Preservation of emulsions-based products with freeze-thaw stability

Abstract

The present disclosure provides methods for maintaining freeze-thaw stability of emulsion-based products during preservation at subfreezing temperature and subsequent warming. The method includes isochoric freezing of an emulsion-based product in a fluid using an isochoric chamber at a subfreezing temperature over a preservation time, where the emulsion-based product maintains freeze-thaw stability during the preservation at the subfreezing temperature. Devices for performing the methods are also provided.

Description

Atty. Dkt: BERK-539WOPRESERVATION OF EMULSIONS-BASED PRODUCTS WITH FREEZE-THAW STABILITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 63 / 695,209, filed September 16, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Number 59-2030-3-001 awarded by the United States Department of Agriculture. The government has certain rights in the invention.TECHNICAL BACKGROUND

[0003] Food preservation is fundamental to addressing the global challenge of food wastage, as food begins to spoil as soon as it is harvested. In particular, the primary goal of preservation of emulsion-based products is to maintain physical stability of such products, thereby extending shelf life and minimizing waste.

[0004] The most common method to preserve emulsions-based products, where emulsion stability cannot be maintained during freezing, is by refrigeration. However, the shelf-life of refrigerated products is much shorter than that of frozen products since the rate of deterioration in quality over time is temperature dependent.

[0005] Attempts have been made to increase the stability of frozen emulsions. For instance, various stabilizers and stabilizer combinations have been added to the foods. The drawback of this approach is that stabilizers frequently are required in such great quantities that the feel of the product in the mouth is altered. Another approach has been to use a frozen concentrate, which serves as a base to which a liquid such as water is added with agitation. Still another approach has been to aseptically prepare the emulsions, which can then be consumed afterAtty. Dkt: BERK-539WO being chilled, shaken and opened. These prior approaches required a reconstitution step that is either difficult or time-consuming.SUMMARY

[0006] Aspects of the present disclosure provide a method for maintaining freeze-thaw stability of an emulsion-based product during preservation. More specifically, the method comprises i) placing an emulsion-based product in a fluid in an isochoric chamber; ii) isochoric freezing the fluid in the isochoric chamber at subfreezing temperature over a preservation time. In some embodiments, the method further comprises warming the fluid in the isochoric chamber above 0 °C. Through the isochoric freezing preservation, the emulsion-based product can maintain freeze-thaw stability during the preservation at the subfreezing temperature and subsequent warming. It is the object of the present disclosure to provide a process or device for preserving native emulsion-based products with freeze-thaw stability to extend the shelflife of such products. The ability to preserve stable food emulsions at subfreezing temperatures will reduce food waste and increase access to national and international markets. The long-term storage advantages of frozen emulsion-based products would also expand production planning options. For example, manufacturing could be scheduled when ingredients are accessible and / or are in season and are thus less expensive.

[0007] Aspects of the present disclosure also provide a device for preserving an emulsionbased product at subfreezing temperature while maintaining freeze-thaw stability of the emulsion-based product. The device comprises an isochoric chamber filled with a fluid containing an emulsion-based product and a temperature control system configured to cool the fluid in the isochoric chamber at subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C.

[0008] In some embodiments, the subfreezing temperature is in the range of from 0 °C to triple point temperature of an aqueous solution. In other embodiments, the subfreezing temperature is in the range of from about -5 °C to about -20 °C.

[0009] In some embodiments, the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa. In other embodiments, the isochoric freezing is at a pressure in the range of about 50 MPa to about 180 MPa.Atty. Dkt: BERK-539WO

[0010] In some embodiments, the method further comprises warming the fluid in the isochoric chamber above 0 °C. In some embodiments, the warming comprises increasing the temperature of the fluid in the isochoric chamber to above 0 °C to about 5 °C.

[0011] In some embodiments, the fluid is water or an aqueous solution.

[0012] In some embodiments, the emulsion-based product is directly placed in the chamber. In other embodiments, the emulsion-based product is placed in a matter container.

[0013] In some embodiments, the isochoric chamber contains a nucleating agent.

[0014] In some embodiments, the emulsion-based product is an emulsion-based food, an emulsion-based pharmaceutical product, or an emulsion-based cosmetic.

[0015] In certain embodiments, the emulsion-based food is selected from a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, and a puree.

[0016] In some embodiments, the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation. In some cases, the medicine produced by emulsion formulation is formulated for oral use, ocular use, topical mucosal use, intramuscular use, or intravenous use. In some cases, the emulsion-based pharmaceutical product is selected from a pharmaceutical cream, a pharmaceutical lotion, a pharmaceutical balm, and a pharmaceutical ointment.

[0017] In some embodiments, the emulsion-based pharmaceutical product is an emulsionbased vaccine. For example, the emulsion-based vaccine is, but not limited to, Novartis's MF59®.

[0018] In some embodiments, the emulsion-based cosmetic product is selected from a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.

[0019] In some embodiments, the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size, the emulsion-based productAtty. Dkt: BERK-539WO during and after isochoric freezing preservation maintains visual appearance, viscosity, color changes, and / or fat globule size.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows a temperature-pressure phase diagram for water and comparison of path of freezing in an isochoric system and an isobaric system.

[0021] FIG. 2 shows the plot of the percentage of ice during isochoric freezing, more specifically the percentage of ice in an isochoric system as the temperature is lowered to the triple point.

[0022] FIG. 3 illustrates a typical isochoric chamber for isochoric freezing.

[0023] FIG. 4 shows how a biological matter can be preserved at subfreezing temperatures while avoiding the formation of ice. As shown in FIG. 4, it is possible to reduce the temperature in isochoric freezing while avoiding damage from the formation of ice by placing the matter to be preserved in the unfrozen part of the volume.

[0024] FIG. 5 shows an isochoric freezing configuration with an isochoric chamber and a matter container for biological matter preservation.

[0025] FIG. 6 depicts the isochoric system comprising an isochoric chamber that is enclosed with a sealing cap. The isochoric chamber can be made of grade 7075 aluminum with a total volume capacity of 2 liters and pressure-rated for up to 110 MPa. The chamber can be connected to an electronic pressure transducer to monitor the pressure inside the chamber. A safety head with a rupture disk is in fluid communication with the interior of the isochoric chamber to ensure that the conditions inside the chamber do not exceed safety standards.

[0026] FIG. 7 shows a conventional freezing process oil in water emulsion.

[0027] FIG. 8 shows the size distribution of fat globules from dairy creams.

[0028] FIG. 9 shows appearance of dairy cream after refrigeration (RF), isochoric freezing (IF) and conventional freezing (CF) for 14 days.

[0029] FIG. 10 shows (A) appearance of conventional frozen cream and (B) isochoric frozen cream after 14 days of preservation.

[0030] FIG. 11 shows CSLM-micrographs of dairy cream, more specifically, (A) CSLM- micrograph of fresh cream, (B) CSLM-micrograph of refrigerated cream at 5 °C, (C) showsAtty. Dkt: BERK-539WOCSLM-micrograph of isochoric frozen cream, and (D) CSLM-micrograph of conventional frozen cream. The fat phase is shown in red, and the protein-rich serum phase is shown in green.

[0031] FIG. 12 shows effects of preservation conditions on apparent viscosity of dairy cream.

[0032] FIGs. 13A-13F show effects of preservation conditions on color parameters of dairy cream.

[0033] FIG. 14 shows (A) fresh cream cheese, (B) isochoric frozen at -15 °C / 150 MPa for one week, and (C) conventionally frozen at -15 °C for one week.

[0034] FIG. 15 shows (A) fresh creamy tomato soup, (B) isochoric frozen creamy tomato soup at -15 °C / 150 MPa for one week, and (C) conventionally frozen creamy tomato soup at -15 °C for one week.

[0035] FIG. 16 shows appearance of sunflower oil in water emulsion after isochoric freezing (IF) and conventional freezing (CF) for 50 days and appearance of fresh control.

[0036] FIG. 17 shows CSLM-micrographs of sunflower oil in water emulsion, more specifically, (A)(B) CSLM-micrograph of fresh control, (C)-(F) CSLM-micrograph of isochoric frozen emulsion, and (G)-(J) CSLM-micrograph of conventional frozen emulsion. The oil phase is shown in red.

[0037] FIG. 18 shows viscosity of sunflower oil in water emulsion after isochoric freezing (IF) and conventional freezing (CF) and viscosity of fresh control.DETAILED DESCRIPTION1. Introduction

[0038] The present disclosure involves emulsions-based products with physical stability during preservation at subfreezing temperatures and subsequent warming. Freeze-thaw stability is an important attribute for many emulsions-based products that could be frozen to extend their shelf-life. Many types of foods are in the form of oil-in-water emulsions, including milks, creams, dressings and sauces, or contain emulsion-based products, such as prepared dishes containing cream or cheese. Some of these foods could benefit from frozen storage to increase their shelf life.

[0039] However, freeze-thaw destabilization of emulsion-based food products has been a major impediment to freezing and seriously hinders the development and sales of frozen foodAtty. Dkt: BERK-539WO products in the market. Emulsion-based foods are highly susceptible to physical instability during the freezing and thawing process. The thawed emulsion can very often be significantly destabilized and completely broken down into an oily and an aqueous phase (e.g., oil on the surface of a defrosted cheese sauce). The mechanism of destabilization has been attributed to the freezing conditions and the difference between the crystallization temperatures of the oil and water phases (Degner et al., 2014). If the oil phase crystallizes before the water phase, destabilization occurs due to partial coalescence. In this process, a crystallized fat droplet collides with another fat droplet and penetrates the fluid region of the second droplet. The liquid from the damaged droplet then flows out and wets the solid fat crystal, creating a linkage between both droplets. This process can be intensified once water crystallizes, and the semisolid crystalline oil droplets are crowded due to exclusion from the space taken by the ice. If the water phase crystallizes before the oil phase, the amount of ice crystals gradually increases in number and the oil droplets become increasingly concentrated into the nonfrozen aqueous phase. The oil droplets begin to come into close contact with each other and lead to droplet flocculation and coalescence, and eventual oiling-off during the thawing process.(FIG. 7) This destabilization involves friction between the emulsion droplets and the rupture of the membranes surrounding individual droplets, allowing some oil-to-oil contact. In addition, other mechanisms can also contribute to emulsion destabilization during freezing and thawing, such as recrystallization and destabilization of the protein adsorption film on the droplets, which results in less effective stabilization of the emulsion.

[0040] A freezing technology, named isochoric freezing, allows the preservation of food products at subfreezing temperatures without ice formation inside the products (Rubinsky et aL, 1995). Therefore, the use of isochoric freezing eliminates the physical destabilization of emulsion-based foods due to ice formation.

[0041] In isochoric freezing, the packed food product is placed inside a rigid chamber filled with a liquid solution, which can just be water, and brought to subfreezing temperatures inside the closed chamber. During freezing, ice forms in the external liquid solution and expands in volume, increasing the pressure inside the closed chamber. However, due to Le Chatelier's principle, part of the volume remains unfrozen. The chamber is designed so the food productAtty. Dkt: BERK-539WO remains in the non-frozen section and the food is preserved at subfreezing temperatures with no internal ice formation.2. Definitions

[0042] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0045] As used herein, the term "isochoric freezing" refers to the freezing of aqueous solutions in a constant volume chamber or in a rigid chamber. In isochoric freezing, the phase transition encompasses the entire volume when cooling below the liquidus line and a substantial portion of the volume remains unfrozen to the triple point temperatures and pressures and is associated with an increase in pressure.Atty. Dkt: BERK-539WO

[0046] As used herein, the term "isobaric freezing" refers to a freezing process at constant pressure. In isobaric freezing, the entire system freezes as soon as the temperature is below the liquidus line intersection with the constant pressure line.

[0047] As used herein, the term "emulsion-based product" refers to any product that exists in an emulsified form, including many dressings, sauces, spreads, dips, creams, and beverages. The emulsion-based product may be, but not limited to, emulsion-based food, medicine produced by emulsion formulation, emulsion-based vaccine, or cosmetic.

[0048] As used herein, the term "emulsion" refers to colloidal dispersions that consist of at least two immiscible fluids (normally water and oil), with one of them being dispersed in the other in the form of small droplets. The principles of emulsion science and technology are commonly employed in the food industry to create a wide variety of emulsified food products, such as beverages, milks, creams, dips, sauces, deserts, dressings, mayonnaise. The nature of emulsions confers these foods with distinct functional attributes, such as desirable appearances, textures, mouthfeels, and flavor profiles.

[0049] Herein, the term "pharmaceutical cream", "pharmaceutical lotion", "pharmaceutical balm", "pharmaceutical ointment" refers to any kind emulsion-based medicine for medical use. The terms "pharmaceutical cream", "pharmaceutical lotion", "pharmaceutical balm", "pharmaceutical ointment" are interchangeably used as "medicated cream", "medicated lotion", "medicated balm", "medicated ointment". The "pharmaceutical cream", "pharmaceutical lotion", "pharmaceutical balm", "pharmaceutical ointment" are used as a medication or pharmaceutically used for treatment or prevention.

[0050] As used herein, the term "freeze-thaw stability" refers to a measure of how well a product can withstand freeze-thaw cycles, without deteriorating. The freeze-thaw stability can be determined by measuring changes of visual appearance, viscosity, color, and / or fat globule size. Emulsion-based foods such as sauces and some beverages need to be frozen before consumption, and their stability is affected by factors like ingredients, homogenization, and freezing and thawing conditions. Freeze-thaw stability can help ensure that a product such as pharmaceuticals is stable enough for preservation or transport.

[0051] The use of the terms "a," "an," and "the," and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construedAtty. Dkt: BERK-539WO to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the embodiments of the disclosure.

[0052] As used herein, the term "about" used in connection with an amount indicates that the amount can vary by 10 % of the stated amount. For example, "about 100" means an amount of from 90-110. Where about is used in the context of a range, the "about" used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and "about" used in reference to the higher amount of the range means that the higher amount includes an amount 10 % higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0053] The term "and / or" as used herein a phrase such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used herein a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.Atty. Dkt: BERK-539WO

[0055] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of this disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to this disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0056] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.3. Method for maintaining freeze-thaw stability of emulsion-based product during preservation

[0057] Aspects of the present disclosure provide a method for maintaining freeze-thaw stability of an emulsion-based product during preservation at subfreezing temperatures and subsequent warming. As such, the present disclosure provides a method for preserving native emulsion-based products with freeze-thaw stability to extend the shelf-life of such products. The ability to preserve stable food emulsions at subfreezing temperatures will reduce food waste and increase access to national and international markets. The long-term storage advantages of frozen emulsion-based products would also expand production planning options. For example, manufacturing could be scheduled when ingredients are accessible and / or are in season and are thus less expensive. In order to maintain freeze-thaw stability of the emulsion-based product, the emulsion-based product is preserved under isochoric freezing conditions.

[0058] In some aspects, the method of the present disclosure comprises i) placing an emulsion-based product in a fluid in an isochoric chamber and ii) isochoric freezing to the fluidAtty. Dkt: BERK-539WO in the isochoric chamber at subfreezing temperature over a preservation time. In some embodiments, the method of the present disclosure further comprises iii) warming the fluid in the isochoric chamber above 0 °C. The emulsion-based product maintains freeze-thaw stability during the preservation at the subfreezing temperature and subsequent warming.3.1. Isochoric freezing for preservation

[0059] The isochoric freezing technique is described in US2007 / 0042337A1 and PCT / US24 / 33896, which are incorporated by reference herein.

[0060] FIG. 1 illustrates the difference between isobaric freezing and isochoric freezing in a temperature-pressure phase diagram for water. It shows the triple point for water after which at lower temperatures, there is no liquid water. It also shows that at constant pressure, as soon as the temperature is below the liquidus line intersection with the constant pressure line, the entire system freezes. In contrast, in an isochoric system, pressure and temperature are interrelated through the liquidus curve in the phase diagram of the aqueous solution. Consequently, only part of the water can freeze to become ice at thermodynamic equilibrium under the pressure and constant volume conditions. This avoids the formation of ice crystals inside the biological matter if the biological matter remains in the unfrozen region. Therefore, isochoric freezing relies on pressure to avoid ice formation inside the preserved biological matter at subfreezing temperatures. See, e.g., Rubinsky, B., Perez, P.A. and Carlson, M.E., "The thermodynamic principles of isochoric cryopreservation", 2005, Cryobiology, 50(2), pp.121- 138, which is incorporated by reference herein.

[0061] In the method of the present disclosure, isochoric freezing relies on Le Chatelier's principle, which states that as water expands upon freezing within a constant volume chamber (isochoric chamber), the enhanced pressures generated hinder further formation of ice. Therefore, the freezing process in an isochoric system follows the liquidus curve in the phase diagram for water depicted in FIG. 1. This minimizes the pressure for the given temperature and its potential harmful effects, such as impacting physicochemical and nutritional properties. In some embodiments, Le Chatelier's principle also allows the transformation of an aqueous liquid solution into two coexisting phases, a solid ice phase and a liquid phase, after lowering the temperature of the liquid solution to subfreezingAtty. Dkt: BERK-539WO temperatures. In this way, a biological matter such as a food product can be stored in the liquid phase inside the chamber at subfreezing temperatures without ice formation inside the products. This controlled freezing process allows food products to be placed within the liquid region of the chamber, preventing ice crystal formation and the according biophysical injury. For example, fruits and vegetables treated in this way can keep their original freshness with minimal changes in physicochemical and nutritional properties. Therefore, in some embodiments, isochoric freezing can help retain the freshness of the biological matter such as harvested fruits and vegetables without substantially impacting their physicochemical and nutritional properties.

[0062] Generally, one of the major mechanisms of damage during preservation of a biological matter in aqueous solutions is the formation of ice. FIG. 2 shows that a substantial percentage of the volume in a liquid state to the triple point and the percentage of ice in an isochoric system as the temperature is lowered to the triple point. Therefore, in isochoric freezing it is possible to reduce the temperature while avoiding damage from the formation of ice by placing the matter to be preserved in the unfrozen part of the volume.3.2. Placing an emulsion-based product in a fluid in an isochoric chamber

[0063] In some embodiments, an emulsion-based product refers to any product that exists in an emulsified form comprising, including many dressings, sauces, spreads, dips, creams, and beverages. In some embodiments, the emulsion-based product basically comprises mixture of oil, water and emulsifying agent. The emulsion-based product comprises an oil-in-water emulsion.

[0064] In some embodiments, the emulsion-based product may be emulsion-based food. In certain embodiments, the emulsion-based food includes, but is not limited to, a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, a puree.Atty. Dkt: BERK-539WO

[0065] In exemplary embodiments, an emulsified sauce includes, but is not limited to, a hollandaise sauce, a barbecue sauce, a ranch sauce, a mayonnaise, a bearnaise, an egg yolk sauce, and an aioli.

[0066] In exemplary embodiments, an emulsified dip includes, but is not limited to, ketchup, mayonnaise, hummus, and a cheddar dip.

[0067] In exemplary embodiments, an emulsified soup includes, but is not limited to, a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

[0068] In some embodiments, the emulsion-based product may be emulsion-based pharmaceutical product. In some cases, the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation. In some embodiments, the medicine produced by emulsion formulation is formulated for oral use, ocular use, topical mucosal use, intramuscular use or intravenous use. In some embodiments, the medicine produced by emulsion formulation contains emulsifiers to blend water-based and oil-based ingredients.

[0069] In exemplary embodiments, the medicine produced by emulsion formulation is, but not limited to, any kind of a pharmaceutical cream, a pharmaceutical lotion, a pharmaceutical balm, a pharmaceutical ointment. Herein, the term "pharmaceutical cream", "pharmaceutical lotion", "pharmaceutical balm", "pharmaceutical ointment" refers to any kind emulsion-based medicine for medical use and they are used as a medication or pharmaceutically used for treatment or prevention.

[0070] In exemplary embodiments, the medicine produced by emulsion formulation is, but not limited to, emulsion-based vaccine. For example, the emulsion-base vaccine is, but not limited to, Novartis's MF59®. The Novartis's MF59® has been licensed as part of the seasonal flu vaccine.

[0071] In some embodiments, the emulsion-based product may be a cosmetic. For example, the emulsion-based cosmetic includes, but is not limited to, a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.Atty. Dkt: BERK-539WO

[0072] In some aspects of the method, the emulsion-based product is placed in an isochoric chamber filled with an aqueous solution. The term "constant volume chamber" is used interchangeably herein with "isochoric chamber" or "rigid chamber". The isochoric freezing technique is described above, and US2007 / 0042337A1 and PCT / US24 / 33896 are incorporated by reference herein.

[0073] In the isochoric freezing, a substantial portion of the volume remains unfrozen which is associated with an increase in pressure. The process of isochoric freezing occurs along the liquidus line in a temperature-pressure phase diagram as the temperature is dropped because the volume in an isochoric chamber is constant.

[0074] In some aspects of the method, the emulsion-based product is placed inside the constant volume chamber filled with an aqueous solution in isochoric freezing. The constant volume chamber is filled with a fluid in such a way as to minimize the amount of air in the chamber. For example, FIG. 3 illustrates an exemplary isochoric chamber system for isochoric freezing. In FIG. 3, the isochoric chamber system comprises a constant volume chamber in pressure vessel 301, a pressure gauge 302, and a rupture disk 303. The constant volume chamber in pressure vessel 301 is seen in cross-sectional view 304. The constant volume chamber in pressure vessel 301 is preferably hermetically sealed, and the pressure therein is monitored with pressure gage 302. Optionally, the constant volume chamber in pressure vessel 301 can be made of stainless steel, but the present invention is not limited. In some embodiments, the isochoric chamber can be also a closed chamber with rigid walls. The constant volume chamber in pressure vessel 301 is filled with fluid.

[0075] In some embodiments, the fluid in the constant volume chamber is aqueous solution or pure water. In the isochoric freezing, if ice nucleates in the constant volume chamber, the pressure of the system increases, and the nucleation can be detected by the pressure gauge. For example, U.S. Patent Publication No. 2007-0042337A1, incorporated by reference herein, which discusses isochoric freezing system and method for cryopreservation of a biological sample.

[0076] In some embodiments, an emulsion-based product can be directly placed in the isochoric chamber. FIG. 4 illustrates an exemplary embodiment of isochoric freezing configuration with an isochoric chamber for preserving a biological mass while avoiding theAtty. Dkt: BERK-539WO formation of ice. In other embodiments, an emulsion-based product can be placed in a container that allows transfer of pressure mass and heat. In still other embodiments, an emulsion-based product can be placed in a container that allows transfer of only pressure and heat but not mass. FIG. 5 illustrates an exemplary embodiment of isochoric freezing configuration with an isochoric chamber and a matter container for preserving a biological mass while avoiding the formation of ice. This is described in PCT / US24 / 33896 and is incorporated by reference herein.

[0077] As generally shown in FIG. 6, the isochoric system comprises an isochoric chamber that is enclosed with a sealing cap. The isochoric chamber can be made of grade 7075 aluminum with a total volume capacity of 2 liters and pressure-rated for up to 110 MPa. The chamber can be connected to an electronic pressure transducer to monitor the pressure inside the chamber. A safety head with a rupture disk is in fluid communication with the interior of the isochoric chamber to ensure that the conditions inside the chamber do not exceed safety standards. This is described in PCT / US24 / 33896 and is incorporated by reference herein.

[0078] In some embodiments, an emulsion-based product may be collected in an impermeable but flexible container. For example, the emulsion-based product is collected in a polyethylene sterile bag. The bag is then sealed with negligible headspace and placed inside the isochoric chamber.

[0079] In some embodiments, the fluid in the constant volume chamber is aqueous solution or pure water with or without organic molecules therein. In some embodiments, the aqueous solution that is primarily water may contain other chemicals so that the freezing point of the water-based solution may be modified as required for a specific application. For example, the water-based solution may contain salt. In certain embodiments, the external solution is water. In some embodiments, the fluid in the container can be the same as the fluid in the isochoric chamber surrounding the container. In other embodiments, the fluid in the matter container can be different from the fluid in the isochoric chamber surrounding the matter container. In other words, the fluid inside the matter container and the fluid outside the matter container can be of different types in osmotic equilibrium with the preserved emulsion-based product. In some embodiments the isochoric chamber is filled with a fluid that does not freeze at the storage temperature, such as a solution of glycerol.Atty. Dkt: BERK-539WO

[0080] In some embodiments, the isochoric chamber contains a nucleating agent. The nucleating agent forms an ice crystal, thereby the emulsion-based product can be preserved in the unfrozen part of the volume. The ice crystal formed by the nucleating agent generates pressures of the isochoric chamber, and the enhanced pressures hinder further formation of ice. In some embodiments, the nucleating agent can be a structural element of the isochoric freezing device. For example, the nucleating agent can be walls of the isochoric chamber itself, or walls of the matter container itself. In other embodiments, the nucleating agent can be any agent that promotes the formation of ice crystal in the isochoric chamber. For example, the ice nucleating agent is, but not limited to, minute solid particles, such as dust or food particles, large molecules, ice-nucleating proteins. In some embodiments, the nucleating agent is placed in the bottom of the isochoric chamber, and the chamber is completely filled with an aqueous solution. In some embodiments, an emulsion-based product is placed in a container in an isochoric chamber that allows transfer of only pressure and heat but not mass and the isochoric chamber is filled with a fluid and nucleation agents that cause the fluid to freeze before the emulsion-based product in the container freezes.3.3. Isochoric freezing the fluid in the isochoric chamber at subfreezing temperature over a preservation time

[0081] After the chamber is prepared and loaded, the chamber is cooled to subfreezing temperatures over a preservation time described in the present disclosure, preferably in a conventional freezer. Once the emulsion-based product is needed, the chamber is gradually warmed above the freezing temperature of the water, allowing the ice in the chamber to melt and the pressure to decrease. The chamber is then opened, and the biological is removed for the intended use.

[0082] In some embodiments, the subfreezing temperature is in the range of from 0 °C to triple point temperature of the fluid in the constant volume chamber. The triple point in the temperature pressure phase diagram depicted in FIG. 1 for water is about -21.9 °C. In other embodiments, the subfreezing temperature is in the range of from lower than 0 °C to about - 20 °C. In certain embodiments, the subfreezing temperature is in the range of about -2 °C to about -20 °C, about -3 °C to about -20 °C, about -4 °C to about -20 °C, or about -5 °C to about -Atty. Dkt: BERK-539WO20 °C. In certain embodiments, the subfreezing temperature is about -1 °C, about -2 °C, about - 3 °C, about -4 °C, about -5 °C, about -6 °C, about -7 °C, about -8 °C, about -9 °C, about -10 °C, about -11 °C, about -12 °C, about -13 °C, about -14 °C, about -15 °C, about -16 °C, about -17 °C, about -18 °C, about -19 °C, about -20 °C, or about -21°C.

[0083] In some embodiments of the method, the isochoric freezing conditions combining the above-described subfreezing temperature and the pressure which occurs at the abovedescribed subfreezing temperature when the closed isochoric chamber is in a state of thermodynamic equilibrium is effective in maintaining freeze-thaw stability in an emulsionbased product during isochoric freezing preservation. Furthermore, the pressures induced by the isochoric freezing of the present disclosure may have effects on avoiding ice crystal formation inside the preserved emulsion-based product at the subfreezing temperature. The applied pressure occurs at the above-described subfreezing temperature when the closed isochoric chamber is in a state of thermodynamic equilibrium. Therefore, the isochoric system provides convenient ways to achieve subfreezing temperature and elevated pressures only by controlling the temperature without the need for mechanical means to elevated pressure.

[0084] In some embodiments, the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa. In other embodiments, the isochoric freezing is at a pressure in the range of about 20 MPa to about 180 MPa, about 30 MPa to about 180 MPa, about 40 MPa to about 180 MPa, about 50 MPa to about 180 MPa, about 60 MPa to about 180 MPa, or about 50 MPa to about 170 MPa. In certain embodiments, the isochoric freezing is at about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, or about 170 MPa. In certain embodiments, the isochoric freezing is at about 59 MPa. In certain embodiments, the isochoric freezing is at about 95MPa. In certain embodiments, the isochoric freezing is at about 170 MPa. The pressure under an isochoric freezing condition occurs at the subfreezing temperature. (FIG. 1)

[0085] In some embodiments, preservation time of the present disclosure is a length of time for preservation under isochoric freezing conditions. In certain embodiments, the preservationAtty. Dkt: BERK-539WO time is a length of time from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, or months to years.

[0086] In certain embodiments, the preservation time is 1 month to 12 months or 1 month to 6 months. In certain embodiments, the preservation time is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0087] In certain embodiments, the preservation time is 1 week to 12 weeks. 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more than 12 weeks.

[0088] In some embodiments, the preservation time is 1 day to 100 days. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 1 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.

[0089] In certain embodiments, the preservation time of the present disclosure is 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.3.4. Warming the fluid in the isochoric chamber above 0 °C

[0090] In some embodiments, the method further comprises warming the fluid in the isochoric chamber above 0 °C. In some cases, the warming time is in the range of 30 minutes to 24 hours, such as, about 3 hours to 24 hours, about 6 hours to 24 hours, about 8 hours to 24 hours, about 12 hours to 24 hours, about 20 hours to 24 hours. In some cases, the warming temperature is above 0 °C to 5 °C. For example, the warming or thawing temperature is aboveAtty. Dkt: BERK-539WO0 °C to 5 °C, above 0 °C to 4 °C, above 0 °C to 3 °C, above 0 °C to 2 °C, or above 0 °C to 1 °C. A conventional warming or thawing process can be applied herein.

[0091] In some embodiments, the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size. In some embodiments, the emulsion-based product maintains freeze-thaw stability during preservation at the subfreezing temperature and subsequent warming. In some embodiments, the emulsion-based product maintains visual appearance, viscosity, color changes, and / or fat globule size stability during preservation at the subfreezing temperature and subsequent warming.

[0092] The emulsion-based product preserved by the method of the present disclosure maintains freeze-thaw stability during the preservation at the subfreezing temperature and subsequent warming.4. Device for preserving an emulsion-based product

[0093] Aspects of the present disclosure provide a device for preserving an emulsion-based product at subfreezing temperature while maintaining freeze-thaw stability of the emulsionbased product, comprising i) an isochoric chamber filled with a fluid containing the emulsionbased product and ii) a temperature control system configured to cool the fluid in the isochoric chamber at subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C. As such, the present disclosure provides a device for preserving native emulsion-based products with freeze-thaw stability to extend the shelf-life of such products.4.1. Isochoric freezing for preservation

[0094] The isochoric freezing technique is described in US2007 / 0042337A1 and PCT / US24 / 33896, which are incorporated by reference herein. The Isochoric freezing is described in "3. Methods for maintaining freeze-thaw stability of emulsion-based product during preservation" and is incorporated into this section.4.2. An isochoric chamber filled with a fluid containing the emulsion-based product

[0095] In some aspects of the method, the emulsion-based product is placed in an isochoric chamber filled with an aqueous solution. The term "constant volume chamber" is usedAtty. Dkt: BERK-539WO interchangeably herein with "isochoric chamber" or "rigid chamber". The isochoric freezing technique is described above, and US2007 / 0042337A1 and PCT / US24 / 33896 are incorporated by reference herein.

[0096] In the isochoric freezing, a substantial portion of the volume remains unfrozen which is associated with an increase in pressure. The process of isochoric freezing occurs along the liquidus line in a temperature-pressure phase diagram as the temperature is dropped because the volume in an isochoric chamber is constant.

[0097] In some aspects of the method, the emulsion-based product is placed inside the constant volume chamber filled with an aqueous solution in isochoric freezing. The constant volume chamber is filled with a fluid in such a way as to minimize the amount of air in the chamber. For example, FIG. 3 illustrates an exemplary isochoric chamber system for isochoric freezing. FIG. 3 is described in "3. Methods for maintaining freeze-thaw stability of emulsionbased product during preservation" and is incorporated into this section.

[0098] In some embodiments, the fluid in the constant volume chamber is aqueous solution or pure water. In the isochoric freezing, if ice nucleates in the constant volume chamber, the pressure of the system increases, and the nucleation can be detected by the pressure gauge. For example, U.S. Patent Publication No. 2007-0042337A1, incorporated by reference herein, which discusses isochoric freezing system and method for cryopreservation of a biological sample.

[0099] In some embodiments, an emulsion-based product can be directly placed in the isochoric chamber. FIG. 4 illustrates an exemplary embodiment of isochoric freezing configuration with an isochoric chamber for preserving a biological mass while avoiding the formation of ice. In other embodiments, an emulsion-based product can be placed in a container that allows transfer of pressure mass and heat. In still other embodiments, a biological matter can be placed in a container that allows transfer of only pressure and heat but not mass. FIG. 5 illustrates an exemplary embodiment of isochoric freezing configuration with an isochoric chamber and a matter container for preserving a biological mass while avoiding the formation of ice. This is described in PCT / US24 / 33896 and is incorporated by reference herein.Atty. Dkt: BERK-539WO

[0100] As generally shown in FIG. 6, the isochoric system comprises an isochoric chamber that is enclosed with a sealing cap. The isochoric chamber can be made of grade 7075 aluminum with a total volume capacity of 2 liters and pressure-rated for up to 110 MPa. The chamber can be connected to an electronic pressure transducer to monitor the pressure inside the chamber. A safety head with a rupture disk is in fluid communication with the interior of the isochoric chamber to ensure that the conditions inside the chamber do not exceed safety standards. This is described in PCT / US24 / 33896 and is incorporated by reference herein.

[0101] In some embodiments, an emulsion-based product may be collected in an impermeable but flexible container. For example, the emulsion-based product is collected in a polyethylene sterile bag. The bag is then sealed with negligible headspace and placed inside the isochoric chamber.

[0102] In some embodiments, the fluid in the constant volume chamber is aqueous solution or pure water with or without organic molecules therein. In some embodiments, the aqueous solution that is primarily water may contain other chemicals so that the freezing point of the water-based solution may be modified as required for a specific application. For example, the water-based solution may contain salt. In certain embodiments, the external solution is water. In some embodiments, the fluid in the container can be the same as the fluid in the isochoric chamber surrounding the container. In other embodiments, the fluid in the matter container can be different from the fluid in the isochoric chamber surrounding the matter container. In other words, the fluid inside the matter container and the fluid outside the matter container can be of different types in osmotic equilibrium with the preserved emulsion-based product. In some embodiments the isochoric chamber is filled with a fluid that does not freeze at the storage temperature, such as a solution of glycerol.

[0103] In some embodiments, the isochoric chamber contains a nucleating agent. The nucleating agent forms an ice crystal, thereby the emulsion-based product can be preserved in the unfrozen part of the volume. The ice crystal formed by the nucleating agent generates pressures of the isochoric chamber, and the enhanced pressures hinder further formation of ice. In some embodiments, the nucleating agent can be a structural element of the isochoric freezing device. For example, the nucleating agent can be walls of the isochoric chamber itself,Atty. Dkt: BERK-539WO or walls of the matter container itself. In other embodiments, the nucleating agent can be any agent that promotes the formation of ice crystal in the isochoric chamber. For example, the ice nucleating agent is, but not limited to, minute solid particles, such as dust or food particles, large molecules, ice-nucleating proteins. In some embodiments, the nucleating agent is placed in the bottom of the isochoric chamber, and the chamber is completely filled with an aqueous solution. In some embodiments, an emulsion-based product is placed in a container in an isochoric chamber that allows transfer of only pressure and heat but not mass and the isochoric chamber is filled with a fluid and nucleation agents that cause the fluid to freeze before the emulsion-based product in the container freezes.

[0104] In some embodiments, an emulsion-based product refers to any product that exists in an emulsified form comprising, including many dressings, sauces, spreads, dips, creams, and beverages. In some embodiments, the emulsion-based product basically comprises mixture of oil, water and emulsifying agent. The emulsion-based product comprises an oil-in-water emulsion.

[0105] In some embodiments, the emulsion-based product may be emulsion-based food. In certain embodiments, the emulsion-based food includes, but is not limited to, a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, a puree.

[0106] In exemplary embodiments, an emulsified sauce includes, but is not limited to, a hollandaise sauce, a barbecue sauce, a ranch sauce, a mayonnaise, a bearnaise, an egg yolk sauce, and an aioli.

[0107] In exemplary embodiments, an emulsified dip includes, but is not limited to, ketchup, mayonnaise, hummus, and a cheddar dip.

[0108] In exemplary embodiments, an emulsified soup includes, but is not limited to, a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

[0109] In some embodiments, the emulsion-based product may be emulsion-based pharmaceutical product. In some cases, the emulsion-based pharmaceutical product is aAtty. Dkt: BERK-539WO medicine produced by emulsion formulation. In some embodiments, the medicine produced by emulsion formulation is formulated for oral use, ocular use, topical mucosal use, intramuscular use or intravenous use. In some embodiments, the medicine produced by emulsion formulation contains emulsifiers to blend water-based and oil-based ingredients.

[0110] In exemplary embodiments, the medicine produced by emulsion formulation is, but not limited to, any kind of a pharmaceutical cream, a pharmaceutical lotion, a pharmaceutical balm, a pharmaceutical ointment. Herein, the term "pharmaceutical cream", "pharmaceutical lotion", "pharmaceutical balm", "pharmaceutical ointment" refers to any kind emulsion-based medicine for medical use and they are used as a medication or pharmaceutically used for treatment or prevention.

[0111] In exemplary embodiments, the medicine produced by emulsion formulation is, but not limited to, emulsion-based vaccine. For example, the emulsion-base vaccine is, but not limited to, Novartis's MF59®. The Novartis's MF59® has been licensed as part of the seasonal flu vaccine.

[0112] In some embodiments, the emulsion-based product may be a cosmetic. For example, the emulsion-based cosmetic includes, but is not limited to, a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.4.3. A temperature control system configured to cool the fluid in the isochoric chamber at subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C.

[0113] The device of the present disclosure comprises ii) a temperature control system configured to cool the fluid in the isochoric chamber of subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C.

[0114] In some embodiments, the temperature control system is any device or system configured to cool the fluid in the isochoric chamber and warm the fluid in the isochoric chamber above 0 °C. In some embodiments, the temperature control system may comprise a cooling system and a warming system. For example, the cooling system is a conventional freezer. For example, the warming system is a conventional heater.Atty. Dkt: BERK-539WO

[0115] Once the emulsion-based product is needed, the chamber is gradually warmed above the freezing temperature of the water, allowing the ice in the chamber to melt and the pressure to decrease. The chamber is then opened, and the emulsion-based product is removed for the intended use.

[0116] In some embodiments, the subfreezing temperature is in the range of from 0 °C to triple point temperature of the fluid in the constant volume chamber. The triple point in the temperature pressure phase diagram for water depicted in FIG. 1 is about -21.9 °C. In other embodiments, the subfreezing temperature is in the range of from lower than 0 °C to about - 20 °C. In certain embodiments, the subfreezing temperature is in the range of about -2 °C to about -20 °C, about -3 °C to about -20 °C, about -4 °C to about -20 °C, or about -5 °C to about - 20 °C. In certain embodiments, the subfreezing temperature is about -1 °C, about -2 °C, about - 3 °C, about -4 °C, about -5 °C, about -6 °C, about -7 °C, about -8 °C, about -9 °C, about -10 °C, about -11 °C, about -12 °C, about -13 °C, about -14 °C, about -15 °C, about -16 °C, about -17 °C, about -18 °C, about -19 °C, about -20 °C, or about -21°C.

[0117] In some embodiments of the method, the isochoric freezing conditions combining the above-described subfreezing temperature and the pressure which occurs at the above-described subfreezing temperature when the closed isochoric chamber is in a state of thermodynamic equilibrium is effective in maintaining freeze-thaw stability in an emulsionbased product during isochoric freezing preservation. Furthermore, the pressures induced by the isochoric freezing of the present disclosure may have effects on avoiding ice crystal formation inside the preserved emulsion-based product at the subfreezing temperature. The applied pressure occurs at the above-described subfreezing temperature when the closed isochoric chamber is in a state of thermodynamic equilibrium. Therefore, the isochoric system provides convenient ways to achieve subfreezing temperature and elevated pressures only by controlling the temperature without the need for mechanical means to elevated pressure.

[0118] In some embodiments, the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa. In other embodiments, the isochoric freezing is at a pressure in the range of about 20 MPa to about 180 MPa, about 30 MPa to about 180 MPa, about 40 MPa to about 180 MPa, about 50 MPa to about 180 MPa, about 60 MPa to about 180 MPa, or about 50 MPa to about 170 MPa. In certain embodiments, the isochoric freezing is at about 50Atty. Dkt: BERK-539WOMPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, or about 170 MPa. In certain embodiments, the isochoric freezing is at about 59 MPa. In certain embodiments, the isochoric freezing is at about 95MPa. In certain embodiments, the isochoric freezing is at about 170 MPa. The pressure under an isochoric freezing condition occurs at the subfreezing temperature. (FIG. 1)

[0119] In some embodiments, preservation time of the present disclosure is a length of time for preservation under isochoric freezing conditions. In certain embodiments, the preservation time is a length of time from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, or months to years.

[0120] In certain embodiments, the preservation time is 1 month to 12 months or 1 month to 6 months. In certain embodiments, the preservation time is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0121] In certain embodiments, the preservation time is 1 week to 12 weeks, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more than 12 weeks.

[0122] In some embodiments, the preservation time is 1 day to 100 days. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, T1 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.Atty. Dkt: BERK-539WO

[0123] In certain embodiments, the preservation time of the present disclosure is 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.

[0124] In some embodiments, the temperature control system is further configured to warm the fluid in the isochoric chamber above 0 °C. In some cases, the warming time is in the range of 30 minutes to 24 hours, such as about 3 hours to 24 hours, about 6 hours to 24 hours, about 8 hours to 24 hours, about 12 hours to 24 hours, about 20 hours to 24 hours. In some cases, the warming temperature is above 0 °C to 5 °C. For example, the warming or thawing temperature is above 0 °C to 5 °C, above 0 °C to 6 °C, above 0 °C to 7 °C, above 0 °C to 8 °C, above 0 °C to 9 °C, or above 0 °C to 10 °C. The warming or thawing conditions are well known in the art. The conventional warming or thawing conditions can be applied herein.

[0125] In some embodiments, the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size. In some embodiments, the emulsion-based product maintains freeze-thaw stability during preservation at the subfreezing temperature and subsequent warming. In some embodiments, the emulsion-based product maintains visual appearance, viscosity, color changes, and / or fat globule size stability during preservation at the subfreezing temperature and subsequent warming.

[0126] In some embodiments, the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size. In some embodiments, the emulsion-based product during preservation under isochoric freezing conditions maintains visual appearance, viscosity, color changes, and / or fat globule size.

[0127] The emulsion-based product preserved by the device of the present disclosure maintains freeze-thaw stability during the preservation at the subfreezing temperature and subsequent warming.5. Examples of Non-Limiting Aspects of the Disclosure

[0128] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure,Atty. Dkt: BERK-539WO each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0129] Aspect 1. A method for maintaining freeze-thaw stability of an emulsion-based product during preservation, comprising: i) placing an emulsion-based product in a fluid in an isochoric chamber; and ii) isochoric freezing the fluid in the isochoric chamber at subfreezing temperature over a preservation time; wherein the emulsion-based product maintains freeze-thaw stability during the preservation at the subfreezing temperature.

[0130] Aspect 2. The method of Aspect 1, wherein the subfreezing temperature is in the range of from 0 °C to triple point temperature of an aqueous solution.

[0131] Aspect 3. The method of Aspect 2, wherein the subfreezing temperature is in the range of from about -5 °C to about -20 °C.

[0132] Aspect 4. The method of any one of Aspects 1-3, wherein the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa.

[0133] Aspect 5. The method of Aspect 4, wherein the isochoric freezing is at a pressure in the range of about 50 MPa to about 180 MPa.

[0134] Aspect 6. The method of any one of Aspects 1-5, wherein the method further comprises iii) warming the fluid in the isochoric chamber above 0 °C.

[0135] Aspect 7. The method of Aspect 6, wherein the warming comprises increasing the temperature of the fluid in the isochoric chamber to above 0 °C to about 5 °C.

[0136] Aspect 8. The method of any one of Aspects 1-7, wherein the fluid is water or an aqueous solution.

[0137] Aspect 9. The method of any one of Aspects 1-8, wherein the emulsion-based product is directly placed in the chamber.

[0138] Aspect 10. The method of any one of Aspects 1-8, wherein the emulsion-based product is placed in a matter container.Atty. Dkt: BERK-539WO

[0139] Aspect 11. The method of any one of Aspects 1-10, wherein the chamber contains a nucleating agent.

[0140] Aspect 12. The method of any one of Aspects 1-11, wherein the emulsion-based product is an emulsion-based food, an emulsion-based pharmaceutical product, or an emulsion-based cosmetic.

[0141] Aspect 13. The method of Aspect 12, wherein the emulsion-based food is selected from a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, and a puree.

[0142] Aspect 14. The method of Aspect 13, wherein the emulsified soup is selected from a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

[0143] Aspect 15. The method of Aspect 12, wherein the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation.

[0144] Aspect 16. The method of Aspect 15, wherein the medicine produced by emulsion formulation is formulated for oral use, ocular use, topical mucosal use, intramuscular use, or intravenous use.

[0145] Aspect 17. The method of Aspect 12, wherein the emulsion-based pharmaceutical product is selected from a pharmaceutical cream, a pharmaceutical lotion, a pharmaceutical balm, and a pharmaceutical ointment.

[0146] Aspect 18. The method of Aspect 12, wherein the emulsion-based pharmaceutical product is an emulsion-based vaccine.

[0147] Aspect 19. The method of Aspect 12, wherein the emulsion-based cosmetic is selected from a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.

[0148] Aspect 20. The method of any one of Aspects 1-19, wherein the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size.Atty. Dkt: BERK-539WO

[0149] Aspect 21. The method of any one of Aspects 1-20, wherein the emulsion-based product during preservation under the isochoric freezing condition maintains visual appearance, viscosity, color changes, and / or fat globule size.

[0150] Aspect 22. A device for preserving an emulsion-based product at subfreezing temperature while maintaining freeze-thaw stability of the emulsion-based product, comprising: an isochoric chamber filled with a fluid containing the emulsion-based product; and a temperature control system configured to cool the fluid in the isochoric chamber at subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C.

[0151] Aspect 23. The device of Aspect 22, wherein the device is configured to perform the method of any one of Aspects 1-21.

[0152] Aspect 24. The device of Aspect 22, wherein the subfreezing temperature is in the range of from 0 °C to triple point temperature on an aqueous solution.

[0153] Aspect 25. The device of Aspect 24, wherein the subfreezing temperature is in the range of from -5 °C to about -20 °C.

[0154] Aspect 26. The device of any one of Aspects 22-25, wherein the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa.

[0155] Aspect 27. The device of Aspect 26, wherein the isochoric freezing is at a pressure in the range of about 50 MPa to about 180 MPa.

[0156] Aspect 28. The device of any one of Aspects 22-27, wherein the temperature control system is configured to warm the fluid in the isochoric chamber from above 0 °C to about 5 °C.

[0157] Aspect 29. The device of any one of Aspects 22-28, wherein the fluid is water or an aqueous solution.

[0158] Aspect 30. The device of any one of Aspects 22-29, wherein the emulsion-based product is directly placed in the chamber.

[0159] Aspect 31. The device of any one of Aspects 22-29, wherein the emulsion-based product is placed in a matter container.

[0160] Aspect 32. The device of any one of Aspects 22-31, wherein the isochoric chamber contains a nucleating agent.Atty. Dkt: BERK-539WO

[0161] Aspect 33. The device of any one of Aspects 22-32, wherein the emulsion-based product is an emulsion-based food, an emulsion-based pharmaceutical product, or an emulsion-based cosmetic.

[0162] Aspect 34. The device of Aspect 33, wherein the emulsion-based food is selected from a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, and a puree.

[0163] Aspect 35. The device of Aspect 34, wherein the emulsified soup is selected from a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

[0164] Aspect 36. The device of Aspect 33, wherein the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation.

[0165] Aspect 37. The device of Aspect 36, wherein the medicine produced by emulsion formulation is formulated for oral, ocular, topical mucosal, intramuscular, and intravenous use.

[0166] Aspect 38. The device of Aspect 33, wherein the emulsion-based pharmaceutical product is emulsion-based vaccine.

[0167] Aspect 39. The device of Aspect 33, wherein the emulsion-based cosmetic is selected from a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.

[0168] Aspect 40. The device of any one of Aspects 22-39, wherein the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size.

[0169] Aspect 41. The device of any one of Aspects 1-40, wherein the emulsion-based product during preservation under the isochoric freezing condition maintains visual appearance, viscosity, color changes, and / or fat globule size.Atty. Dkt: BERK-539WOEXAMPLES

[0170] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0171] All publications and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0172] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention.Example 1.1. Materials and Methods1.1. Isochoric system

[0173] In this study, dairy cream (36 % fat) was used as a food emulsion model to evaluate the potential use of isochoric freezing to preserve emulsion-based products with freeze-thaw stability. Dairy cream is an emulsion of milk fat droplets surrounded by adsorbed protein in a continuous aqueous skimmed milk phase. The effects of refrigeration, isochoric and conventional freezing were compared on the physical stability and physico-chemical characteristics of dairy cream.

[0174] For isochoric freezing, homogenized and pasteurized dairy cream (36% fat) was processed in 1.5 liter pressure chambers from BioChoric Inc. (Bozeman, MT, USA). The chambers were made of grade 7075 aluminum with pressure rated to 220 MPa. The chambers were connected to an electronic pressure transducer (Stork Solutions Ltd, Hampshire, UK) toAtty. Dkt: BERK-539WO monitor the pressure over time and cooled using a chest freezer (Magic Chef Model #HMCF9W3, MC Appliance Corporation, Wood Dale, IL).1.2. Experimental protocol

[0175] For the preservation experiments, three bags with 75 mL of cream in each bag were processed for each preservation condition. The bags were heat-sealed with negligible headspace and immediately processed before each experiment. For the refrigerated treatment (RF), the bags were stored for 14 days at 5 °C. For isochoric freezing (IF), the bags for each freezing temperature were placed inside an isochoric chamber filled with water. The chamber was cooled to a preset temperature inside the chest freezer for 14 days. The processing conditions selected were -10 °C / 95 MPa, -15 °C / 150 MPa and -20 °C / 170 MPa. For conventional freezing (CF), the bags were frozen in the chest freezer at the same temperatures as those used for the IF experiments. After IF and CF, the bags were slowly thawed at 5 °C overnight.1.3. Particle size

[0176] Particle size was determined using a laser scattering particle size distribution analyzer (Partica LA-960, Horiba Scientific, Kyoto, Japan). The refractive index of cream fat was 1.46 and the refractive index of water was 1.33. The following parameters were determined: mean (Dv43), median (Dv50), DvlO and Dv90 (diameter at which 10% and 90% of the sample was comprised of particles with smaller diameters, respectively).1.4. Visual appearance

[0177] After each treatment, 25 mL of cream sample was placed in a vial and left at room temperature for 24 hours to allow creaming to occur. A high-resolution digital camera (Nikon-7000) under constant lighting was used to capture images of the cream samples.1.5. Microscopic analysis

[0178] Cream samples were stained with 0.2 % (w / v) Nile Red and 0.01 % (w / v) Fast Green. An aliquot of 5pL of the stained cream was visualized under a Leica-TCS-SP8-confocal microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using LAS-X software.1.6. Viscosity

[0179] A DHR-3 rheometer (TA Instrument, New Castle, DE) was used to measure the viscosity of the dairy cream at 20 °C. A concentric cylinder geometry consisting of a bob (28.04Atty. Dkt: BERK-539WO mm in diameter and 21.1 mm in length) and cup (30 mm diameter) was used in the tests. Eight mL of cream was transferred into the cup and the sample was equilibrated at 20 °C for 5 minutes. The shear rate was then increased from 0.1 to 500 s ~1. Three replicates were performed for each sample.1.7. Color measurements

[0180] The color of the cream samples was measured using a tristimulus colorimeter (CM508D, Konica-Minolta Inc., Ramsey, NJ, USA) with a sample holder (CM-A128) and an 8 mm diameter target mask (CM-A195). Cream (10 mL) was pipetted into the sample holder and the color of each sample was measured three times. Results were expressed as L* (lightness), a* (redness / greenness) and b* (yellowness / blueness) in the CIE Lab system. These values were used to calculate the Chroma (C*), Hue angle (h*) and color difference (AE*) according to the following equations:2. Results and Discussion2.1. Fat globule size distribution

[0181] The fat globule size profiles of the fresh, refrigerated and isochoric frozen cream exhibited a Gaussian shape (FIG. 8). For isochoric frozen cream, the distribution slightly shifted to smaller globule sizes and narrower distributions. In comparison, conventional frozen samples showed a bimodal distribution with much larger particle sizes and broader distributions.

[0182] Table 1. Fat globule size parameters (pm).Atty. Dkt: BERK-539WO

[0183] Table 1 shows the fat globule size parameters describing particle size and distribution (mean, median, DvlO and Dv90). The droplet mean size for fresh cream was 5.3 pm. The globule size parameters slightly increased in value after 14 days of refrigerated storage. For example, the droplet size increased to 5.8 pm. The increase in globule size might be due to Ostwald ripening in which large globules grow at the expense of smaller globules when they diffuse through the liquid phase. The increase in the globule size parameters was avoided when samples were stored under isochoric conditions. All isochoric frozen samples exhibited smaller droplet mean sizes, ranging from 3.7 to 4.2 pm, indicating the efficiency of isochoric freezing to retain emulsion stability during frozen storage. Table 1 also shows that the fat globule size parameters slightly decreased in value with a decrease in temperature / increase in pressure. In contrast to our results, previous authors had reported that high pressures at 450 MPa and 25 °C for 30 mins did not change the size of cream fat globules. The different results may be due to the different pressure, temperature and time conditions used to process the dairy cream (Dumay et al., 2013).

[0184] Conventional freezing significantly increased fat globule size parameters, which was probably due to partial and complete coalescence. Fredrick, Walstra, & Dewettinck, (2010) reported that creams can be destabilized by partial coalescence in which the crystals in the fat globules form a network and a few crystals may even protrude from a globule into the continuous phase. Such a protruding crystal can, upon collision with another globule, pierce its interfacial layer.

[0185] Consequently, oil-oil contact can be established, leading to a partial merging of the droplets and coalescence if the fat globules are melted during the thawing process. Table 1 shows that the mean droplet size increased 27 times for CF -10 °C compared with the fresh sample. The droplet size for CF samples decreased in value with a decrease in freezingAtty. Dkt: BERK-539WO temperature. This was due to the formation of smaller ice crystals from faster freezing rates at lower temperatures, which prevented the growth of large and more destructive crystals. Also, studies have shown that recrystallization processes can be retarded by maintaining frozen foods at temperatures of -18 °C or less (Degner et aL, 2014). However, the mean droplet size was still 17 times larger for CF -20 °C than for fresh cream samples.2.2. Visual Appearance

[0186] FIG. 9 shows the appearance of fresh and preserved dairy cream for 14 days. Minor phase separation or creaming was noted in the fresh non-frozen cream caused by the density differences between the dispersed phase and the dispersion medium. The cream fat globules possess a lower density than the skim phase (Ma & Barbano, 2000) and tend to rise to the surface. Creaming eventually leads to a separation phase, characterized by a clear phase at the bottom of the sample vial.

[0187] Refrigerated samples showed similar phase separation as fresh cream samples. Isochoric frozen creams also showed some clarification. However, they were more stable than fresh cream, which was probably due to the presence of smaller globule sizes (FIG. 8). Fat globules tend to rise under the influence of gravity at a rate described by Stokes' Law in which creaming rate is proportional to particle size. Also, a decrease in fat globule size and therefore, an increase in the number of fat droplets limit phase separation when the proteins adsorbed from the continuous phase to the newly formed oil surface protected the droplets from flocculation and coalescence. In contrast, conventional frozen samples showed an increased volume of clarification, indicating a destruction or destabilization of the fat emulsion. Webb and Hall (1935) reported that freezing of cream causes a gradual precipitation of the caseinate system and an immediate destruction of the fat emulsion. The level of clarification was greater at lower freezing temperatures. Also, the cream appeared partially coagulated with an increased viscosity and the presence of visible lumps (FIG. 10).2.3. Confocal microscopy

[0188] FIG. 11 depicts representative micrographs of the microstructure of dairy cream from confocal scanning laser light microscopy (CSLM). The micrographs of the refrigerated emulsions revealed individual oil droplets (red) dispersed in a continuous aqueous phase containing protein (green). Micrographs of isochoric frozen samples showed no significantAtty. Dkt: BERK-539WO flocculation or coalescence of fat globules. In comparison, micrograph of conventionally frozen cream showed much larger fat globule sizes, indicating that extensive coalescence occurred in the sample. The conventionally frozen sample also had irregularly shaped aggregates that contained clusters of partially crystalline fat droplets. In addition, the serum in CF samples was drained from the thawing mass, leaving behind a mixture of fat and adsorbed casein. These results were consistent with the measured particle size distributions that shifted toward larger size ranges and increasing average particle sizes (FIG. 8).3.4. Rheology

[0189] The viscosity of fresh cream first decreased in value with an increase in shear rate, indicating shear-thinning behavior. After a lag phase, the viscosity then rises suddenly due to shear-induced partial coalescence, indicating that network formation occurs rapidly as soon as fat droplets are close enough to partially coalesce. The oil in water emulsion can also be transformed into a ternary system with the incorporation of air during the rheology experiment (Fredrick, Walstra, & Dewettinck, 2010).

[0190] The refrigerated cream had a higher viscosity than fresh cream, though the difference was not statistically significant (p >.05) (FIG. 12). This might have been due to aging of the cream that caused the fat globules to agglomerate and become more resistant to shear forces. The isochoric samples also showed a slight increase in viscosity values compared to fresh cream, although the differences were not significant (p > .05). The increase in viscosity can be attributed to a decrease in droplet size and therefore, an increase in the number of fat droplets in the water phase. This reduced the speed of droplet movement and resulted in an increase in the amount of protein on the oil droplet surface, leading to increased viscosity (Long et aL, 2012). In addition, these results were consistent with those reported by Donsi et al. (2011), who evaluated the rheological behavior of dairy cream under pressure (400-500 MPa for 5-10 min at 25 °C) and found qualitatively similar non-Newtonian flow behavior.

[0191] In contrast, conventionally frozen cream displayed more variable and considerable higher viscosity values due to partial coalescence and agglomeration of semicrystalline solid droplets. These droplets resisted shear forces better than the fat in water emulsion present in fresh cream. In addition, the water enclosed in the voids of the agglomerates no longer behave as free water, leading to increased viscosity values. FIG. 12Atty. Dkt: BERK-539WO also shows that conventionally frozen cream lost the important ability of dairy cream to form a foam by incorporating air during whipping.3.5. Color

[0192] Isochoric freezing did not significantly affect the color parameters of fresh cream (FIGs. 13A-13F). In comparison, conventional freezing resulted in a darker (lower L* values) and more intense green color (lower a* values) compared with fresh cream.

[0193] The chromatic difference values (AE*) confirmed differences in color of the frozen cream. AE* is classified as not noticeable (0-0.5), slightly noticeable (0.5-1.5), noticeable (1.5-3.0), well visible (3.0-6.0) and great (6.0-12.0) to the human eye (Cserhalmi et aL, 2006). According to this classification, the color differences in the isochoric frozen samples are "not noticeable" or "slightly noticeable". In comparison, the color differences in conventional frozen samples are considered "noticeable" or "well visible".3. Conclusions

[0194] Cream frozen under isochoric conditions did not result in the usual fat separation that occurred during the thawing of conventionally frozen dairy cream due to the absence of ice crystal formation. Isochoric freezing led to the reduction in fat droplet size, which increased the physical stability of the cream. In addition, isochoric freezing helped to preserve the cream's viscosity and color.4. Other emulsion-based products with freeze-thaw stability

[0195] Further, isochoric freezing at -15 °C was applied to preserve cream cheese for one week. See FIG. 14. The cream cheese from the isochoric chamber showed a smooth, dense texture similar to refrigerated cream cheese. In comparison, conventionally frozen cream cheese had a crumbly, grainy texture. FIG. 14A shows fresh cream cheese, FIG. 14B shows isochoric frozen cream cheese at -15 °C / 150 MPa for one week, and FIG. 14C shows conventionally frozen cream cheese at -15 °C for one week.

[0196] Isochoric freezing at -15 °C was also applied to creamy tomato soup for one week. See FIG. 15. The appearance and organoleptic properties of these products resembled that of the fresh soup. In comparison, conventionally frozen creamy tomato soup showed phase separation, with a distinct liquid portion and a lumpy solid portion with unpleasant appearance, instead of a homogenous mixture. FIG. 15A shows creamy tomato soup, FIG. 15BAtty. Dkt: BERK-539WO shows isochoric frozen at -15 °C / 150 MPa for one week, and FIG. 15C shows conventionally frozen at 15 °C for one week.Example 2.1. Materials and Methods1.1. Isochoric system

[0197] For isochoric freezing, sunflower oil in water emulsions (respectively 10 % sunflower oil in water emulsion and 20% sunflower oil in water emulsion) were processed in 1.5 liter pressure chambers from BioChoric Inc. (Bozeman, MT, USA). For this study, 10% oil was emulsified in water, which is hereinafter referred to as "10 % sunflower oil in water emulsion"; and 20% oil was emulsified in water, which is hereinafter referred to as "20 % sunflower oil in water emulsion". A ratio of the oil and surfactant (soy lecithin powder) was maintained at 2.5:1 for all formulations. The chambers were made of grade 7075 aluminum with pressure rated to 220 MPa. The chambers were connected to an electronic pressure transducer (Stork Solutions Ltd, Hampshire, UK) to monitor the pressure over time and cooled using a chest freezer (Magic Chef Model #HMCF9W3, MC Appliance Corporation, Wood Dale, IL).1.2. Experimental protocol

[0198] For the preservation experiments, three bags with 100 mL of sunflower oil in water emulsion in each bag were processed for each preservation condition. The bags were heat-sealed with negligible headspace and immediately processed before each experiment. For isochoric freezing (IF), the bags for each freezing temperature were placed inside an isochoric chamber filled with water. The chamber was cooled to a preset temperature inside the chest freezer for 3 days. The processing conditions selected were -5 °C / 59 MPa, -20 °C / 170 MPa. For conventional freezing (CF), the bags were frozen in the chest freezer at the same temperatures as those used for the IF experiments. After IF and CF, the bags were slowly thawed at 5 °C overnight and left in in an environmental chamber (Thermo Scientific Forma, Model 3961) at 23 °C for 1 and 50 daysAtty. Dkt: BERK-539WO1.3. Particle size

[0199] Particle size of thawed emulsions after IF or CF was determined using a laser scattering particle size distribution analyzer (Partica LA-960, Horiba Scientific, Kyoto, Japan). The following parameters were determined: mean, median (Dv50), DvlO and Dv90.1.4. Visual appearance

[0200] After each treatment, 15 mL of the sunflower oil in water emulsion were placed in a vial and left at 23 °C for 24 hours and 50 days. A high-resolution digital camera (Nikon- 7000) under constant lighting was used to capture images of the emulsions.1.5. Microscopic analysis

[0201] The sunflower oil in water emulsion (150 pl) was stained with 0.1 mL of Nile Red. A small droplet of the stained oil in water emulsion was visualized under a Leica-TCS-SP8- confocal microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using LAS-X software.1.6. Viscosity

[0202] A DHR-3 rheometer (TA Instrument, New Castle, DE) was used to measure the viscosity of the emulsions at 20 °C. A concentric cylinder geometry consisting of a bob (28.04 mm in diameter and 21.1 mm in length) and cup (30 mm diameter) was used in the tests. Eight mL of sunflower oil in water emulsion was transferred into the cup and the sample was equilibrated at 20 °C for 5 minutes. The shear rate was then increased from 0.1 to 500 s1. Three replicates were performed for each sample.2. Results and Discussion2.1. Particle size

[0203] Table 2. Particle size parameters (pm).Atty. Dkt: BERK-539WO

[0204] Table 2 shows the fat globule size parameters describing particle size and distribution (mean, median, DvlO and Dv90). The droplet mean size for fresh control samples was 1.6 pm. The globule size parameters increased in value after 3 days of conventional freezing. For example, the droplet size increased to 39 pm at -5 °C and increased to 23 pm at - 20 °C through the conventional freezing. The increase in the globule size parameters was avoided when samples were stored under isochoric conditions. The isochoric frozen 10 % sunflower oil in water emulsion samples exhibited smaller droplet mean sizes, ranging from 1.6 to 2.3 pm, and isochoric frozen 20 % sunflower oil in water emulsion samples exhibited smaller droplet mean sizes, ranging from 4.2 to 4.6 pm, indicating the efficiency of isochoric freezing to retain emulsion stability during frozen storage.

[0205] Conventional freezing significantly increased fat globule size parameters, which was probably due to partial and complete coalescence.

[0206] Consequently, oil-oil contact can be established, leading to a partial merging of the droplets and coalescence if the fat globules are melted during the thawing process. Table 2 shows that the mean droplet size of 20 % sunflower oil in water emulsion increased 24 times for CF -5 °C compared with the fresh sample. The droplet size for CF samples decreased in value with a decrease in freezing temperature. This was due to the formation of smaller iceAtty. Dkt: BERK-539WO crystals from faster freezing rates at lower temperatures, which prevented the growth of large and more destructive crystals. Also, studies have shown that recrystallization processes can be retarded by maintaining frozen foods at temperatures of -18 °C or less (Degner et al., 2014). However, the mean droplet size of 20 % sunflower oil in water emulsion was still 14 times larger for CF -20 °C than for fresh cream samples.2.2. Visual Appearance

[0207] FIG. 16 shows the appearance of fresh and preserved sunflower oil in water emulsion after 1 and 50 days at 23 °C. Following CF, no oil layer separation was observed, yet CF emulsions appeared notably yellower than control and IF emulsions on day 1. On day 50, CF emulsions exhibited clear phase separation. In comparison, IF 10% samples exhibited similar creaming as control samples. IF 20% was more stable than control samples that showed a clear phase separation with an oil phase at the top of the sample vial.2.3. Confocal microscopy

[0208] FIG. 17 depicts representative micrographs of the microstructure of sunflower oil in water emulsion from confocal scanning laser light microscopy (CSLM). The micrographs of the control emulsions revealed individual oil droplets (red) dispersed in a continuous aqueous phase. Micrographs of isochoric frozen samples showed no significant flocculation or coalescence of fat globules. In comparison, micrograph of the conventionally frozen emulsions showed much larger fat globule sizes, indicating that extensive coalescence occurred in the sample. The conventionally frozen emulsions also had irregularly shaped aggregates that contain clusters of partially crystalline fat droplets.2.4. Rheology

[0209] The conventionally frozen emulsion had a higher viscosity than the control and isochoric frozen emulsion (FIG. 18). The conventionally frozen emulsion displayed more variable and considerable higher viscosity values due to partial coalescence and agglomeration of semi-crystalline solid droplets. The isochoric samples showed a slight increase in viscosity values compared to the control, although the differences were not significant.3. Conclusions

[0210] Sunflower oil in water emulsion frozen under isochoric conditions did not result in the usual fat separation that occurred during the thawing of conventionally frozen sunflowerAtty. Dkt: BERK-539WO oil in water emulsion due to the absence of ice crystal formation. Isochoric freezing led to the reduction in fat droplet size, which increased the physical stability of the emulsion. In addition, isochoric freezing helped to preserve the emulsion's viscosity.

[0211] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Atty. Dkt: BERK-539WOCLAIMSWhat is claimed is:

1. A method for maintaining freeze-thaw stability of an emulsion-based product during preservation, comprising: i) placing an emulsion-based product in a fluid in an isochoric chamber; and ii) isochoric freezing the fluid in the isochoric chamber at subfreezing temperature over a preservation time, wherein the emulsion-based product maintains freeze-thaw stability during the preservation at the subfreezing temperature.

2. The method of claim 1, wherein the subfreezing temperature is in the range of from 0 °C to triple point temperature of an aqueous solution.

3. The method of claim 2, wherein the subfreezing temperature is in the range of from about -5 °C to about -20 °C.

4. The method of any one of claims 1-3, wherein the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa.

5. The method of claim 4, wherein the isochoric freezing is at a pressure in the range of about 50 MPa to about 180 MPa.

6. The method of any one of claims 1-5, wherein the method further comprises iii) warming the fluid in the isochoric chamber above 0 °C.

7. The method of claim 6, wherein the warming comprises increasing the temperature of the fluid in the isochoric chamber to above 0 °C to about 5 °C.

8. The method of any one of claims 1-7, wherein the fluid is water or an aqueous solution.Atty. Dkt: BERK-539WO9. The method of any one of claims 1-8, wherein the emulsion-based product is directly placed in the chamber.

10. The method of any one of claims 1-8, wherein the emulsion-based product is placed in a matter container.

11. The method of any one of claims 1-10, wherein the chamber contains a nucleating agent.

12. The method of any one of claims 1-11, wherein the emulsion-based product is an emulsion-based food, an emulsion-based pharmaceutical product, or an emulsion-based cosmetic.

13. The method of claim 12, wherein the emulsion-based food is selected from a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, a mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, and a puree.

14. The method of claim 13, wherein the emulsified soup is selected from a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

15. The method of claim 12, wherein the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation.

16. The method of claim 15, wherein the medicine produced by emulsion formulation is formulated for oral use, ocular use, topical mucosal use, intramuscular use, or intravenous use.Atty. Dkt: BERK-539WO17. The method of claim 12, wherein the emulsion-based pharmaceutical product is selected from a pharmaceutical cream, a pharmaceutical lotion, a pharmaceutical balm, and a pharmaceutical ointment.

18. The method of claim 12, wherein the emulsion-based pharmaceutical product is an emulsion-based vaccine.

19. The method of claim 12, wherein the emulsion-based cosmetic is selected from a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.

20. The method of any one of claims 1-19, wherein the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size.

21. The method of any one of claims 1-20, wherein the emulsion-based product during preservation under the isochoric freezing condition maintains visual appearance, viscosity, color changes, and / or fat globule size.

22. A device for preserving an emulsion-based product at subfreezing temperature while maintaining freeze-thaw stability of the emulsion-based product, comprising: an isochoric chamber filled with a fluid containing the emulsion-based product; and a temperature control system configured to cool the fluid in the isochoric chamber at subfreezing temperature and warm the fluid in the isochoric chamber above 0 °C.

23. The device of claim 22, wherein the device is configured to perform the method of any one of claims 1-21.

24. The device of claim 22, wherein the subfreezing temperature is in the range of from 0 °C to triple point temperature on an aqueous solution.Atty. Dkt: BERK-539WO25. The device of claim 24, wherein the subfreezing temperature is in the range of from -5 °C to about -20 °C.

26. The device of any one of claims 22-25, wherein the isochoric freezing is at a pressure in the range of about 0.1 MPa to about 200 MPa.

27. The device of claim 26, wherein the isochoric freezing is at a pressure in the range of about 50 MPa to about 180 MPa.

28. The device of any one of claims 22-27, wherein the temperature control system is configured to warm the fluid in the isochoric chamber from above 0 °C to about 5 °C.

29. The device of any one of claims 22-28, wherein the fluid is water or an aqueous solution.

30. The device of any one of claims 22-29, wherein the emulsion-based product is directly placed in the chamber.

31. The device of any one of claims 22-29, wherein the emulsion-based product is placed in a matter container.

32. The device of any one of claims 22-31, wherein the isochoric chamber contains a nucleating agent.

33. The device of any one of claims 22-32, wherein the emulsion-based product is an emulsion-based food, an emulsion-based pharmaceutical product, or an emulsion-based cosmetic.

34. The device of claim 33, wherein the emulsion-based food is selected from a cheese, a cream cheese, a quark cheese, a cottage cheese, a skyr, a sour cream, a dairy cream, aAtty. Dkt: BERK-539WO mascarpone, an eggnog, a custard, an emulsified sauce, a cream sauce, an emulsified soup, a gravy, a spread, a pudding, dip, a salad dressing, a vinaigrette, a mayonnaise, a whipped topping, and a puree.

35. The device of claim 34, wherein the emulsified soup is selected from a chowder soup, a cream soup, a tomato soup, a bean soup, a chicken soup, a beef soup, and a vegetable soup.

36. The device of claim 33, wherein the emulsion-based pharmaceutical product is a medicine produced by emulsion formulation.

37. The device of claim 36, wherein the medicine produced by emulsion formulation is formulated for oral, ocular, topical mucosal, intramuscular, and intravenous use.

38. The device of claim 33, wherein the emulsion-based pharmaceutical product is emulsionbased vaccine.

39. The device of claim 33, wherein the emulsion-based cosmetic is selected from a facial lotion, a facial cream, a facial sunscreen, a facial mask, an eye cream, a body lotion, a body cream, a body milk, a body sunscreen, a hand cream, a hair mask, a shaving cream, a makeup foundation, and an emulsion-based cleansing.

40. The device of any one of claims 22-39, wherein the freeze-thaw stability is determined by measuring visual appearance, viscosity, color changes, and / or fat globule size.

41. The device of any one of claims 1-40, wherein the emulsion-based product during preservation under the isochoric freezing condition maintains visual appearance, viscosity, color changes, and / or fat globule size.

Images