to receive a container, 4 for example a straw, ampoule or bag, as discussed above, for the material. Ampoules may be made of 6 glass, plastics or any other suitable material;
7 suitable plastics ampoules include those sold under the 8 trade mark CRYOTUBES. For the case of straws or 9 ampoules to be held in a solid insulating block, the holding means may simply comprise holes drilled or 11 otherwise formed in the block. Several containers may 12 be received in the same hole. It may be that the 13 insulating block has more than one components, which 14 can is used in a single operation of the device: the components may be stacked, one upon the other, with the 16 cylindrical core being extended appropriately such that 17 it accommodates the entire depth of the stacked 18 insulator block components.

In use, the heat sink (in the preferred embodiment, the 21 brass core) will first be cooled, for example by 22 placing it in a cold environment. The insulating means 23 and the material to be frozen can then be positioned 24 around the heat sink, so that the cold environment at least partially surrounds the insulating means. The 26 material to be frozen will therefore be cooled at the 27 first heat extraction rate by the combined effects of 28 the heat sink and the cold environment until the 29 temperature of the heat sink equilibrates the temperature of the adjacent portion of the insulating 31 means; thereaf~er, the material to be frozen will be 32 cooled at the second heat extraction rate solely by the 33 effect of the cold environment, the temperature at any - . . . . ................................ .
. ~ ., . ~: . , --.: , . . . .

W O 91/07085 PC~r/GB90/01783 ~99~ ; 46 1 time being dependent upon the properties of the cold 2 environment and the thermal properties and dimensions 3 of the insulating means and the heat sink. (The ~ temperature profile is predictablP using the computer simulations involved in the design of this piece of 6 equipment, and can be adjusted to suit a required 7 application by varying the parameters considered 8 above.) The thermal characteristics of the heat sink and the 11 insulating means, the position of the holding means 12 within the insulating means and the nature of the cold 13 environment will be chosen so that heat is extracted 14 from the material to be frozen at the first extraction rate for the appropriate length of time, ie while 16 latent heat is being extracted from the material and 17 the temperature of the material is not substantially 18 falling.

According to a further aspect of the invention, there 21 is provided a method of freezing matèrial comprising a 22 liquid, the method comprising providing material to be 23 frozen within insulating means, at least partially 24 surrounding a cold heat sink with the insulating means, ~S and providing a cold environment at least partially 26 surrounding the insulating means.
~7 28 The cold environment may be defined by a container 29 which may be well insulated (ie having lower heat conductivity than the insulating means) for example 31 provided by vacuum insulation. The environment may 32 therefore be defined by a Dewar flask or a dry shipper.
~3 34 A further application of nucleation of aqueous :

-47 20S8~3 l solutions by acoustics would be the controlled,2 simultaneous nucleation of multiple samples during the 3 cooling phase of freeze-drying. A possible scenario is 4 the freeze-drying of vaccines, where several thousand small glass vials would be cooled, frozen and dried in 6 the freeze-drying apparatus in a single run.
7 Undercooling of the samples during the cooling phase of 8 freeze-drying is, to some extent, inevitable and g without any attempt at synchronised nucleation the ice formation points of individual vials (or other sample 11 container) will vary by several degrees. This will 12 lead to variations in processing time, sample quality 13 as drying begins and inconsistencies in the quality of 14 the completed, dried batch of samples. The problem could be solved if a source of acoutstics was 16 appropriately configured and placed within the 17 freeze-dryer to be used to bring about controlled 18 nucleation and ensure that it coccurred at a required 19 temperature, and uniformly between the samples.
21 In the foregoing discussion, reference has primarily 22 been made to systems in which liquid water is frozen to 23 ice. However, it will be appreciated that the 24 invention is not limited to water based systems.
26 Other preferred features of each of the aspects of the 27 present invention are as for the other aspects mutatis 28 mutandis.

For a be~ter understanding of the invention, and to 31 show how it may be put into effect, preferred 32 embodiment of the invention will now be described by 33 reference to the accompanying drawings, in which:

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

,, .

W09l~07085 PCT/GB90/01783 ~I
9g~ ~

2 FIGURE 1 is a graph showing how the temperature of ~ a biological sample varies against time as it is 4 cooled through its freezing point;
6 FIGURE 2a shows a vertical sectional view through 7 a device which is a "passive freezer" embodiment 8 of the invention;
9 FIGURE 2b shows an exploded perspective view of a further passive freezer embodiment;
11 ?
12 FIGURE 2c shows an exploded perspective view of a ~-13 still further passive freezer embodiment; :;:

FIGURE 3 shows five temperature cooling curves for 16 material cooled in accordance with the invention; . ~.

18 FIGURE 4 shows a temperature cooling curve for -19 plum slices frozen in accordance with Example 1 of the invention and a comparative temperature 21 cooling curve for plum slices frozen by a 22 conventional blast freezing apparatus;
23 ;
24 FIGURE 5 shows a temperature cooling curve for -i.
strawberry halves frozen in accordance with 26 ~xample 2 of the invention and a comparative :
27 temperature cooling curve for matched strawberry 28 halves frozen by a conventional blast freezing .
29 apparatus:
. ~:
31 FIGURE 6 shows a schematic process for the .
3323 manufacture of ice cream.

. :

~, .

.
.'~ :

,- .
49 2~ 3 1 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 against 4 temperature for a bovine embryo being cooled through its freezing point towards its cryopreservation 6 temperature in liquid nitrogen. The embryo is kept in 7 bovine embryo culture medium 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 itself. 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 not 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 released, 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 - - ~

W O 91/07085 PC~r/GB90/01783 9~3 occurring to the embryo. In this context, 2 "unacceptable" means the recorded rate of cooling 3 differs tby being more rapid) from the rate recommended 4 or used in conventional practice to achieve successful cryopreservation; an unnaceptable 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 significantly from the rate recorded during operation of the protocol: hence the requirement 11 to control cooling rate.

13 Such difficulties can be avoided by means of the 14 present invention, part of one embodiment of 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 1 comprises a vertically arranged, circular-22 sec~ioned 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 boss 7 integral with a base plate 9. The base plate 9 ? 7 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 o~ 27mm, to correspond with the brass core 3, 31 a height of 20mm, and is bevelled outwardly towards the 32 base plate 9 at 45'. In use, the brass core 3 is 33 firmly attached to the boss 7 and base plate 9. ~-, . . - . . .

.. : , :: - . - .- . ..... : , . , , : .--WO 91/07085 PCI`/GB90/01783 51 ~89~3 2 An insulating block 11, generally in the for~ of a 3 hollow circular-sectioned cylinder is configured to 4 slide and fit over the brass core 3 and to seat snugly 5 in the boss 7 and base plate 9. The insulating block 6 11 is also constructed from laminated polystyrene and 7 it has a maximum height of 180mm and a diameter of 8 200mm. Its hollow has a diameter of 2.7cm to g correspond with the brass core.

11 A first series of twelve holes 13 are formed in the 12 insulating block 11. They extend vertically 13 downwardly, parallel to the axis of the brass core 3 14 and are symmetrically arranged about the core's axis.
15 Each hole 13 in the first series is 3mm in diameter and 16 extends down from the uppermost surface of the 17 cylindrical block 11 to a depth of 140mm. The axis of 18 each of the holes 13 lies 35mm from the axis of the 19 brass core 3 or 21.5mm from the periphery of the brass 20 core 3.

22 Second, third and fourth series of twelve holes lie, in 23 register, radially outwardly from the first series;
24 representative holes are indicated by reference 25 numerals 15, 17 and 19, respectively. The axis of the 26 holes of the second series 15 lie 50mm radially 27 outwardly from the axis of the brass core 3, and the 28 corresponding distances for the third and fourth series 29 17 and 19 are 65mm and 80mm; otherwise the holes of the second, third and fourth series 15, 17 and 19 are as 31 for the first series 13.

33 The purpose of each series of holes 13, 15, 17 and 19 t'~

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

W O 91/0~085 PC~r/GB90/01783 ~ Q r~ ~ 9 9 52 1 is to hold plastics straws (not shown) conventionally 2 used for the cryopreservation of mammalian embryos and 3 gametes. Such straws are available from IMV, L'Aigle, 4 France, and are internally coated with cholesterol, as taught in EP-A-0246824. Instead of coating straws (or 6 any other container) with cholesterol, crystals of an 7 appropriate nucleator, including cholesterol, can be 8 added to the contents. Appropriate nucleators are 9 available from Cell Systems Limited under the trade ;~
marks CRYOSEED or XYGON.

12 On top of the insulating block 11, and covering the top 13 of the brass core 3 and the first to fourth series of 14 holes is an insulating cover plate 21 in the form of a disc of 200mm diameter to correspond to the insulating 16 block 11. The cover plate 21 is constructed of 17 laminated polystyrene and is 20mm thick. -19 In use, the brass core 3 and base plate 9 are first placed in a cold environment, for example in a dry 21 shipper. (A dry shipper is a well insulated container 22 resembling a large Dewar flask lined with absorbent 23 material containing liquid nitrogen; because the ~.
24 nitrogen is absorbed, there is little or no free liquid 2S in the shipper.) The brass core 3 is allowed to 26 equilibrate with the cold environment, whereafter the 27 insulating block 11, containing twelve straws in the 28 first series of holes 13, each containing a bovine 29 embryo, is positioned round the brass core 3 to seat on the base plate 9. The cover plate 21 is then placed on 31 the insulating block 19, and the device 1 is left to 32 cool.

~." .. ~ .. .. . ...... ... . . . .. . .

woslJo7o85 PCT/GB90/01783 r~:
53 2Q~9 1 Initially, the straws are cooled 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 the 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 cryopreserv~tion straws containing 8 bovine embryo culture medium plus 10 % v/v glycerol as 9 a cryoprotectant.) The first heat extraction rate is applied while 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 the straws. However, when the temperature reaches the 17 nucleating point (Tn) the sample temperature rises as 18 shown at ~ 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 11. Therefore, the continued heat 23 extraction is solely towards the periphery of the 24 insulating block 11, 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 0.32-C/min with this configuration.

33 Figure 2b shows a further embodiment of a passive ,.~,~ ,. "................. - - , - , .

, .. . .

.. . .. .

W O 91/07085 PC~r/GB90/01783 ~, ~ '3 5~

1 freezer, broadly similar to that shown in Figure ~a, 2 but including a handle assembly 101 and locating lugs 3 103 on an insulating block 105 adapted to extend 4 through a cover plate 107 and to engage apertures in a locating disc 109 of the handle assembly 101. A
6 locating lug 111 on the cover plate 107 locates in a 7 spigot 113 of the handle assembly 101. The insulating 8 block 105 is made of acetal and has sample placement 9 holes 106 adapted to receive 2.5ml ampoules for cryopreservation of, for example, mammalian cell lines.
11 The insulating block 105 is seated on a bevelled boss .
12 115 on a base 117 and surrounds a brass core 119. All 13 components other than the brass core are made of 14 acetal. Salient dimensions of the device of Figure 2b 15 are as follows: :

17 .18 ACETAL CONSTRUCTION - .
19 cryo-ampoules [c2.5ml]
21 DIMENSION [mm]
22 Component Diameter:Depth/height 1 Lid 107 200 : 40 24 2 Block 105 200 : 140 3 Locating lugs 103 [2~ 15 : 52 4 Brass rod 119 57 : 140 26 5 Base 117 200 : 20 28 Machined holes 30 6 Sample placement holes 106 13 : 50 31 7 Countersink for boss 115 5 32 3 Centre of sample placement hole 106 to perimeter of 33 block 105 44 ^- . - . . .. .

:
., ... ,. -W091/0708~ PCT/G~90/01783 ~>~ 55 2~8~3 1 9 Centre of locating lug 103 to perimeter of block 105 22.5 2 10 Hole for brass rod 119 57 : 140 4 Note 1: the height of the locating lugs 103 does not include threaded portion inserted into block -6 dimensions not critical 8 Note 2: the height of the brass rod 119 does not 9 include locating lug on base - dimensions not critical 12 Note 3: the base 117 has three small acetal feet 13 mounted, equally spaced, at the periphery. Feet 5mm 14 high x 5mm diam. Size of boss to locate brass rod and block not critical.

17 This construction, when used in conjunction with a 18 liquid nitrogen-containing dry shipper, allows a 19 cooling rate of -l-C/min.
21 A different embodiment, essentially similar in 22 construction to that shown in Figure 2b but for use in 23 connection with crycpreservation straws (eg for bovine 24 embryos), has the acetal component parts replaced with 25 PTFE parts. The salient dimensions are as follows: ~ ;

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

3 plastic straws 4 [0 25/0 5ml]

DIMENSION [mm]
6 Component Diameter:Depth/height 1 Lid 107 200 : 20 9 2 Block 105 200 : 160 3 Locating lugs 103 [2] 35 : 10 4 Brass rod 119 22 : 160 11 5 Base 117 200 : 20 13 Machined holes 15 6 Sample placement holes 106 3 : 133 7 Countersink for boss 115 5 16 8 Centre of sample placement 17 hole 106 to perimeter of block 105 63 18 9 Centre of locating lug 103 19 to perimeter of block 105 30 20 ~0 Hole for brass rod 119 22 : 160 22 No~e 1: the height of the locating lugs 103 does not 23 include threaded portion inserted into block -24 dimensions not critical 26 Note 2: the height of the brass rod 119 does not 27 include locating lug on base - dimensions not critical 29 Note 3: the base 117 has three small acetal feet mounted, equally spaced, at the periphery. Feet 5mm 31 high x 5mm diam. Size of boss to locate brass rod and 32 ~lock not critical.

34 This construction, when used in conjunction with a liquid nitrogen-containing dry shipper, again allows a 36 cooling rate of -0.3 C/min.

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

WO9l/0708~ PCT/GB90/01783 ~i - .
57 ~89~3 2 Figure 2c shows a still further embodiment of a passive 3 freezer. The construction is a modification of that 4 shown in Figure 2b, and like components have been given the same reference numerals. The principal difference 6 is that in the Figure 2c construction the insulating 7 block 105 has been replaced with two half height blocks 8 105a and 105b; this allows for more of ampoules to be 9 present (up to 15). Salient dimensions of the device

10 of Figure 2c are as follows: ~-

11 -

12

13 ACETAL CONSTRUCTION

14 cryo-ampoules ~c2.5ml]
16 DIMENSION [mm]
17 Component Diameter:Depth/height -1 Lid 107 200 : 40 19 2 Block 105a 200 : 70 3 31Ock 105b 200 : 70 21 4 Locating rods 103 [2] 15 : 123 5 Brass rod 119 S7 : 120 22 6 Base 117 200 : 20 23 Note 1: the height of the locating lugs 103 does not 24 include threaded portion inserted into block - --di~ensions not critical 27 Note 2: the height of the brass rod 119 does not 28 include locating lug on base - dimensions not critical Note 3: the base 117 has three small acetal feet 31 mounted, equally spared, at the periphery. Feet 5mm 32 high x 5mm diam. Size of boss to locate brass rod and . . ,,. , . . . ~ -WO9l/07085 PCT/GB90/01783 ~ 58 1 block not critical.

4 Machined holes _ - .
6 7 Sample placement holes 106 13 : 50 ~ .
7 8 Countersink for boss 115 5 8 9 Centre of sample hole 106 to perimeter of block 105 44 9 10 centre of locating lug 103 -10 to perimeter uf block 105 22.5 11 11 Hole for brass rod 119 57 : 120 12 It must be noted that the configuration described here 13 in detail are only a few of a great number of possible 14 configurations, depending upon the cooling rate required and the type of sample holder (for example 16 straw or ampoule) to be cooled.

18 The variables can be:

i the diameter of the insulator (although in , 21 practice it may be convenient to use a 22 standard diameter for a range of products for 23 manufacturing and marketing reasons);

ii the depth of the insulating block; :.

27 iii the diameter of the metal core;

29 iv the number, size and placement of the holes for the samples; and . :~

32 . :

WO9l/0708~ PCT/GB90/01783 -:~ 59 2 ~ 9 3 l v the materials of the insulating block and 2 metal core.

4 The invention will now be illustrated by the following examples, which relate to active, as well as passive, 6 systems. Unless otherwise stated, all examples of 7 active systems in accordance with the invention (ie 8 those active examples other than comparative examples) 9 were carried out in a PLANAR KRYO 10/16 controlled rate freezing machine. (The expression PLANAR KRYO lO/16 is 11 a trade mark). Temperature was measured with T type 12 thermocouples connected to a SQUIRREL data logger (1200 13 series). (~he word SQUIRREL is a trade mark.) Data were 14 transferred to an IBM-compatible computer for storage and analysis. In order to compare different treatments, 16 the time the sample is at the latent heat plateau, 17 defined here as the exotherm time (ET), is used; this 18 is further defined by the final temperature eg ET-5 or 19 ET-10 being the time from the exotherm to -5-C or -lO-C
respectively. Application of acoustics was either from ~1 a Branson model 250 sonicator operating at 20kHz, a 22 Branson Model 2200 ultrasonic cleaner, a Lucas-Dawe 23 series 6266 immersible transducer, a Telesonics tube 24 resonator type TR connected to a ultrasonic generator type USR-20 (20kHz) or a HILSONICS acoustic driver, 26 model IMG 400 (Hilsonic Ltd, Merseyside, England).

28 Example 1 This example shows that plums freeze better when using 31 an efficient latent heat removal protocol of the 32 invention, even in the absence of acoustics, as 33 compared to conventional methods. Korean dark skinned W O 91/07085 PC~r/GB90/01783 ~ 9 60 ~`- `

1 plums (Tesco foodstores) were sliced into 4.5mm slices 2 and were frozen by a method in accordance with the 3 present invention. For comparison purposes, plum slice 4 were also frozen by conventional methods. The methods used are as follows.
7 1. Slices were frozen by a method in accordance 8 with the invention. The initial environment g temperature was -75-C, which was held for 2 minutes. The environment temperature was then 11 warmed to -30 C at lO C/min. The temperature 12 reduction in the plum slice was significantly 13 faster than in the blast freezer treatment (2, 14 below), with a measured exotherm (ET-10) of 80 seconds (Figure 4).

17 2. (This is a comparison method.) Slices were 18 placed in a commercial blast freezer operating at 19 -40-C; the measured exotherm (ET 10) was 554 seconds (Figure 4). They were then transferred to 21 a commercial deep freeze operating at -20-C.

23 3. (This is a comparison method.) Material was 24 immersed directly into liguid nitrogen and transferred to a commercial deep freeze. The 26 sample cooled quic~ly throu~h its exotherm;
27 however the final temperature attained was below 28 -lOO-C.

Sensory evaluation of frozen/thawed material was made 31 against fresh plum slices. Frozen plums were removed 32 from the freezer ~5 minutes before evaluation and laid 33 on a plate with cling film to cover them. The plums ~ . ~ . . . .... . . . . . . .

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

61 2~gg3:

1 were placed on paper plates before panellists singly, 2 on demand, according to a statistically randomised 3 design. The panellists were instructed to assess the 4 flesh only and to discard the skin of the fruit.
S Malvern water was used as a mouth wash between samples.
6 24 replicate tastings of each sample were carried out.
7 The assessment took place under purple lighting to 8 disguise any colour differences.
g Results 12 Adjusted mean scores for the whole trial are shown 13 below; the scores are on a scale of 1-10.
14 Texture: 1 2 3 4 16 Firmness 5.46 3.46 6.08 7.83 17 Wetness 6.46 7.75 5.67 2.92 18 Crispness 5.42 4.00 6.33 6.79 19 Fibrous/Chewiness 6.25 5.29 6.71 7.42 Particulateness 5.25 4.71 5.75 6.88 21 Juiciness 6.92 7.46 6.08 3.79 23 Flavour:

Overall strength 6.33 6.88 6.04 3.75 26 Sweetness 4.79 4.88 4.38 3.63 27 Sharp/Acidic 4.79 4.71 5.00 2.96 28 Bitterness 2.83 2.96 2.88 2.25 Key: 1 = Present invention; 2 = Blast frozen; 3 =
31 Liquid nitrogen; 4 = Fresh ,. ~ - . . , ~ . . - - . - ,.... - : -WO 91/07085 PCl /G890101783 ~3 -, 2 ~

1 Discussion 3 Present invention vs. Fresh.
The fresh sample is significantly firmer, drier, more 6 fibrous/chewy than the sample frozen by the invention.
7 In flavour terms the fresh sample is lower in flavour 8 overall, less sweet and less sharp/acidic than the 9 plums frozen by the invention.
Present invention vs. blast freezing.

12 The plums frozen by the present invention are 13 significantly firmer and more fibrous/chewy than the 14 blast frozen plums. The remaining parameters show no significant differences.
16 Present invention vs. liquid nitrogen freezing.

18 There were no significant differences for any - 19 parameters.
21 _xam~le 2a 23 This example shows that strawberries freeze better when 24 using an efficient latent heat removal protocol of the invention, even in the absence of acoustics, as 26 compared to conventional methods. Spanish class 1 27 strawberries (Sainsburys Foodstores) were halved and 28 frozen by the following methods:

l) Simulat~on of blast freezing in a Planar 31 controlled rate freezer, with a rate of cooling of 32 the gas temperature of l-C/min. The measured 33 exotherm was 660 seconds (Figure 5).

~^........ - - ~ . - , .
.. , ,. , , ., ~ , . , -, W O 91/07085 PC~r/GB90/01783 _ ~,, 2~89~3 1 2) Frozen by a method in accordance with the 2 invention. The initial environment temperature was 3 -50-C for 7 minute with rewarming at lO-C/minute 4 to -30~C. The measure exotherm in the matched strawberry half to treatment 1 was 280 seconds 6 (Figure 5).
8 3) Strawberries were frozen by immersion into 9 liquid nitrogen.
11 Results.

13 Following freezing in liquid nitrogen many strawberries 14 fractured. Strawberries blast frozen and immersed in liquid nitrogen displayed significant leaXage of 16 cellular contents. For those frozen by the present 17 invention leakage was less pronounced and the 18 strawberries were significantly firmer. The exudate 19 was less pigmented than following blast freezing or liquid nitrogen freezing, clearly demonstrating that 21 less intracellular damage occurred following the 22 current method.

24 Sensory evaluation of the frozen/thawed material was made against fresh strawberries. Frozen strawberries 26 were removed form the freezer 45 minutes before 27 evaluation. 25 independent replicate tastings of each 28 sample were carried out.

.

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

?,~6 64 PCI/GB90/01781 1 Texture:
2 Treatment , 4 Rating 1 2 3 6 Excellent 7 Very Good 0 3 8 Good 2 6 2 9 Fairly Good 3 7 10 Moderate 12 6 9 11 Poor 7 3 2 12 Very Poor 1 0 14 Key: 1 = Blast Freezing; 2 = Invention; 3 = linuid N2 Flavour:
16 Treatment 18 Rating 1 2 3 Excellent - - -21 Very Good - 1 3 22 Good 3 7 5 .,.
23 Fairly Good 4 5 3 24 Moderate 8 8 4 Poor 5 2 8 26 Very Poor 5 3 2 28 Key: 1 = Blast Freezing; 2 = Invention; 3 = liquid N2 Tbere appeared to be little effect of storage time, 31 within the range of from 1 to 30 days, on the quality 32 of the material frozen by the method in accordance with 33 the present invention.

-WOgl/07085 PCT/GB90/01783 ~!.'.:, 2 ~ 3 ~ 9 3 .

2 Both the type of strawberry and the degree of ripeness3 also determined the quality on thawing; the 4 observations here are not intended to be exclusive but rather to be a guide to the trends observed. The best 6 results were obtained with slightly under-ripe class 1 7 Spanish strawberries. Poorer results were obtained with 8 riper class 1 strawberries of the same type. Good 9 results were achieved with slightly under-ripe class 2 Carmel strawberries (from Israel). With ripe class 1 11 Carmel strawberries and class 1 Kenyan strawberries 12 (Sainsburys Foodstores) poorer results were obtained.
13 It must be emphasised that with such riper starting 14 material the results following the method in accordance with the present invention outlined above was always 16 superior to blast freezing or liquid nitrogen freezing 17 of the same material.

19 ExamDle 2b 21 This example shows that even better results are 22 obtained when strawberries are frozen using an 23 efficient latent heat removal protocol, with the 24 application of acoustics. Strawberries (Californian guadalupe) were obtained in bulk from a retail outlet 26 and sorted to discard all over- or under-ripe material.
27 The selected strawberries were washed and then halved.
28 The separated halves of each fruit were collected 29 together to provide two populations of 280, essentially matched strawberry halves.

32 The strawberries were frozen in batches of 70 halves.

- . - : . - . ~ , W O 91/070B5 PC~r/G B90/01783 ;, 2Q~89~3 66 1 A 12"x12" (30.5cm x 30.5cm) acoustic plate (22.5 kHz, 2 220V, Hilsonic Ltd, Birkenhead, UX) was precooled to 3 -70 C in a CryoMed 2700 freezer and the strawberry 4 halves loaded on to it, which resulted in a temperature rise to -50'C. The material was cooled according to 6 the following protocol: tl) providing an initial 7 environment te~perature at -58-C for one minute: (2) 8 warming at lO C/minute to -48-C.

Sample temperature was monitored using type T
11 thermocouples embedded in the mid-point of 12 representative strawberry halves, connected to a 13 microprocessor data-logger (Grant Instruments, 14 Cambridge, UR). When the samples reached -20-C they were transferred to storage at -30-C for 5 days.
16 Samples were thawed by exposure to room temperature for 17 90 minutes before sensory evaluation.

19 When an acoustic treatment was applied a pulse of 2 sec every 30 sec was used throughout the entire cooling 21 cycle.

23 Subsequently thawed strawberries were subjected to a 24 sensory evaluation panel, with the following results:

WO91/0708~ PCT/GB90/01783 ~'' 6'/ ~6~993 2 Characteristic - acoustics + acoustics sig. dif. in mean t 3 scores due to , ;
4 acoustic treatment I ;
Berry colour 5.6 6.2 nsd 6 1=dull red 7 9~bright red 8 Free liquid on plate 4.3 3.4 0.01 9 l=small amount 10 9=large amount 11 Firmness 3.2 4.5 0.01 l=soft 12 g=firm 13 Mushiness 6.2 4.9 0.01 14 l=not mushy ~ ,

15 9=very mushy

16 Overall appearance 5.4 6.4 0.05

17 1=dislike extremely

18 9=like extremely

19 Overall Texture 4.2 5.5 0.01 l=dislike extremely

20 9=extremely

21 Overall flavour 5.0 6.0 nsd

22 l=dislike extremely

23 9=like extremely

24 Overall opinion 4.6 5.8 0.10

25 1=dislike extremely

26 9=like extremely

27 ExamDle 3a

28

29 This example shows that a blanched vegetable, celery, freezes better when using an efficient latent heat 31 removal protocol of the invention, as compared to 32 conventional methods, and that even better results are 33 obtained in the additional presence of acoustics.

Celery was obtained from a retail outlet. Celery 36 samples were cut into 0.6cm (~ inch) pieces, and 250g 37 were blanched per run at 90-C (190-F) for 2 minutes., ,, . . .~ . . . -.- ~ . . -;. - . . .
:. : : . .. . . . , ~ ~ ... : -; : .. . . -W O 91/07085 PC~r/G B90/01783 ~3 68 ~ ¦

1 There was a loss of 10% material on blanching. The 2 samples were rinsed with cold water to bring them to 3 room temperature (20C). The celery samples were then 4 frozen in accordance with the invention using the ~ ¦
5 following protocol: ¦
7 (1) The initial environment temperature was 8 maintained at -75-C for 2 minutes;
9 : .
(2) The environment temperature was then warmed 11 to -30C at lO-C per minute. This protocol was 12 followed with and without the application of acoustics.
13 When acoustics was applied, an ultrasound frequency of 14 22.5kHz was used, and the power level was 220 watts, applied over an area of 929cm2 (144 square inches), 16 resulting in a power level of 0.24W/cm2. The 17 ultrasound was not applied continuously, but rather was 18 applied for 3 seconds every 30 seconds.

As a control, the blanched celery was also blast frozen 21 at an environment temperature of -40-C. The samples 22 were removed when they reached -30-C. After treatment, 23 some of the frozen celery samples were stored at -30-C
24 and some were subjected to a standard temperature abuse protocol.

27 The resulting samples were evaluated in a balanced, 28 sequential order by a tasting panel consisting of 42 29 panelists, who had been pre-screened to have a positive attitude towards evaluating frozen celery slices that 31 had been thawed. A serving consisted of 6 slices of 32 celery that had undergone a given treatment. The 33 celery had been thawed at ambient temperature for 60 ,, - - ..... - .. ~.

- : . ~ - -. - , ~., : ~ , ''~. , . ~ . ~
, WO9l/07~85 PCT/GB90/0178~ ' ~' 69 2~5~9~3 l minutes prior to serving; this was sufficient to 2 eliminate any ice crystals, yet still to be sli~htly 3 chilled. The panelists were instructed to evaluate all 4 slices having undergone a given treatment before rating the attributes, so that the rating would reflect the 6 majority of slices.
8 The results showed that the efficient latent heat 9 removing protocol in accordance with the ivention resulted in better firmness, less mushiness and a 11 better overall impression of freshness of flavour than 12 the control, blast-frozen samples. Further, when 13 acoustics was also applied, it was not only found that 14 the samples offered textural advantages over the control samples, but it was also found that they held 16 up better under temperature abuse than the control 17 samples. An additional advantage of the invention 18 displayed was the reduction in the time taken for the 19 sample temperature to be reduced from ambient to the storage temperature (-30-C). Using prior art blast 21 freezing techniques, the time taken to reach -30'C is 22 in the order of 20 minutes. Using an efficient latent 23 neat removal protocol in accordance with the invention, 24 this time is reduced to about 8.2 minutes. A further improvement to about 5.2 minutes, is seen with the 26 additional application of acoustics.

28 ExamDle 3b Celery sticks were purchased from a local supermarket 31 (Tesco foodstores), washed and cut into lcm sections.
32 They were blanched for 3 minutes at 80-C, then flushed 33 with cold water. Samples were frozen according to W09l/07085 PCT/GB90/0l783 ~6~9~ 70 1 three methods:

3 ~1) Simulated blast freezing (Planar Kryo 10 set 4 at -40'C);

6 (2) According to the invention, using an initial 7 environment temperature of -50 C, with a hold time of 8 8 minutes, and then warming to -20 C at a rate of 9 lO-C/min.

11 (3) As in (2) with the addition of acoustics 12 supplied from a 20cm x 20cm plate equilibrated at -50-C
13 (25kHz, 260W power, 2 seconds per 30 seconds pulse 14 time).
16 On thawing, texture of the three samples was assessed 17 according to a subjective assay, the results of which 18 were as follows: :

Scored 0-5 (O=poor, 5=excellent) 23 The average taste panel scores for each treatment were:

Treatment (1) - 2.5 26 Treatment (2) - 3.0 27 Treatment (3) 4.0 29 Example 4a 31 Small new potatoes of less than 4 cm in diameter 32 (Sainsbury's Foodstores) were frozen by a number of 33 treatments, as described below, and evaluated on '~ 71 2~6,~993 1 thawing. Potatoes were neither cooked nor blanched 2 before freezing.
4 1) The potatoes were 'blast frozen' as for strawberries in Example 2a above; on thawing the 6 potatoes were very soft, leaked cell water and 7 were considered unacceptable after cookiny.
9 2) The potatoes were frozen by liquid nitrogen immersion; they invariably fractured during ll freezing.

13 3) The potatoes were frozen by a method in 14 accordance with the present invention by (l) providing an initial environment temperature of 16 -80-C for 1 minute, (2) war~ing at 10-C/minute to 17 -20 C. on thawing, the potatoes were intact and 18 retained their original texture with no leakage.
19 On boiling, the potatoes were acceptable. -23 Exam~le 4b Small new potatoes (3-5cm length, var. M.Bard, Tesco 26 foodstores), were cooked in boiling water for 15 27 minutes, then flushed with cold water until cool. 200g 28 batches were frozen to -30-C by the following methods;

30 (l) Simulated blast freezing (-40-C) in a Planar -

31 Xryo 10 freezer.

32

33 (2) According to the invention, using a Planar WO9l/07085 PCT/GB90/01783 ` , 1 Kryo 10 freezer. The initial temperature was -50 C, 2 which was held for 6.5 minutes; the temperature was 3 then allowed to rise a a rate of lO C per minute until 4 -20'C was reached.
6 (3) As (2) with the addition of ultrasound, 7 supplied over 20cm x 2cm at 360W, 25kHz, and various 8 pulsing lengths, as described below.

The lengths of latent heat plateaus in the various 11 treatment were measured. Following thawing, batches 12 were assessed by a taste panel, and quantitative drip 13 loss by halving tubers, wrapping in gauze in a funnel, 14 and placing a 31b (1.36kg) weight on the sample for 20 minutes. Smears of sample material were mounted on a 16 microscope slide, and observed using light microscopy.

18 The results are given below.

(1) Lengths of latent heat plateaus (LHP's) in various 21 cooling txeatments, were as follows:

23 LHP length (minutes) 24 Treatment 1 8 Treatment 2 6.5 26 Treatment 3 2s in 15s 7.0 27 2s in lOs 5.0 28 2s in 5s 4.0 According to sensory evaluation, the treatments were 31 ranked for texture in the following order;

33 Treatment 3 2s in 5s > Treatment 3 2s in lOs >

WO9l/07085 PCT/GB90/01783 -` 73 206~93 1 Treatment 3 2s in 15s > Treatment 2 > Treatment 1.
3 ~2) Fluid extrusion.
TreatmentFluid Extruded 9 3 2s in 40s 7 11 (3) Microscopy 13 Cells from Treatments 1 and 3 were compared. Blast 14 frozen cells showed a loss of organized cell structure lS and contents, with extensive folding of the cell 16 membrane. By contrast, cells frozen by Treatment 3 17 (acoustics), showed good retention of cellular 18 integrity, and less folding of the cell membrane.
19 ~:
20 Example 5 ;

22 Two types of asparagus obtained from Sainsbury's 23 Foodstores, which were Peruvian and Thai in origin 24 respectively, were frozen by a number of methods as described below and evaluated following steaming of the 26 thawed product.

28 1) Both types of asparagus were blast frozen as 29 described in Example 2a. The subsequently thawed product had poor taste and texture and scored 31 4/20.

33 `~

~5~9~3 74 1 2) ~oth types of asparagus were frozen in liguid 2 nitrogen. The spears fractured and, on thawing, 3 had very poor taste and texture; they scored 2/20.
3) Both types of asparagus were frozen by a method 6 accordance with the present invention by (1) 7 providing an initial environment temperature of 8 -80-C for 1 minute and (2) rewarming to -20-C at 9 15-C/minute. On thawing, the taste of the spears was improved, as was their texture on cooking;
11 they scored 10/20.

13 2xample 5b Raw asparagus spears (produce of Thailand, purchased at 16 Sansibury's foodstore) were trimmed to 6 inch (15cm) 17 lengths, and frozen by:

19 (1) Simulation of blast freezing in a Planar controlled-rate freezer, set at -40 C.
21 (2) Frozen in a KRYO 10 series chamber Model 22 10-16 controlled rate freezer by Planar Biomed, Sunbury 23 on Thamesj England, in accordance with the invention 24 optimised by computer modelling. The initial environment temperature was -50-C, which was held 12 26 minutes, and the temperature was then increased at a 27 rate of 10-C per minute until -20-C was reached.

29 (3) Frozen as in (2) with addition of acoustics (22.5kHz, 360W power, 2 seconds per 20 seconds).
31 Acousrtics was supplied by a HILSONIC acoustic driver 32 model IMG 400 (Hilsonic Ltd, Merseyside, England) 33 coupled through an ISOPAR M liquid filled chamber to an , ., . . , --- . . - - . :, . :

W09l/07085 PCT/GB90/01783 ,^ 5 2~89~3 :

1 8" x 8" (20cm x 20cm) plate forming the floor or Ine 2 freezer chamber. Following freezing, the samples were 3 thawed to ambient temperature over 6 hours. The spears 4 werethen cooked for 4 minutes in boiling water, and the s three frozen treatments compared with an unfrozen 6 sample using a taste panel.

8 The panel recorded average scores (0 - 5, o=poor, 9 5=excellent):

11 Unfrozen - 5 12 Method (1) - 1.5 13 Method (2) - 2.5 14 Method (3) - 3 16 Exam~le 6a 18 Single cream is an example of a oil in water emulsion.
19 Single pasteurised cream was obtained from Sainsbury's Foodstores. Following freezing and thawing of this 21 product, separation of the cream solids from the 22 liquids occurs. Freezing damage may be assessed by the 23 loss of liquid through a small mesh filter. 10 ml 24 aliquots were placed in glass universals and frozen by a variety of methods, as described below:

27 1) Blast freezing, as described in Example 2: on 28 thawing the cream is discoloured yellow, curdled.
29 The liquid loss is 34%;
31 2) Liquid nitrogen immersion, as described in 32 Example 2a; on thawing the cream does not 33 visually separate but becomes very viscous. The .

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

W09l/07085 PCT/GB90/01783 ~993 76 1 liquid loss is 12%; and 3 3) Freezing by a method of the present invention, 4 with an initial environment temperature of -80-C
for 1 minute, followed by warming at 15-C/minute 6 to -20-C. On thawing the cream does not visually 7 separate; ther~ is an increase in viscosity but 8 not as pronounced as with liquid nitrogen g freezing. The liquid loss is 10%.
11 4) Freezing as for method (3) except that 12 ultrasound was applied for 0.1 seconds for every 13 1C cooling of the cream from O to -20C. This 14 combination of acoust~c nucleation and efficient removal of latent heat consistently, in five 16 independent trials, further reduced the drip loss 17 by 10-16% of that observed in method (3).

19 It can be seen, therefore, that the present invention gives results which are appreciably better than blast 21 freezing and which are also better than the more 22 expensive and relatively inconvenient process of 23 freezing by liquid nitrogen immersion.

Example 6b 27 Single cream (Tesco foodstores) was divided into 100ml 28 batches, either in freezer bags supported by metal 29 frames or in metal moulds.
31 The cream was frozen according to the following 32 methods:

. . . ~ .- . . -WO 91/07085 pcr/GB9o/ol78~
~? 77 2 Q ~ ~ 9 9 ~

(1) Simulated blast freezing (-40-C) using a 2 Planar Kryo 10.

4 (2) According to the invention, involving rapid freezing by immersing samples in a Planar Kryo 10 - 6 controlled rate freezer initially at -80 C (hold 10 7 minutes), then warmed to -20-C at lO C per minute, with 8 the addition of acoustics throughout the cycle (300W
9 over 20cm x 20cm, 22kHz, 2 seconds every 60 seconds pulsing).

12 (3) According to the invention, using a Planar 13 Xryo 10 freezer at -50-C, holding 15 minutes, with the 14 addition of acoustics throughout the cycle as in (2).
16 Sensory analysis of the three tratments post-thaw, 17 indicated as follows: -19 (1) Separation of the cream had occurred, resulting in liquid loss, very grainy, and buttery 21 tasting.

23 (2) Very good texture, no fluid loss.

(3) No fluid loss, but texture not as good as in 26 (2).

~8 Example 7 30 Mayonnaise is an example of a water in oil emulsion.
31 Commercial mayonnaise, such as Hellman's, appears to be 32 stable following a wide range of freezing methods. This 33 probably reflects the degree of physico-chemical

34 stabilisation of the product. Home-prepared mayonnaise .. ~ - ~ ~ , . , WO 91/07085 PCr/GB90/01783 ~, 9~3 78 1 and nsn-stabilised commercial mayonnaise such as Kite 2 wholefood mayonnaise separate following freezing and 3 thawing. Such mayonnaises were frozen in 10 ml aliquots 4 in glass universals by the following methods:
6 1) Blast freezing, as in Example 2a; total 7 separation of the oil occurred on thawing;

9 2) Liquid nitrogen immersion, as in Example 2a;
total separation of oil occurred on thawing; and 12 3) Freezing by a method in accordance with the 13 present invention, in which the mayonnaise was 14 cooled at 20-C/minute from O C to -50-C, held at -50'C for 2 minutes, warmed at 15-C/minute to 16 -20-C. on thawing, there was good retention of 17 texture with little or no separation of 18 constituents.

ExamDle 8 22 Prepared prawn and mayonnaise sandwiches were obtained 23 from Tesco and Sainsbury's Foodstores and singly frozen 24 by a variety of methods, as follows:
26 1) Blast freezing as described in Example 2a; on 27 thawing there was a total separation of the 28 mayonnaise: the oil component seeped throuqh the 29 lower slice of bread and the product was totally unacceptable;

32 2) Liquid nitrogen immersion as described in 33 Example 2a; fracturing of the sandwich occurred - ,.... ,: . ~ . .. ,., : .- ~. -' W09l/0~085 PCTtGB90/01783 ~, 79 2Q~9~3'`''''''"' ! ~

l and on thawing there was total separation of 2 mayonnaise as in (1) above;
3 , , 4 3) Freezing by a method in accordance with the s present invention, in which each sandwich is 6 cooled at 20 C/minute to -50-C, held isothermally 7 at that temperature for 30 minutes and then warmed 8 at 10-C/minute to -20-C. On thawing the product g was acceptable. There was little or no separation of the mayonnaise, good retention of prawn quality 11 and no fracturing of the bread.

13 Exam~le 9 Fillets of fresh Scottish smoked salmon (Sainsbury's 16 foodstore) were frozen according to two methods:
17 i 18 (1) Simulation of blast freezing in a Planar Xryo 19 10 controlled-rate freezer st at -40~C.
(2) ln accordance with the ivnention, using 21 thermal modelling and ultrasonics application. The 22 initial environment temperature was -50-C, which was 23 held for 4 minutes, and the temperature was increased 24 at a rate of 10-C per minute until -20-C was reached.
Ultrasonic acoustics was supplied at 360W over 20cm x 26 20cm, 22.5kHz and 2 seconds per 40 seconds pulsing.
27 `
28 Following thawing, samples were tested by a panel for 29 texture and taste. The panel recorded average scores of:

'~' ..

WO9l/07085 PCT/GB90/01783 3 ~ ~

1 Unfrozen : 5 2 Method (1) : 1 3 Method (2) : 3 (0-5, o=poor, 5=excellent).
7 Example 10 9 25ml ice pops (similar to sorbets) were obtained from a local supermarket (Tesco Foodstores), and frozen 11 according to two methods;

13 (1) By processing according to the invention by 14 holding first at -50-C for S minutes and then increasing the temeprature at lO-C/min until -20-C was 16 rached in the sample, as detected by a thermocouple.

18 (2) As (l), with the addition of ultrasound l9 delivered from a 20cm x 20cm plate equilibrated at -50-C, powered by a 260W, 22.5kHz generator, 2 seconds 21 per 40 seconds pulsing. There results were as follows:
22 Cooling profiles in the two treatments varied, with 23 acoustic treatment considerably reducing latent heat 24 plateaus, and freezing time to -20-C. An assessment of crystal size by eye indicated smaller ice crystals were 26 present in the sample frozen with acoustics compared to 27 the sample frozen without. In addition, the ice pops 28 frozen with acoustics were harder to the bite and 29 crispier in texture than those without acoustics.
31 Example ~1 33 Cream cheese (Kraft General Foods) was sliced into , .. .. . .. . .... . . ... .. . . .. .

W091/07085 PCT/GB90/0178~
.
81 2~68~3 1 ~ inch (1.3cm) cubes, and samples frozen according to 2 the following methods:

4 (1) Simulated blast feezing in a Planar Kryo lO
5 controlled rate freezing apparatus held at -40-C:
7 (2) According to the invention, again using a 8 Planar Kryo 10 apparatus but using a hold time at -50 C
9 for 5 minutes then warming at lO-C/min to a temperature of -20 C.
1 1 i,, .
12 (3) As (2), with the addition of ultrasound, 13 supplied at 360W over 20cm x 20cm, 25kHz, 2 seconds per 14 30 seconds pulsing.
16 When thawed, the samples were analysed by a taste panel 17 on a 0-5 ranking (0=poor, 5=excellentj. The average 18 scores were:

Unfrozen : 5 21 Method (1) : 3 22 Method (2) : 3.5 23 Method (3) : 4.0 24 ~-ExamDle 12 ~7 Lean beef was obtained from a local butcher and sliced 28 into approximately 1" (2.5cm) cubes. Four samples of 29 375g each were frozen according to the following methods:

32 (l) Using a -20-C chest freezer .. . . ~ . . : .

W O 91/07085 PC~r/GB90/01783 ~ ~5~93 (2) Simulation of blast freezing (-40 c, Planar 2 ~ryo 10).

(3) According to the invention, in a Planar Kryo ~ 10 controlled rate freezer kept initially at -50 C for 6 15 minutes and then warmed at a rate of lO~C/min until the temeprature reached -20~C. Acoustics (360W over ~ 20cm x 20cm, 25kHz, 2 seconds per 30 seconds pulsing) a was supplied.

11 Following incubation at -20 C overnight, sample~ were 12 thawed, and fluid loss rrom the samples assayed over 6 ~3 hours.

(1) 14ml 16 (2) 3ml 17 ~3) 2.5ml . 9 , ~xam~le 13 22 'nhis example demonstrates that acoustics imporve an ~3 olherwise conventional blast freezing process.
2~
3eiaian strawberries were purchased from a local 26 ,upermarket (Tesco Foodstores), washed, halved and 7 iivided into lOOg batches.

29 3a~ches were frozen according to the following methods:
'O
31 (1) Simulation of blast freezing in a Planar Kryo 32 '0 controlled-rate freezer, set at -40-C.

Wo 9l/0708~ PCr/GB90/01783 f~'' 83 2~689.93 (2) As (1), with the additionof a 20cm x 20cm ultrasonics plate equilibrated at -40 C, supplied by an 3 external generator with 360W, 25kHz, with pulsing of 2 seconds every 30 seconds, 2 seconds every 60 seconds and 2 seconds every 120 seconds. ' (3) As (2) with 260W power.
9 ~ollowing freezing, samples were assayed for drip loss 10 over a 6 hour period. -12 The ~esults obtained were as follows:
;3 14 Freezing Method ! Drip loss (ml) 16 ' 260W power 1 360W power 17 (1) 1 12 1 14 18 (2) 2s in 30s j 13 1 18 19 25 in 60s 1 10 1 15 2s in 120s ! 12 ! 12 21 , j_ ~
22 ~' ?3 These results indicate that improved freezing can be 2~ obtained when blast freezing/acoustics are combined, 25 providing pulsing intervals are optimized.

27 _xamDle 14a 2~ ;
29 This example demonstrates that acoustics improves an 30 otherwise conventional chest freezing process.

32 :~oneydew melons wee frozen to -20 C according to two 33 methods:

. .: .. .

WO 91/07085 PCr/GB90/01783 ~,~6Q,9~3 84 ~ '.

(1) In a chest freezer set at -20 C.
4 (2) On a 2 Ocm x 2 Ocm ultrasonics plate 5 equilibrated at -20'C powered by a generator providing 6 22.5kHz frequency, 260W power, at on/off intervals of 2 , seconds every 4 0 seconds .
9 (3) As (2) with a fluid-filled plate, incorporating a glycol-filled layer.
11 ~ ..
12 Upon ~hawing, the treatments were assayed by a taste i3 ?anel, which scored for texture on a range of O (poor) 14 - 10 (excellent).
16 Treatment ( 1 ) 2 17 Treatment ( 2 ) 4 . 5 18 Treatment ( 3 ) 3 . 5 ~0 Example 14b 22 ~oneydew melons (Tesco Foodstores) were halved and, 23 using a 3cm diameter scoop, samples were removed, mixed 24 and 200g portions frozen by the following methods:
26 (1) Simulation of blast freezing in a Planar Kryo 27 10 controlled-rate freezer, set at -40-C.

2 9 ( 2 ) ~rozen in accordance with theinvention . The environment temperature was initially -50-C, with a 31 holding time of 16 minutes, and the temperature was 32 raised at a rate of lO~C per minute to -20-C.

34 ( 3 ) Frozen as in ( 2 ), with the addition of . -: . . . . . . . . : ~

-` 85 2~8gg3 7 acoustics (22.5kHz, 260W over 20cm x 20cm, 2 seconas 2 per 30 seconds).
4 Following freezing,~ the samples were maintained at S -20 c overnight, then thawed for 6 hours. The fluid 6 lost from each sample was recorded:
8 (1) 31mls 9 ~2) 15mls 3) 13mls 12 Exam~le 15 14 A typical ice cream mix without preservatives was frozen in a chest freezer at -SO C with and without the 16 application of acoustics. 13 samples (25 to 27ml) were 17 placed in stainless steel cylindrical moulds (length 18 12cm, mean diameter 2.2cm) and immersed in a 30% w/v 19 solution of calcium chloride in a Branson (Shelton, Connecticut, USA) Model 2200 ultrasonic cleaner. The 21 ultrasonic cleaning bath was placed in the chest 22 rreezer and the bath solution was maintained at -40-C.
23 ?or the samples under test, acoustics was applied at 70 24 ~o 80~ of the maximum power level (120W) at a frequency of 47kHz. The frequency was pulsed for 4S seconds 26 every 30 seconds. The samples were removed when a 27 temperature of -30-C was reached. The control and 28 experimental samples of the frozen ice cream mix were 29 divided into halves, with one part being stored at -30-C and the other being subjected to accelerated 31 ~hermal abuse.

33 A significant improvement in quality was observed in a W09l/07085 PCT/GB90/01783 8 6 ~ 1, blind taste test for the ice cream that had been 2 subjected to acoustics during the freezing process.
Additionally, the time taken to reach -30-C was significantly less, when acoustics was applied.
~ Freezing could therefore be achieved more rapidly with 6 the application of acoustics. ~ , 7 , ' 8 Example 16 This example demonstrates that the acoustics aspect of 11 this invetion has application during the cooling phase 12 of a rreeze-drying (lyophylisation) operation.

14 O.Sml of distilled water was placed in each of 20 conventional glass freeze-drying vials and cooled to 16 -4-C without 4reezing. The vials were placed on a 17 precooled (-5-C) 20cm x 20cm acoustic plate (Hilsonic 18 Ltd) and immediately subjected to 2 seconds of 25kHz 19 acoustics at 320W. The contents of each of the vials 20 nucleated instantly, demonstrating the feasibility of ~ .
21 nucleating undercooled aqueous or other solutions in ,2 ~lass vials, using an acoustic source that was 23 _onfigured such that it could also be used as the',shelf 2~ ~pon which the vials were standing.
6 _xample 17 - Bacterial Cells 28 aacteria were harvested from culture slopes in lOml of ~9 nutrient broth + 10% v/v glycerol and the resulting ~ , suspended bacterial population measured into lml '1 aliauots in polypropylene CRYOTUBES [2ml]. CryoSeedsTN , 32 cholesterol crystals rCell Systems, Cambridge] were 33 added to each tube tO ensure reproducible ice : . . . . : .
: ~ : :' . - ...................... .. -' ' :
. .

W O 91/01085 PC~r/GB90/01783 1 nucleation.

3 The tubes were transferred either to a Planar Xryo 10 4 conventional programmable freezer [Planar Products, Sunbury on Thames, Middx] or to a passive freezing 6 device as described above in relation to Figure 2b and 7 configured to be cooled at l-C per minute. The tubes 8 were cooled to -70-C, when they were removed and 9 plunged into liquid nitrogen. Samples temperatures were monitored using a Type T thermocouple/electronic 11 thermometer combination with the probe immersed in one 12 of the samples.

14 The tubes were thawed by immersion in water at 25 C and the samples spirally-plated onto nutrient broth to 16 provide a viable cell count.
17 Bacterium % viable cells rmeans 18 of duDlicate cultures 19 Planar freezer Passive freezer 21 Escherichia coli82.45 82.70 22 Sta~hvlococcus aureus 80.70 81.45 23 Neisseria meninqitidis 63.85 59.45 24 ~aemo~hilus influenzae 59.50 70.65 "ibrio cholerae 75.70 72.45 27 The results show that the passive freezer of this 28 invention enables good results to be obtained even with 29 a small and portable piece of equipment.
31 _xample 18 -Bovine embryos 33 Bovine embryos at the 4-cell stage of development were 34 ncubated in ovum culture medium + 10% v/v glycerol and , ~. - : . . . , ~ ~ . .

- - . . : . . -.. .

:~ ' WOsl/0708~ PCT/GB90/01783 ~ Q)~, 88 1 then loaded individually into 0.25ml plastic straws.
2 XYGONTM cholesterol was incorporated into 5 straws 3 which were cooled in the passive freezer as described in relation ~o Figure 2, config~red to provide a ~ -0.3C/min cooling rate, before plunging into liquid 6 nitrogen. The remaining 5 straws were cooled in a 7 Planar R206 controlled rate freezer and seeded manually 8 at -6-C.

~he cooling profile for this machine was:

12 cool @ 5.0 C per min from 20 to -5 C
13 cool @ 0.2 -~ -6 C
14 seed during the second step cool Q 0.5 C per min from -6 to -32-C
16 plunge into liquid nitrogen 18 Embryos were thawed by immersion of the straws in water '9 at 30-C, rinsed in several washes of culture medium ~ith decreasing concentrations of cryoprotectant and 21 ncubated in culture medium overnight.
~2 23 ~f ~he five embryos frozen in the passive freezer, four 24 were in excellent condition after culture and the fifth was still of an acceptable ~uality for transplanting.
26 ~he em~ryos cooled in the Planar freezer were scored as ~7 (three) excellent and (two) still viable but not 28 acceptable for transplanting.
.,9 _xam~le l9 - Mammalian Cell Lines 32 .~ range of cultured mammalian cells were suspended in 33 ~1% ~BS culture medium with 10% v/v D~SO, placed in '4 '.5ml plastic ampoules and then frozen in the passive .,. . .: - . -- : . :. . .. .: .
: : . . : .......... . . ........ . . .

.... ~ . , WO9l/07085 PCT/GB90/01783 . 9 2 ~ S ~ 9 9 ~ !

l freezer described above in relation to Figure 2b and 2 configured to cool at l.O C per min. The cells were 3 removed from the freezer when the samples had reached 4 -18-C and were plunged directly into liquid nitrogen ~ for a minimum storeage period of 24h.
7 Recovered cells were cultured ln vltro and viable cell 8 counts taken, based on the mean of two ampoules. :

10 Cell Line % Viability 13 Rat ribroblast 16 Monkey kidney cells 18 3T3-Li 95 19 ~ouse fibroblast 22 Examplè 20 - Potatoes; Blanching and Distribution 24 During freezing of halved new potatoes, in the absence of ultrasound treatment, to -40'C, oxidation occurred 26 due to enzyme activity, resulting in extreme brownlng 27 af the cut surface of the potatoes. When acoustic .28 .reatment was applied from a 12" x 12" ultrasound plate 29 (22.5kHz, 360W, 2s per 30s on/of cycle) during freezing to -40-C, as above, oxidative activity was 31 significantly reduced, such that little browning of the 32 cut tissue occurred. This is evidence that ultrasound 33 treatments give rise to the formation of ice crystals ~ . :
.
.: : - .. : :
:,: . - , ,-: :. : .
:, ~,, ~ :, - ~ . . : -WO 91tû7085 PCr/GB90/01783 ~ ~,r~9 ~ so l which are less damaging, in size, configuration and 2 intracellular location, than those which form during 3 conventional blast freezing. Such ice crystal 4 formation limits the membrane breakdown and loss of cellular compartmentation which is known to give rise 6 to damage-induced oxidative phenol-releasing enzyme 7 activity (Matile, pH. (1976) Vacuoles. In: "~lant 8 Biochemistry" (3rd Edition), Eds. Bonner, J. and g Varner, J.E. pp. 1~9-224. Academic Press). This evidence suggests that acoustic treatments can be used 11 to replace, partially or totally, the blanching process 12 which is commonly employed prior to the freezing of 13 most vegetables. The occurrence of oxidative enzyme 14 activity in sliced vegetables during freezing may be '5 used as an accurate monitor of cytological damage 16 during freezing treatments.

18 _xample 21 - Potatoes: Simulated Distribution _g Conditions ~1 Eollowing freezing at the freezing plant, frozen 22 foodstuffs are generally subject to temperature ~3 luctuations during distribution through retail outlets ^4 _o consumers. ~he severity of temperature fluctuations varies according to geographic region, and in some 26 ~nstances, thawing and re-freezing of the product can 27 occur. In order to test whether ultrasound-treated 28 Crozen material retains its superiority over 29 conventional freezing through severe distribution s~ress, halved new potatoes, prepared as in Example 20 31 above with and without ultrasound, were treated as ~ -32 ~ollows:- -W091/0~085 PCT~GB90/01783 ~ 91 2~58993 ;.~.. -1 (1) Retained for 24 hours at -20 c;
(2) Thawed from -20-C after initial storage for 430 minutes, then re-frozen by replacing at -20^C (one freeze/thaw cycle); and 7(3) Repeating (2) for a sample which has ~3previously experienced one freeze/thaw cycle, 9ie giving two consecutive freeze/thaw cycles.

11 The treatments were compared after 12 hours further ;2 storage at -20~C.

14 Prior to thawing, samples were inspected, and given an '5 index of oxidative deterioration according to a scale 16 of 0 to 5, where 0 = No oxidative damage (potato 17 surface is creamy white) and 5 = severe oxidative 18 damage (potato surface is dark-brown/black). The 19 results are shown in the following table.
Gl ConditionsNo AcousticsWith Acoustics 23 -20-C stored 2.5 o 24 1 F/T cycle 3.5 1 2 F/T cycle 4.5 2 27 Thus, whilst oxidative damage occurs increasingly in 28 Doth treatments following freeze/thaw cycles, the 29 improving effects of acoustics treatments are retained through freeze/thaw cycles. It would be expected that 31 this would model a severe distribution chain stress.
~2 WOgl/07085 PCT/GB90/01783 .. (~; j .
2 ~9g3 92 1 Exam~le 22 - Ice Cream in Domestic Ice Cream Maker 3 Thawed WALL'S vanilla ice cream mixture was frozen in l litre batches accordin~ to the method given below.
(The word WALL'S is a trade mark.) 7 Using a GELATTERIA domestic 2 litre ice cream maker, 8 1 litre of the ice cream mixture was added and 9 processed in accordance with the manufacturer's directions using continuous scrape stirring. (The word 11 GELATTERIA is a trade mark.) One batch was prepared 12 without ultrasound treatment, being frozen to -5-C in 13 the scrape freezer, then hardening overnight at -20 C.
14 In a second batch, ultrasound was supplied to the ice cream mix via an ultrasound probe (BRANSONS - trade 16 mark) at 20kHz, 360W. (20 to 25kHz and 200 to 500W are 17 the preferred ranges). Ultrasound was applied in a 18 10%/90% on/off cycle in which each on time was 5 l9 seconds and each off time was 45 seconds. (Application -ould be of any intermittent pattern or applied 21 _ontinuously.) Ultrasound was applied over the time 22 ~hen the tempera~ure was falling from +5DC to -5-C, 23 ~lthough it could have been applied for longer. After 24 reaching -5-C, samples were transferred to a -20-C
_hest _reezer for overnight hardening. A taste panel 26 was instituted to taste the samples; the panel's 27 instructions were to evaluate the samples on the basis 28 of good texture and iciness alone according to the 29 scheme 0 = poor, crystalline texture, much iciness and ~ = creamy texture, no discernment of ice crystals.
31 The mix that had been frozen without application of 32 ultrasound scored 2, and the mix that had been frozen 33 with application OI ultrasound scored 3.

W09l/07085 PCT/GB90/01783 ~, 93 2~ q3 2 Example 23 - Ice Cream in Commercial Pilot Plant 3 Mixture Thawed NESTLE's ice cream mixtures were frozen using a 6 commercial pilot plant 3 gallons scrape freezer. (The 7 word NESTLE's is a trade mark.) Batches were prepared 8 as in Example 20, with the same acoustic probes g suspended to a depth of 2 inches (5.lcm) into the ice cream sample. In addition, ice cream mixture was 11 placed into moulds in contact with a pre-cooled glycol 12 bath. Care was taken to ensure that the glycol was not 13 in contact with the ce cream. Ultrasound was supplied 14 in two ways:
16 (1) An acoustic probe was dipped into each mould, 17 whose volume was approximately 20 cm2.
18 During cooling, a total of 10 pulses of 19 duration 0.5 seconds was supplied at power 120 W. (The power could range for example 21 from 50 W to 360 W.) 23 (2) The acoustic probe was dipped into the glycol 24 outside the moulds. Power was increased to ~5 300 W.

~7 Frozen ice cream was hardened at -20-C overnight as in 28 Example 22, then subjected to a tasting panel 29 instructed as in Example 22. Each batch frozen in conjunction with acoustics scored 3, whereas a control 31 batch 4rozen without acoustics scored 2.

~3 These results indicate that implementation of : . ~. . . ~ . - . . , ~ . , W091/0~085 PCT/GB90/01783 ~ I
~Q5a993 94 1 ultrasound, using ultrasound probes, stirrers, plates 2 or encased transducers (for example lining ageing and/or freezing tanks) cause improvement and possibly 4 more efficient processing in ice cream manufacture, s particularly at the stages of maturation/ageing, 6 initial freezing (and aeration, if applied) and 7 hardening. , 9 xam~le 24 - Margarine 11 Margarine samples containing sunflower or corn oil and 12 sold under the trade marks FLORA, PROMISE, TOUCH OF
13 3UTTER SPREAD and CHIFFON SOFT were melted in a 45 C
14 -~ater bath until each sample was 1 to 2 C above its ~elting point. (The melting points lie in a range of 16 approximately 35 to 43 C.) 5ml aliquots were dispensed 17 into scintillation vials. Some samples were allowed to 13 remain at room temperature for several hours. Other 19 samples were given acoustic pulses from an ultrasound ?robe (BRANSONS delivering 360W at 20kHz). Three 21 ~ulses were given to each vial when the samples reached 22 28O~, 26-C and 24 C. All the samples were then ~3 ~ain~ained at room temperature for several hours, after 24 -~hicA they were stored overnight in a 4-C refrigerator.
26 _nspection of samples indicated that some product 27 samples untreated with ultrasound (the FLORA and 8 ~HIFFON SOFT products) remained unset, whereas the 29 others had set, although in those cases severe emulsion breakdown had occurred. Inspection of samples treated -1 ~ith ultrasound indicated the emulsion sta~ility was 32 retained in all samples and that normal setting had '3 occurred.
` .

WO91/0708~ PCl/GB90tO1783 . I
2068993`

2 These results indicate that ultrasound can be applied 3 to margarine emulsions during tempering and 4 crystallisation to improve the efficiency and possibly reduce the timing of each stage. Ultrasound may be 6 applied by means of a probe, stirrer, plate or encased 7 transducer, for example.

9 Exam~le 25 - Chocolate 11 If chocolate is solidified without any attention to 12 crystal seeding, the texture will be granular and the 13 colour poor, since large cocoa butter crystals will be 14 produced. Such crystals will also give rise to "bloom"
over a storage period of weeks, since unstable crystals 16 impart an undesirable greyish colour to the product.
17 The traditional means of overcoming these problems is 18 by "tempering", which involves the seeding of liquid 19 chocolate. Cocoa butter exhibits polymorphism, with six crystalline forms of different melting points.
21 Correct tempering involves seeding the liquid with B' 22 crystals (melting point 27.5-C, which then propagate 23 and transform rapidly into stable ~ crystals. This 24 generally involves maintaining the chocolate for 30 minutes at 27.5-C. Bad tempering can give rise to 26 unstable crystalline forms, which cause the undesirable 27 forms above.

29 As in other examples, ultrasound an be used to produce small crystalline nuclei during the crystallisation 31 process. Ultrasound can be applied to molten chocolate 32 via a probe, stirrer, plate or encased transducer, for 33 example. The frequency applied may range from 20kHz to .

. .
: . , - . . ~ :

WO9l/07085 PCT/GB90/01783 ~3 ` 9 6 ~ :

1 30kHz for example. Exemplary power levels are 260 IO
2 360W. Ultrasound may be applied continuously or in 3 pulses. If ultrasound is applied at or around 27.5C, 4 numerous B' crystals can be produced in a short time, thus reducing or removing the need to temper.
7 Similar benefits may be obtained during a step of 8 moulding the chocolate or enrobing it around a centre, 9 although in these applications the frequency and power of the applied ultrasound may be altered. It is even 11 possible that the freque~cy could be increased to the 12 megahertz level. Moulding and enrobing call for 13 optimally tempered chocolate, since the chocolate is 14 heated to 32-C for handleability; sufficient seeding crystals must nevertheless remain. Chocolate tempered 16 by ultrasound may produce an increased number of such 17 seeding crystals following tempering. Alternatively or 18 additionally, re-heated chocolate could again be 19 supplied with ultrasound prior to or at the point of enrobing.

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

Claims

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, and subjecting the material being frozen to sound waves.

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, the method comprising subjecting the material being frozen to sound waves.

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 foodstuff comprises ice cream and/or chocolate and/or margarine.

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

13. 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.

14. 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.

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

16. A method as claimed in claim 13, 14 or 15, wherein the sound waves are at a frequency of at least 16 kHz.

17. A method as claimed in any one of claims 13 to 16, wherein the sound waves are pulsed.

18. A method as claimed in any one of claims 13 to 17, wherein the sound waves are applied at a power level of less than 2 W/cm2.

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

20. A method as claimed in any one of claims 1 to 19, wherein the material is being freeze-dried.

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

22. 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, 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, and means for applying sound waves to the material being frozen.

23. 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, means for holding, within the insulating means, material to be frozen, and means for applying sound waves to the material being frozen, the device being adapted to withstand a temperature at which the material is frozen.

24. A device as claimed in claim 23, wherin the heat sink comprises metal.

25. A device as claimed in claim 23 or 24, wherein the insulating means comprises plastics material.

26. 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, providing a cold environment at least partially surrounding the insulating means and applying sound waves to the material being frozen.

27. 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.

28. A method of lyophilising material by a process comprising freezing and drying the material, characterised in that sound waves are applied to the material being frozen.

29. A method of making ice cream, the method comprising applying sound waves to the ingredients at a stage when solid material is crystallising or about to crystallise.

30. A method as claimed in claim 29, wherein sound waves are applied during one or more of (a) ageing;
(b) freezing; and (c) hardening.

31. A method of making margarine, the method comprising applying sound waves to the ingredients at a stage when solid material is crystallising or about to crystallise.

32. A method as claimed in claim 31, wherein the sound waves are applied during tempering.

33. A method of making chocolate, the method comprising applying sound waves to the ingredients at a stage when solid material is crystallising or about to crystallise.

34. A method as claimed in claim 33, wherein the sound waves are applied during tempering.

35. A method as claimed in claim 33, wherein sound waves are applied at a time and temperature to promote formation of .beta.' and/or .beta. crystals of cocoa butter.

36. A method as claimed in claim 33, 34 or 35, wherein the chocolate is moulded and/or enrobed, and the sound waves are applied at the time of, or shortly before, moulding and/or enrobing.