Low temperature annealing in the freeze-drying of coffee

Abstract

There is provided a method for the manufacture of a freeze-dried coffee powder, the method comprising: (i) providing an aqueous coffee extract comprising at least 40 wt.% soluble coffee solids; (ii) foaming and cooling the aqueous coffee extract to form a foamed viscous coffee extract; (iii) freezing the foamed viscous coffee extract to form a slab of frozen coffee; (iv) fragmenting the slab of frozen coffee to form a plurality of frozen coffee granules; and (v) subliming water from the plurality of frozen coffee granules to form a freeze-dried coffee powder, wherein, after step (iv) and before step (v), the method comprises a low temperature annealing step whereby the frozen coffee granules remain within a temperature range of from -15 to -30°C for at least 20 minutes, or within a temperature range of from -12 to -30°C for at least 75 minutes.

Description

[0001] Low temperature annealing in the freeze-drying of coffee

[0002] The present invention relates to an improved process for providing a freeze-dried coffee powder. In particular, it relates to a process for providing a freeze-dried coffee powder having a darker colour profile, where the dark colour is obtained by including a low temperature annealing step prior to subliming water from a plurality of frozen coffee granules. The low temperature annealing step can also lead to an increase in the collapse temperature, resulting in higher throughput, increased yield and an improvement in energy efficiency of the process.

[0003] Freeze-dried coffee is well known in the art and the products are generally considered to be a premium soluble coffee product. The drying process is typically gentler than spray-drying, with a reduced loss of volatile flavour components due to the low temperatures used. Accordingly, while it is generally more expensive to produce freeze-dried coffee than spray- dried coffee, due to the energy costs associated with the freeze-drying process, the product is generally more desirable.

[0004] A conventional freeze-drying system for preparing soluble coffee is well known in the art. Such a system takes an aqueous soluble coffee extract, typically having from 40 to 50wt% soluble coffee solids, and cools it down. The cooled coffee extract is then typically foamed, which provides a number of benefits. These include improved dissolution, colour control and the density of the final freeze-dried product. The foamed, cooled coffee extract is then loaded onto cooling trays and frozen to a temperature of less than minus 40°C and typically less than minus 50°C. The frozen material is ground to increase its surface area and then subjected to a freeze-drying process with added heat under a reduced pressure. An exemplary freeze-drying process is disclosed in EP3448166.

[0005] There exists a prejudice amongst consumers who consider soluble coffee products, such as freeze-dried coffee, to be inferior to the coffee produced in cafes or produced in beverage machines. In particular, consumers prefer coffee beverage powders which possess a darker colour, resembling coffee produced by ground or roasted coffee. Therefore, it is desirable to provide a freeze-drying process that results in darker coffee powders.

[0006] Moreover, the freeze-drying process can fail if the coffee reaches a “collapse temperature” at which the grains start to melt and fuse to form a “muesli-like” product, or large lumps that do not dry. Without wishing to be bound by theory, it is believed that the amorphous phase of the frozen coffee contains an excess of water, so that its glass transition temperature is unusually low. A low collapse temperature can occur if the extract is too concentrated.

[0007] Therefore, it is also desirable to increase the collapse temperature to enable the use of more concentrated extracts

[0008] Various previous attempts have been made to provide alternative freeze-drying processes that address the above problems. US 2022 / 0295815 A1 describes cooling a coffee extract before adding gas hydrates to the extract to create high-pressure closed bubbles. This creates a slurry. The slurry is then mixed with another coffee solution, foamed and freeze- or spray-dried to form a dried coffee. The dried coffee is ground to provide an instant coffee powder. WO 2020245318A1 provides a similar disclosure.

[0009] WO 2004 / 047753 describes a freeze-drying microscope, wherein the rate of sublimation can be increased by annealing. The size of solvent crystals can be increased by freezing a sample followed by warming the sample to within a few degrees below the melting point of the solvent.

[0010] GB1282584 A relates to a process of freeze-drying to produce coffee with a good aroma and darker colour by adding back aromatised oil. The process involves percolating roasted and ground coffee to obtain a coffee extract, cooling the extract to below its eutectic point and freeze-drying the frozen extract to produce a freeze-dried coffee. The composition is cooled between -4°C and -12°C during freeze-concentration.

[0011] US3682650A relates to a process for making dark-coloured freeze-dried coffee by holding the coffee extract at between -4°C and -12°C for an extended period to increase the size of ice crystals to a size of at least 0.5mm before the final freezing and freeze-drying.

[0012] US11229221 B1 relates to a process for manufacturing a freeze-dried coffee. The process involves adding gas to a coffee extract comprising 40 to 55 wt.% coffee solids and depressurising the extract to form a foamed coffee extract. The extract is then cooled to below -40 °C to form a frozen coffee extract that is ground into a powder and dried.

[0013] US3573929 provides a method for compacting and extruding fines from a conventional freeze-drying process.

[0014] GB1302289 provides a freeze-drying method with control over the initial freezing step between -2 and -7°C. DE1964709 disclose a process in which frozen coffee extract is surface-coated with a spray- dried coffee powder.

[0015] “Effect of freezing on microstructure and reconstitution of freeze-dried high solids hydrocolloid-based systems”, Malik et al. Food Hydrocolloids 83 (2018) 473-484, looks at the effects of temperature cycling steps during the freezing of coffee slabs. It is concluded that the slow, stepwise cycling between -40°C and -20°C changes the porosity and affects the final dissolution characteristics of the product. This document focuses on the internal pore structure of the product.

[0016] Accordingly, the inventors sought to provide an improved method of producing a freeze-dried coffee product with a darker colour profile, or at least to tackle problems associated therewith in the prior art, or provide a commercially viable alternative thereto.

[0017] According to a first aspect there is provided a method for the manufacture of a freeze-dried coffee powder, the method comprising:

[0018] (i) providing an aqueous coffee extract comprising at least 40 wt.% soluble coffee solids;

[0019] (ii) foaming and cooling the aqueous coffee extract to form a foamed viscous coffee extract;

[0020] (iii) freezing the foamed viscous coffee extract to form a slab of frozen coffee;

[0021] (iv) fragmenting the slab of frozen coffee to form a plurality of frozen coffee granules; and

[0022] (v) subliming water from the plurality of frozen coffee granules to form a freeze-dried coffee powder, wherein, after step (iv) and before step (v), the method comprises a low temperature annealing step whereby the frozen coffee granules remain within a temperature range of from -15 to -30eC for at least 20 minutes, or within a temperature range of from -12 to -30eC for at least 75 minutes.

[0023] The present disclosure will now be described further. In the following passages different aspects / embodiments of the disclosure are defined in more detail. Each aspect / embodiment so defined may be combined with any other aspect / embodiment or aspects / embodiments unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. It is intended that the features disclosed in relation to the product may be combined with those disclosed in relation to the method and vice versa. The present inventors were investigating the primary energy costs of the freeze-drying process while seeking to provide a way of reducing the energy costs. The energy costs can be divided into those steps performed before the cold room, and those performed in the cold room. The steps before the cold room include pre-cooling, foaming and the further cooling to the temperatures where the extract is loaded onto a tray or conveyor belt. These account for roughly one third of the energy costs.

[0024] During this investigation the inventors separately discovered US6428833 which describes a process for obtaining a freeze-dried coffee extract in pellet form. This process had a number of advantages, but was also associated with a number of deficiencies which the inventors then sought to address.

[0025] In more detail, US6428833 describes a process in which coffee extract with a 35 to 45wt% solids content is foamed to a foam weight of 450 to 750 g / l and then dropped onto a cooled belt for the formation of pellets. The pellets are frozen on the belt to temperatures below minus 30eC and then freeze-dried. The product is preferably dropped onto the cooled belt in quantities such that, after freezing, pellets with a diameter of 4 to 7 mm, preferably approximately 6 mm, and with a height of 2.5 to 3.5 mm, preferably 3 mm are obtained.

[0026] The US6428833 method has a number of advantages over conventional freeze-drying processes. Clearly, the process avoids the formation of fines and avoids the need for a grinding step. The pellets dropped onto the cooled belt can be frozen to temperatures between -30 and -40eC within 2 to 3 minutes. In comparison with conventional processes, an approximately 10-fold saving in time is consequently achieved in this stage. Since the product does not have to be comminuted, and therefore no frictional heat is generated, it is sufficient to freeze the pellets at -30 to -40eC. Energy is therefore also saved in this stage. Overall, therefore, this method provides considerable energy savings.

[0027] US6428833 provides comparative examples comparing the benefits of the process over a conventional freeze-drying approach. These examples look at three different qualities of coffee A-C. In each instance the new pellets are formed in only a few minutes. They are cooled to a warmer temperature than is conventional and there are no fines produced. In this way the product is produced with a much lower energy cost.

[0028] US6428833 is aware of concerns relating to the colour of the product obtained. It discusses how the colour of the product can be controlled by having a staged vacuum drying time. Firstly an initial drying time to form a shell and a controlled pressure profile. This is said to give “an attractive appearance” and if the pressure is too low (below 0.8mbar) then “the pellets acquire a light or mottled appearance”. The inventors understand that such drying conditions cause colour darkening via slightly collapsing the coffee. Collapse is, however, not desired since it results in the loss of a consistent internal structure and thereby could reduce dissolution rate in the final product

[0029] When the inventors reproduced the method of US6428833 using a modern drying profile, however, they found that the product appearance was overly light. Rather than a conventional dark coffee colour, the pellets were light, creamy or beige in colour. This did not meet with consumer expectations. Furthermore, the disclosed drying process at higher pressures (less reduced) gives rise to a significant loss of aroma.

[0030] In developing an approach to providing a pellet which overcame these defects, the inventors discovered that the same process steps could be applied back to conventional freeze-dried coffee product. This provided a small process change with a discernible benefit to both the darker product colour (associated with a consumer perception of a richer coffee flavour) and also improved aroma.

[0031] Accordingly, the inventors arrived at a method for the manufacture of a freeze-dried coffee powder. The freeze-dried coffee powder is an instant coffee powder, such that on addition of hot water a coffee beverage is reconstituted. The instant coffee powder is primarily formed from soluble coffee solids which dissolve on reconstitution. However, when the product also comprises significant amounts of insoluble coffee oil and / or insoluble roast and ground coffee particles, which disperse, the term instant coffee is more appropriate.

[0032] Preferably the plurality of frozen coffee granules has a particle distribution having a D90 of less than 10 mm, preferably less than 5 mm, more preferably less than 3 mm. This is a normal particle size distribution for a freeze-dried coffee, but it distinguishes over alternative product formats such as tablets.

[0033] The method comprises a first step (i) of providing an aqueous coffee extract comprising at least 40 wt.% soluble coffee solids. Preferably the aqueous coffee extract has 45 to 65 wt.% soluble coffee solids, preferably 48 to 52 wt.% soluble coffee solids. The higher the solids content the less water needs to be removed during freeze-drying. However, at very high solids there are associated difficulties with the preceding foaming and cooling steps. The method comprises a further step of (ii) foaming and cooling the aqueous coffee extract to form a foamed viscous coffee extract. This step may involve combined foaming and cooling, so the foaming and cooling may be performed sequentially in either order.

[0034] Coffee foaming processes are well known in the art and any suitable process may be used. This includes gas injection into the extract and also systems which simultaneously foam and produce ice (e.g. scraped surface heat exchanger). The intention of the foaming step is to affect the density of the coffee extract and hence the bubble structure of the finished product. One approach to foaming is to introduce a gas under pressure into the pumped coffee extract. The pressurised coffee extract can then be depressurised with associated foaming and bubble formation before the following steps.

[0035] A suitable pressure for the extract into which the gas is added is from 10 to 400 Bar, preferably 20 to 150 Bar and most preferably 30 to 50 Bar. Suitable gases for addition include nitrogen and carbon dioxide. Preferably the foamed coffee extract has a density of from 400 to 900g / L, preferably 650 to 750g / L.

[0036] The method comprises a further step of (iii) freezing the foamed viscous coffee extract to form a slab of frozen coffee. This may be achieved by loading the extract onto trays and then freezing the trays in a cold room. Preferably step iii) comprises extruding the viscous coffee extract onto a freezing belt or into freezing trays. The freezing step is performed to cool the coffee extract from its supplied temperature (typically +10 to -5eC) and cooling it to a temperature where it is entirely frozen, suitable for fragmenting. Suitable target temperatures are at most -30eC, preferably at most -40eC and most preferably -35 to -55eC. The temperature has to be at most -30eC and preferably colder such that the energy introduced during fragmentation does not lead to localised heating and melting of the coffee.

[0037] The method comprises a further step of (iv) fragmenting the slab of frozen coffee to form a plurality of frozen coffee granules. The fragmentation may be performed using conventional machinery and is generally followed by a sieving or sifting step to recover and recycle fines material. The fragmentation increases the surface area for ready sublimation drying, and also gives the characteristic angular form of freeze-dried coffee granules. Fragmentation may involve milling or grinding the coffee. The fragmentation is typically performed in a cold room to ensure that the product temperature does not increase. The fragmentation provides a large and consistent surface area which then benefits from the annealing step to darken the powder appearance. The method comprises a further step of (v) subliming water from the plurality of frozen coffee granules to form a freeze-dried coffee powder. Preferably step v) is performed with an initial temperature of less than -40 °C, most preferably -40 to -55eC. This step is performed until the suitably dry product is obtained.

[0038] All of the foregoing steps are conventional in the production of a freeze-dried coffee powder. The inventors new process requires that the process further involves a low temperature annealing step after fragmentation and before sublimation, whereby the frozen coffee granules remain within a temperature range of from -15 to -30eC for at least 20 minutes. This step was found to provide improved aroma and a more desirable darker colour. Critically, this is after the fragmentation step which is performed at a colder temperature, so it is required that the fragments are allowed to warm to the preferred temperature range. For some warmer temperatures it was found that the benefits could still be achieved, provided annealing temperatures were long enough, such as a temperature of from -12 to -30eC for at least 75 minutes.

[0039] Preferably the low temperature annealing step is performed at a temperature of from -15 °C to -28 °C, preferably -18 to -25eC. Preferably the low temperature annealing step is within a temperature range of from -15 to -30eC and performed for at least 30 minutes, preferably from 30 minutes to 5 hours, more preferably 1 to 3 hours. When the temperature is from -12 to -30eC, the annealing step is from 75 minutes to 3 hours, preferably 2 to 3 hours.

[0040] Preferably the low temperature annealing step is performed at a temperature at least 10eC, preferably at least 15eC, warmer than the freezing temperature achieved in step (iii). An exemplary freezing profile is shown in Figure 1.

[0041] Preferably the low temperature annealing step is performed at a substantially constant temperature. For example, preferably the temperature during the holding step does not deviate from a target value by more than ±5eC throughout the holding step, preferably less than ±3eC and more preferably less than ±1eC. For the avoidance of doubt, during this holding time the product is preferably not under pressure or under vacuum, since this holding step is performed before the lyophilisation begins. Rather, the holding step is preferably at atmospheric pressure.

[0042] It is preferred that there is a single annealing step That is, there is a single step in which the frozen coffee body is allowed to warm before having the water sublimed away, rather than having the temperature of the frozen coffee repeatedly cycled. During the annealing step the frozen coffee typically remains on the trays or on a belt onto which it is loaded following fragmentation. The frozen coffee is preferably not subjected to any additional agitation or coating steps. In this way the surface of the final powder consists of coffee from the coffee extract.

[0043] The key part of this process is that there is an annealing step performed in the above discussed window on the fragmented particles. It is known that slow-cooling around the freezing point of a coffee extract can help the formation of larger crystals and this is discussed at length in EP3448166. As explained therein, it is critical that for a four degree Celsius window, extending from 1eC above the freezing point to 3eC below the freezing point, the freezing rate is very slow. This window preferably takes at least 20 minutes and, thereafter, the Patent teaches that the cooling can be much faster down to a suitable freeze- drying start temperature. Indeed, it specifically contemplates fast cooling out of the window as no further benefits were perceived.

[0044] In EP3448166 this slow cooling is performed to provide a product with a reduced internal connectivity and hence produce a foaming product. This is very different to the present case, where the slow cooling is performed to change the product colour (and in a different target temperature range). Specifically, it appears that the annealing at this colder temperature has a particular effect on the surface ice-crystal structure, such that the colour of the product is especially affected. This is why the process has to be performed on the exposed particle surfaces after fragmentation.

[0045] We note that the cooling profile of the other steps, i.e. pre-fragmentation and after the herein-described annealing step can be performed as desired. Preferably the cooling rate after the annealing step involves fast cooling to the temperature specified for grinding and sublimation (preferably -35 to 55eC). The temperature profile before the annealing can be as described in EP3448166 or may be a faster cooling rate, as is conventional.

[0046] It is preferred that the coffee product comprises up to 15wt% of roast and ground coffee additive, preferably from 1 to 10wt% and most preferably 3 to 5wt%. These would contribute to the coffee solids in the final product. A preferred roast and ground coffee additive is a microground coffee which is well known in the art. Examples of products with microground coffee include Millicano. A general definition of a microground roasted coffee is one having a D50 of less than 40 pm and a D90 of less than 80 pm. The presence of such an additive improves the flavour and aroma and gives a higher perceived product quality. It is preferred that the roast and ground coffee is homogeneously distributed within the coffee product particles (i.e. within a matrix of soluble coffee material).

[0047] Without wishing to be bound by theory, it is believed that the colour of the freeze-dried product is determined by its surface structure and, specifically in this case, the surface pore configuration. It is well known in the art that the colour of freeze-dried coffee can be changed by foaming the coffee extract before drying. An increased number of larger surface pores gives rise to a darker coloured product via a light scattering phenomenon. It is generally understood that the surface of conventional freeze-dried granules are dominated by pores derived from gas bubbles and this is why changing the foam structure is generally used to achieve darker powders.

[0048] The inventors have now found that they can also affect the surface appearance by controlling the ice-crystal surface pores on the freeze-dried coffee. Whereas ice pores are generally smaller than the gas bubbles in the product and therefore less involved in the light scattering, the inventors have found that a slow cooling or annealing step can help the ice crystals grow and mature such that there is a meaningful effect on the colour. Without wishing to be bound by theory, it is considered that this is especially true in high concentration systems where less water is present to become ice.

[0049] The inventors have found that annealing the ice in fragmented frozen coffee sheets has a colour impact. The benefits of the invention are particularly observed when forming a product from a higher-solids concentrate (i.e. less ice present) which is desirable to reduce energy costs. Typically the annealing can lead to a reduction in the colour (as measured in La) of at least 2, or at least 3 and typically at least 4 La. La colour measurements can be performed using a Dr Lange LK 100 unit (Hach-Lange GmbH). The standard operating procedure of the unit involves spreading a layer of the sample flat in a standard sample holder before measurement.

[0050] The inventors have now found that the surface ice-crystal structure, and hence the final product colour, can be best controlled by having a holding step performed on the already- frozen and fragmented product. The holding temperature is within a temperature window colder than that taught in EP3448166 and it is surprising that this is key to providing the desired appearance. The inventors have found that performing the cold-annealing step allows the provision of a product that is darker than normally achievable using a conventional freeze-drying, avoiding the need for new equipment or a change in procedure. Furthermore, there is an improved aroma content. According to a preferred embodiment, the method comprises:

[0051] (i) providing an aqueous coffee extract comprising at 45-55 wt.% soluble coffee solids;

[0052] (ii) foaming and cooling the aqueous coffee extract to form a foamed viscous coffee extract;

[0053] (iii) freezing the foamed viscous coffee extract to form a slab of frozen coffee at a temperature of at most -40eC;

[0054] (iv) fragmenting the slab of frozen coffee to form a plurality of frozen coffee granules;

[0055] (v), low temperature annealing the frozen coffee granules at a temperature within a range of from -15 to -30eC for at least 20 minutes, preferably 30 minutes to 2 hours, and

[0056] (v) subliming water from the plurality of annealed frozen coffee granules to form a freeze-dried coffee powder.

[0057] Figures

[0058] The invention will now be described further in relation to the following non-limiting figures, in which:

[0059] Figure 1 shows an illustrative temperature profile during the cooling of the present invention.

[0060] Examples

[0061] The invention will now be described further with reference to the following non-limiting examples.

[0062] Example 1

[0063] Concentrated coffee extract (48 wt%) was aerated and frozen to -7eC simultaneously in a continuous freezer. This produced a semi-liquid frozen slurry which was loaded into trays. These trays were placed into a cold room at -50eC until fully frozen. After some time, the frozen coffee within the trays was removed, broken up by granulation and sieved to remove fine dust. The frozen granules were separated. These were in the shape of typical freeze- dried instant coffee although were still frozen. The frozen granules were loaded into metal trays suitable for freeze drying. Some of the loaded trays containing frozen granules were placed in a cabinet freezer at a fixed temperature of -20eC to facilitate annealing. Samples were removed from the cabinet freezer periodically. After removal from the cabinet freezer, the sample trays were returned the -50eC cold room to arrest the annealing process within the granules. Samples were taken from the cabinet freezer after 30, 60 and 90 minutes respectively.

[0064] Afterwards, the 3 trays containing frozen granules that had been annealed for different durations were loaded into a freeze dryer. At the same time, a reference tray containing frozen granules kept at -50eC (having experienced no annealing) was loaded to the same dryer. The dryer was then operated.

[0065] The dried products were then analysed for colour, moisture and solubility in hot water. Results are given below.

[0066] The annealing process at -20eC was consistent with darkened product colour (reduced La). Moisture and solubility remained acceptable.

[0067] Example 2

[0068] The method described in Example 1 was repeated with an annealing temperature of -12eC rather than -20eC. Samples annealed for 30, 60 and 90 minutes respectively plus a reference (which was kept at -50eC) were dried and analysed analogously to those in Example 1 . Results are shown in the table below.

[0069] Again, the annealed samples were darker in colour than the non-annealed reference. While moisture was consistently acceptable, solubility in some samples did not meet the target criteria. This trial was considered less successful therefore than that in Example 1 .

[0070] The term “comprising” as used herein can be exchanged for the definitions “consisting essentially of” or “consisting of”. The term “comprising” is intended to mean that the named elements are essential, but other elements may be added and still form a construct within the scope of the claim. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting of” closes the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith.

[0071] The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.

[0072] For the avoidance of doubt, the entire contents of all documents acknowledged herein are incorporated herein by reference.

Claims

Claims1 . A method for the manufacture of a freeze-dried coffee powder, the method comprising:(i) providing an aqueous coffee extract comprising at least 40 wt.% soluble coffee solids;(ii) foaming and cooling the aqueous coffee extract to form a foamed viscous coffee extract;(iii) freezing the foamed viscous coffee extract to form a slab of frozen coffee;(iv) fragmenting the slab of frozen coffee to form a plurality of frozen coffee granules; and(v) subliming water from the plurality of frozen coffee granules to form a freeze-dried coffee powder, wherein, after step (iv) and before step (v), the method comprises a low temperature annealing step whereby the frozen coffee granules remain within a temperature range of from -15 to -30eC for at least 20 minutes, or within a temperature range of from -12 to -30eC for at least 75 minutes.

2. The method according to claim 1 , wherein the aqueous coffee extract has 45 to 65 wt.% soluble coffee solids, preferably 48 to 52 wt.% soluble coffee solids.

3. The method according to claim 1 or claim 2, wherein the foamed coffee extract has a density of from 400 to 900 g / L, preferably 650 to 750 g / L.

4. The method according to any preceding claim, wherein the coffee extract is foamed in step (ii) with nitrogen and / or carbon dioxide.

5. The method according to any preceding claim, wherein step iii) comprises extruding the viscous coffee extract onto a freezing belt.

6. The method according to any preceding claim, wherein step iv) is performed with an initial temperature of -30 to -40 °C, preferably -35 to -40 °C.

7. The method according to any preceding claim, wherein step v) is performed with an initial temperature of -30 to -50 °C, preferably -40 to -50 °C.

8. The method according to any preceding claim, wherein the plurality of frozen coffee granules has a particle distribution having a D90 of less than 10 mm, preferably less than 5 mm, more preferably less than 3 mm.

9. The method according to any preceding claim, wherein the low temperature annealing step is performed at a temperature of from -15 °C to -28 °C, preferably -18 to - 25eC.

10. The method according to any preceding claim, wherein the low temperature annealing step is performed at a temperature of from -15 °C to -30 °C and is performed for at least 30 minutes, preferably from 30 minutes to 5 hours, more preferably 1 to 2 hours.11 . The method according to any preceding claim, wherein the low temperature annealing step is performed at a temperature at least 10eC, preferably at least 15eC, warmer than the freezing temperature achieved in step (iii).

12. The method according to any preceding claim, wherein the low temperature annealing step is performed at a constant temperature and preferably the method consists of a single said annealing step.

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