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Title:
PROTEIN-STABILISED OLEOGELS
Document Type and Number:
WIPO Patent Application WO/2016/062685
Kind Code:
A1
Abstract:
The invention provides a method for the production of an oleogel comprising a protein and an oil, the method comprising: (i) providing a water protein composition, wherein the composition comprises protein and water and 0-10 wt.% of one or more other components, and wherein the protein comprises a globular protein; (ii) exchanging in a solvent exchange process at least part of the water by an oil, wherein an intermediate solvent is applied wherein water is solvable; and (iii) providing said oleogel, wherein the oleogel comprises 0.5-30 wt.%> of said protein, 70-99.5 wt.%> of said oil, and 0-10 wt.%> of said one or more other components.

Inventors:
SCHOLTEN, Elke (P.O. Box 557, 6700 AN Wageningen, NL-6700 AN, NL)
DE VRIES, Auke (P.O. Box 557, 6700 AN Wageningen, NL-6700 AN, NL)
Application Number:
EP2015/074209
Publication Date:
April 28, 2016
Filing Date:
October 20, 2015
Export Citation:
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Assignee:
STICHTING TOP INSTITUTE FOOD AND NUTRITION (Nieuwe Kanaal 9A, 6709 PA Wageningen, NL-6709 PA, NL)
International Classes:
A23D9/00; A21D13/08; A23D9/007; A23D9/02; A23J1/06; A23J1/20; A23J3/00; A23J3/08; A23J3/12
Domestic Patent References:
WO2014004018A12014-01-03
Foreign References:
EP1836897A12007-09-26
US20120289610A12012-11-15
US20070128258A12007-06-07
GB690888A1953-04-29
EP0051943A21982-05-19
EP1836897A12007-09-26
US20120289610A12012-11-15
US20070128258A12007-06-07
GB690888A1953-04-29
Other References:
ASHOK R. PATEL ET AL: "Biopolymer-Based Structuring of Liquid Oil into Soft Solids and Oleogels Using Water-Continuous Emulsions as Templates", LANGMUIR, 22 August 2014 (2014-08-22), XP055161642, ISSN: 0743-7463, DOI: 10.1021/la502829u
SANGHOON KO ET AL: "Preparation of sub-100-nm [beta]-lactoglobulin (BLG) nanoparticles", JOURNAL OF MICROENCAPSULATION., vol. 23, no. 8, 1 December 2006 (2006-12-01), GB, pages 887 - 898, XP055235307, ISSN: 0265-2048, DOI: 10.1080/02652040601035143
ASHOK R. PATEL ET AL., LANGMUIR, 22 August 2014 (2014-08-22)
SCHMITT ET AL., FOOD HYDROCOLLOIDS, vol. 25, 2011, pages 558 - 567
Attorney, Agent or Firm:
ELLENS, Andries (Agro Business Park 20, 6708 PW Wageningen, NL-6708 PW, NL)
Download PDF:
Claims:
CLAIMS:

1. A method for the production of an oleogel comprising a protein and an oil, the method comprising:

(i) providing a water protein composition, wherein the composition comprises protein and water and 0-10 wt.% of one or more other components, and wherein the protein comprises a globular protein;

(ii) exchanging in a solvent exchange process at least part of the water by a lipid material comprising an oil, wherein an intermediate solvent is applied wherein water is solvable; and

(iii) providing said oleogel, wherein the oleogel comprises 0.5-30 wt.% of said protein, 70-99.5 wt.% of said oil, and 0-10 wt.% of said one or more other components.

2. The method according to claim 1, wherein the composition comprises 0.5- 30 wt.%) protein and 70-99.5 wt.% water, wherein the oil is a vegetable oil, and wherein the 0-10 wt.% of one or more other components comprise one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a vitamin, a gelling agent, and a surfactant.

3. The method according to any one claims 1 and/or 2, wherein the water protein composition comprises a hydrogel, and wherein the water protein composition comprises at least 8 wt.% globular protein. 4. The method according to claim 3, wherein the hydrogel has a volume in the range of 1-1000 μιη3.

5. The method according to claim 3, wherein the hydrogel has a volume in the range of 1000 μιη3 - 1 mm3.

6. The method according to claim 3, wherein the hydrogel has a volume larger than 1 mm3, and wherein after the solvent exchange process the thus obtained oleogel is further processed into smaller gel particles.

7. The method according to any one claims 1-2, wherein the water protein composition comprises protein aggregates, and wherein the water protein composition comprises at least 2 wt.% globular protein. 8. The method according to any one of the preceding claims, wherein the globular protein comprises one or more of whey protein, albumin, immunoglobulin, and soy protein, wherein the oil comprises one or more of sunflower oil and canola oil, wherein the intermediate solvent comprises one or more solvents selected from the group consisting of acetone, tetrahydrofuran, ethanol, propanol, butanol, and DMSO.

9. The method according to any one of the preceding claims, wherein in a first stage of the solvent exchange process, a first series of exchanges are executed wherein the protein composition is exposed to a first series of liquids that decrease in water content to a last liquid in the first series substantially comprising the intermediate solvent, and subsequently in a second stage of the solvent exchange process, a second series of exchanges are executed wherein the protein composition is exposed to a second series of liquids that decrease in intermediate solvent content to a last liquid in the second series substantially comprising the oil. 10. An oleogel comprising 0.5-30 wt.% protein, 70-99.5 wt.% lipid material comprising oil, and 0-10 wt.% of one or more other components, wherein the protein is available in the lipid material as one or more of protein aggregates and protein particles.

11. The oleogel according to claim 10, wherein the 0-10 wt.% of one or more other components comprise one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a vitamin, gelling agent, and a surfactant, and wherein the globular protein comprises one or more of whey protein, albumin, immunoglobulin and soy protein, wherein the oil comprises a vegetable oil. 12. The oleogel according to any one of claims 10-11, comprising an organic solvent in an amount of up to 1 wt.% and water in an amount of up to 3 wt.%.

13. A food product or non-food product comprising an oleogel phase according to any one of claims 10-12.

14. The food product according to claim 13, wherein the oleogel phase is a continuous phase.

15. The food product according to claim 13, wherein the oleogel phase is a discontinuous phase with oleogel regions having one or more dimensions in the range of up to 10 mm.

16. A protein aggregate material comprising over 70 wt.% globular protein, wherein the protein aggregate materials comprises protein comprising particles having a volume averaged particle size selected from the range of 100-1000 nm.

17. The protein aggregate material according to claim 16, having a packing density selected from the range of 0.05-0.5 g/cm3. 18. A method for the production of a protein aggregate material, the method comprising:

(i) providing a water protein composition, wherein the composition comprises protein and water and 0-10 wt.% of one or more other components, wherein the water protein composition comprises protein aggregates, and wherein the protein comprises a globular protein;

(ii) exchanging in a solvent exchange process at least part of the water by a non-polar solvent, wherein optionally an intermediate solvent is applied wherein water is solvable, to provide a protein aggregate dispersion in a non-polar solvent;

and

(iii) providing a protein aggregate material by separating in a separation stage the protein aggregates from the non-polar solvent.

19. The method according to claim 18, wherein the intermediate solvent comprises one or more solvents selected from the group consisting of acetone, tetrahydroiuran, ethanol, propanol, butanol, and DMSO, wherein the non-polar solvent is selected from the group consisting of C6-10 alkanes, and wherein the separation stage includes one or more of air-drying and freeze-drying.

20. The method according to any one of the preceding claims 18-19, wherein the water protein composition comprises protein aggregates is obtainable by solubilizing globular proteins in water, heating the thus obtained protein solution to provide the protein aggregates, and applying shear.

Description:
Protein-stabilised oleogels FIELD OF THE INVENTION

The invention relates to a method for the production of an oleogel. The invention further relates to an oleogel obtainable with such method, as well as (food) products comprising such oleogel and/or based upon such oleogel, and a protein powder. BACKGROUND OF THE INVENTION

Oleogels, for instance for use in food products, are known in the art. WO2014004018, for instance, describes a method of preparing an edible oleogel comprising combining an edible triacylglycerol oil or triacylglycerol fat and ethyl cellulose in a weight ratio from 99: 1 to 80:20 to form a mixture, and heating and agitating the mixture at a temperature within the range from 80 to 300 °C to form an oleogel, wherein the heating and agitating is conducted under an inert atmosphere; an oleogel obtainable by said method, its use to prepare food products and to food products comprising said oleogel. EP1836897 describes a solid product comprising oil-droplets having a diameter in the range of 0.1 to 100 microns, cross-linked proteins at the interface of said droplets and any polar, low molecular compound in between the cross-linked protein interfaces. US2012/289610 describes oil thickeners and in particular oil gels. Embodiments relate for example to a an oil composition containing at least one complex comprising an oil dispersible emulsifier and protein fibres and preparations comprising such oil compositions; the use of such complexes as oil thickeners, and a method to produce the complexes. Ashok R. Patel et al, Langmuir, 22 August 2014 describes biopolymer-based structuring of liquid oil into soft solids and oleogels using water- continuous emulsion as templates. US2007/128258 describes oil-in-water emulsion gels and medical articles containing an omega-3 oil are disclosed. Also described are methods of preparing and using such emulsion gels and medical articles. GB690888 describes a composition for use as a dielectric sealing material.

SUMMARY OF THE INVENTION

As WO2014004018 also indicates, structure in solid fat-containing food products is provided by the network of crystalline triacylglycerols. However, these triacylglycerols contain high levels of saturated fatty acids. Instead of using naturally highly saturated solid fats, oils comprising triacylglycerols having high levels of unsaturated fatty acids are also transformed to more solid products by hydrogenation or partial hydrogenation. "Trans fats" which are unsaturated fats with trans-isomer unavoidably emerge as a by-product from partial hydrogenation of unsaturated oils into saturated fats. Research into the role fats and oils play in human health has indicated that consumption of saturated fatty acids, even more of trans fatty acids is associated with elevated cholesterol levels and cardiovascular diseases. Therefore, it is desirable to develop healthier alternatives to triacylglycerols containing saturated or partially hydrogenated fatty acids. WO2014004018 teaches a process for making an oleogel including heating and agitating under an inert atmosphere such as under an inert gas atmosphere or in ambient air under vacuum (reduced pressure), which may complicate such process for making an oleogel.

Hence, it is an aspect of the invention to provide an alternative method for the production of an (oleo)gel, which preferably further at least partly obviates one or more of above-described drawbacks. Yet, it is also an aspect of the invention to provide an alternative (oleo)gel, which preferably further at least partly obviates one or more of above-described drawbacks.

In a first aspect, the invention provides a method for the production of an oleogel comprising a protein and an oil, the method comprising: (i) providing a water- protein composition, wherein the composition comprises protein, water, and optionally one or more other components (especially wherein the water protein composition comprises 0.5-30 wt.% protein and 70-99.5 wt.% water and 0-10 wt.% of one or more other components), and wherein the protein especially comprises a globular protein; (ii) exchanging in a solvent exchange process at least part of the water by a lipid material comprising an oil, wherein in a specific embodiment the oil is a vegetable oil, wherein an intermediate solvent is applied wherein water is solvable (and wherein the oil is solvable); and (iii) providing said oleogel. It surprisingly appears that with substantially only proteins and oil an oleogel can be obtained, starting from a hydrogel or protein aggregates (see also below). Hence, with the present method an oleogel can be obtained based on food safe ingredients, which can replace fat and may therewith be healthier than the same product but based on fat. Further, with the present method no additional surfactants are necessary and no severe heat treatment (oxidation) may have to take place. Also, with the present method an oleogel can be obtained that can be used for cosmetics or (other) personal care products, but also e.g. for drug delivery (e.g. a pharmaceutical product), etc. The oleogel thus provided can be a GRAS (Generally Recognized As Safe) material. Further the oleogel can be used as biocompatible oleogel. Though proteins appear to have gelation and water holding capacities in aqueous systems, it is now also found that proteins may have these properties in oil-continuous (apolar) systems. In this invention, we use a solvent exchange process to provide an oleogel wherein proteins also have gelation and oil holding capacities for oil-continuous gels (apolar phase).

The term "lipid material comprising an oil" and similar terms especially refers to fats and oil, wherein the former at least includes a portion of oil. The term oil refers to a lipid that is liquid at room temperature, especially at the place of making the oleogel. For the sake of simplicity, each lipid that is liquid at 20 °C is at least considered to be an oil. Herein, the term lipid especially refer to triglycerides. Hence, the term "lipid material comprising an oil" especially refers to triglyceride material comprising an oil. The oil content (of the oleogel) is especially at least 20 wt.%, even more especially at least 30 wt.%, such as at least 40 wt.%, even more especially at least 50 wt.%, such as at least 60 wt.% or even at least 70 wt.%.

Hence, in a further aspect the invention also provides an oleogel obtainable with such method. The invention especially provides in an embodiment an oleogel comprising 0.5-30 wt.% protein, 70-99.5 wt.% lipid material comprising (vegetable) oil, and 0-10 wt.% of one or more other components (all weight percentages on a dry weight basis). The oleogel may be part of a food product or a non-food product (both are herein indicated as "product"). For instance, it may be integrated or embedded in such products, or even may substantially for such food product or non-food product.

Hence, in a further aspect the invention also provides a food product and/or a non-food product comprising an oleogel phase as defined herein. The term "phase" or "oleogel phase" indicates that the (food) product includes a region (or phase) with said oleogel. The food or non-food product, i.e. "product", may include next to the oleogel, as obtainable with the herein described method, also other components. For instance, when combining the oleogel with water, a water comprising oleogel may be obtained, comprising up to e.g. 70 wt.% water (see also above). For instance, the oil to water ratio in the oleogel based product may for instance be in the range of 20:80 to 40:60. Hence, in an embodiment a product is provided comprising the herein described oleogel and water. In yet a further embodiment, the product may include oleogel and one or more of the above indicated other components. Alternatively or additionally, the product may include a pharmaceutical (drug), with e.g. the pharmaceutical being embedded in the oleogel.

In yet a further aspect, the invention also provides a protein aggregate material comprising over 50 wt.% globular protein, such as at least about 60 wt.% protein, even more especially at least 70 wt.% protein, wherein the protein aggregate materials comprises protein comprising particles, especially having a volume averaged particle size selected from the range of 100-2000 nm, especially 100-1000 nm, yet even more especially 150-500 nm. These particle sizes especially refers to the particles suspended in e.g. hexane or in an oil, or to the primary particles in a powder. In a powder the aggregate particles may aggregate to larger particles. However, such larger particles substantially comprises aggregates of protein aggregate particles having a volume averaged particle size selected from the range of 100-2000 nm, especially 100-1000 nm, yet even more especially 150-500 nm. Such protein aggregate material may have a packing density selected from the range of about 0.05-0.5 g/cm 3 , such as 0.05-0.3 g/cm 3 , such as 0.05-0.2 g/cm 3 . Such material may be dispersed in a liquid lipid material, especially an oil, to provide the herein described oleogel. Packing density may especially be evaluated according to ASTM D7481.

In yet another aspect, the invention also provides a method for the production of (such) protein aggregate material, the method comprising: (i) providing a water protein composition, wherein the composition comprises protein and water and 0- 10 wt.% of one or more other components, wherein the water protein composition comprises protein aggregates, and wherein the protein comprises a globular protein; (ii) exchanging in a solvent exchange process at least part of the water by a non-polar solvent, wherein optionally an intermediate solvent is applied wherein water is solvable, to provide a protein aggregate dispersion in a non-polar solvent; and (iii) providing a protein aggregate material by separating in a separation stage the protein aggregates from the non-polar solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Fig 1 : displays images of oleogels prepared at different volumes. A: WPI (whey protein isolate) macrogel with a volume > 1 cm 3 , B: oleogels with a volume of roughly 1 mm 3 , C: oleogel paste consisting of oleogels of 1-1000 μιη 3 , D: oleogel paste consisting of protein aggregates; see further also Fig. 14;

Fig. 2: displays microscopy images (SEM and CLSM) of the different types of oleogels. A: Cryo-SEM pictures of a WPI macrogel (macro WPI oleogel, 91% oil); B: CLSM images of ± 1 μιη 3 particles (macro-sized WPI particles suspended in oil after solvent exchange. Stains used are FITC and Nile Red. Protein particles appear as bright spots); C: CLSM images of an oleogel paste consisting of protein aggregates WPI aggregates ~ 250 nm suspended in oil imaged by CLSM after solvent exchange. Stain used: Pvhodamine. Protein aggregates appear as bright spots);

Fig. 3: displays light microscope pictures of WPI aggregates prepared by heat treatment at 4 wt.% protein at pH 5.7 in water, dispersed in sunflower oil prepared using acetone drying (Fig. 3a) or short solvent exchange procedure (Fig. 3b). The bar indicates a length scale of 50 μιη;

Fig. 4: Solvent exchange steps, from water to acetone to oil; ten stages I- X are schematically depicted: I: 70% demi water and 30% acetone; on the bottom a hydrogel; II: 50 % demi water and 50%> acetone; III: 30%> demi water and 70%> acetone; IV: 100% acetone; V: 100% acetone; VI: 30% oil and 70 % acetone; VII: 50% oil and 50 % acetone; VIII: 70% oil and 30 % acetone; IX: 100% oil; X: 100% oil; on the bottom oleogel; Figs 4-1 and 4-X schematically depict a holder than can be used to host the hydrogel / oleogel (three white boxes; rectangle below the boxes schematically indicates a magnetic stirrer); of course, the invention is not limited to such embodiment);

Fig. 5: displays the oil binding capacity as a function of the gel strength of the WPI gel (x-axis Modulus (M) in kPa);

Fig. 6: displays the acetone concentration as a function of drying time (at RT) (x-axis time (t) in hours (h); y-axis concentration (c) acetone in ppm;

Fig. 7: Displays the oil loading (Q oi i) for 10% WPI gels at pH 7 and 9; S indicates a standard, A indicates albumin and I indicates immunoglobulin;

Fig. 8: displays the frequency sweep of the oleogel prepared with protein aggregates (WPI). Filled squares: G', open squares: G", triangles: tan5; on the x-axis the frequency (f) in herz (hz) is inidcated and on the left y-axis the modulus (M) in Pascal (Pa);

Fig 9: displays the stickiness of the different cookie doughs, after 50%> compression; Fig. 10: displays the results of a sensory evaluation of the cookies; Fig. 11 : displays the breaking force and the maximum force at fracture of a sausages; Fig. 12: displays the sensory evaluation of the sausages;

Figs. 13 a- 13b: display SEM graphs WPI aggregates dried from hexane (13a) and commercial WPI powder, respectively; Fig. 14: display another example of particle sizes of WPI powder in water, acetone and hexane, with the respective SEM graphs; and

Figs. 15a- 15b: display the particle size of whey protein aggregates, formed by heat treatment at 85°C for 15 min of a 4% whey protein isolate (WPI) solution at pH 5.7. Particle size is measured in sunflower oil after the solvent exchange procedure, where the water is first exchanged to acetone, followed by an exchange for sunflower oil (Fig. 15a); and the particle size distribution of whey protein aggregates, formed by heat treatment at 85°C for 15 min of a 4% whey protein isolate (WPI) solution at pH 5.7. Particle size is measured in sunflower oil after re-dispersing dried aggregate powders using different drying media: (dashed:) air drying a WPI aggregate suspension from the solvent water; (dotted:) air drying a WPI aggregate suspension from the solvent acetone, where the water was exchanged for acetone; and (line:) air drying a WPI aggregate suspension from the solvent hexane, where the water was exchanged for acetone and the acetone was exchanged for hexane.

The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The term "oleogel" is known in the art and refers to a gel having an oil as a continuous phase. The percolation structure (in this invention) is based on the proteins. Hence, in the absence of gelling agents or surfactants that are necessary in the art to create oleogels herein the oleogel is substantially based on oil and protein, with the latter forming the percolating structure. This structure can e.g. be seen with SEM (scanning electron microscopy) or CLSM (confocal laser scanning microscopy). In embodiments the oleogel may essentially consists of protein and oil or a lipid material based on oil (see also below), though in other embodiments the one or more of gelling agents and surfactants may also be comprised by the oleogel.

The oil, or other lipid based material, may comprise any oil, but may for food applications especially be selected from vegetable oils, such as one or more selected from the group consisting of coconut oil, corn oil, cottonseed oil, flax seed oil, linseed oil, olive oil, palm oil, peanut oil, safflower oil, soybean oil, sunflower oil, etc. Additionally or alternatively, the oil may include one or more of grape seed oil, sesame oil, argan oil, rice oil, rice bran oil, algal oil, canola oil, maize kern oil, and echium oil. Additionally or alternatively, the oil may include one or more of squid oil and fish oils such as salmon oil and halibut oil. Hence, the term "oil" may also refer to a plurality of different oils. A vegetable oil is a triglyceride extracted from a plant. Optionally, the oil may also be modified, such as by hydrogenation. In a specific embodiment, the (vegetable) oil comprises one or more of sunflower oil and canola oil, etc.. In yet another embodiment, the (vegetable) oil comprises one or more of palm oil and olive oil. In yet another embodiment, paraffin oil may be used as oil. In an embodiment, especially when a macro gel is applied, the oil may be selected from one or more of sunflower oil, canola oil, maize kern oil, rice oil, flax seed oil and olive oil. In yet another embodiment, especially when protein aggregates are applied the oil may be selected from one or more of sunflower oil, maize kern oil, rice oil, flax seed oil and olive oil. Other lipid based materials, other than oil, may refer to semi- so lid fats, such as chicken fat, pork fat, or other animal fats.

The protein may comprise any protein. However, especially good results were obtained with globular proteins. Examples of suitable proteins are hemoglobin, immunoglobulin, (bovine serum) albumin, ovalbumin, a-lactoglobulin, β-lactoglobulin, soy protein, etc.. Hence, as source for the protein (see also below), e.g. one or more of pig blood serum albumin, pig blood serum immunoglobulin, hen egg ovalbumin, whey protein isolate, whey protein concentrate, soy protein isolate, soy protein concentrate, etc., may be applied. Other suitable proteins may be derived from one or more of soy (see above), wheat, pea, rice, lupine, potato, canola, etc. Yet other suitable proteins may be derived from e.g. egg white, such as ovalbumin (see above). Using gelatin, (an example of) a non-globular protein, no oleogel was obtained. The term "protein" may also refer to a plurality of different proteins. In a specific embodiment, the globular protein comprises one or more of whey protein, albumin and immunoglobulin.

The oil and the protein (in the oleogel) may be available in a weight ratio of 100:0.5 - 60:40. The one or more other components in the oleogel may come along with the starting materials (see also below). The one or more other components (in the oleogel) may e.g. comprise one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a bioactive compound (such as a vitamin), a gelling agent (such as a polysaccharide), and a surfactant. Even more especially, the one or more other components (in the oleogel) may e.g. comprise one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a vitamin, a gelling agent (such as a polysaccharide), and a surfactant. The bioactive compound may e.g. include one or more of a vitamin, a mineral, an antioxidant, etc. Especially, these one or more other components may be available in the oleogel in amount of 10 wt.% or less (dry weight), especially in amount of 5 wt.% or less (dry weight), such as in amount of 2 wt.% or less, such as 1 wt.% or less (dry weight). Hence, in an embodiment the 0-10 wt.% of one or more other components comprise one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a vitamin, a bioactive compound, a gelling agent (such as a polysaccharide), and a surfactant.

Especially, the oleogel does not (necessarily) include one or more of a surfactant and a (additional) gelling agent, as the percolating structure is formed by the protein without the need of such surfactant or gelling agent. Examples of surfactants include e.g. nonionic, cationic, amphoteric, and polymeric surfactants, etc. Typical gelling agents include natural gums, cellulose, starches, pectins, agar-agar and gelatin. Often they are based on polysaccharides. For instance, in an embodiment the oleogel does not include ethyl cellulose as a gelling agent, or only up to an amount of 0.5 wt.% (dry weight), especially only up to an amount of 0.2 wt.% (dry weight). As indicated above, the one or more other components are not necessarily available.

Water is not considered one or more of the other components. The oleogel, as obtained with the herein described method, may include e.g. up to about 5 wt.% water, such as up to about 3 wt.% water, like up to about 1 wt.% water, relative to the total weight of the oleogel. A water content up to these values may add to the gel strength. The amount of water that may be included in the oleogel may amongst others depend upon the presence of a salt (see also below) in the starting materials. Hence, the oleogel as such is substantially free of water, i.e. equal to or less than 5 wt.% water.

Of course, the oleogel may be applied in water containing systems. In such applications, the oleogel may absorb water and the water content can be much higher. For instance, the total weight of such oleogel may include up to 70% water, i.e. up to 70 wt.% relative to the total weight may be water, the other 30% essentially including lipid (including oil) and protein (see further also below at the (non-) food product comprising the oleogel. Hence, the invention also provides a water comprising oleogel, comprising at least 30 wt.% lipid (including oil) and protein, and in the range of 5-70 wt.% water.

Herein, substantially only the above-mentioned protein is used as gelling agent. In this way, an oil continuous phase is obtained with the protein as gel former. As indicated above, the protein forms a percolating structure in the micrometer range, in which the building blocks are proteins (1-20 nm) or protein aggregates (5-1000 nm, such as in the range of at least 20 nm, like in the range of 30-500 nm, like especially 150-500 nm) forming either a fine stranded or a course network. Hence, the lipid material (oil) essentially represents the continuous phase of the oleogel. Therefore, in the oleogel the protein is available in the lipid material as one or more of protein aggregates (5-1000 nm, especially 150-500 nm volume average particle size) and protein particles (1-20 nm).

In general, the present oleogel can be made in two ways. One may first create a hydrogel by forming a space spanning network of the proteins and exchange the aqueous phase into an oil phase in a solvent exchange process, thereby providing the oleogel. In an alternative method, one may first create protein aggregates (5-1000 nm) and exchange in a solvent exchange process the aqueous phase for an oil phase, thereby initiating further network formation of the protein aggregates, providing the oleogel.

Hence, the starting composition especially comprises water and protein. The amount of protein may be as high as the solubility of the protein (or proteins), but the amount may even be higher (i.e. over saturated). In general, the (starting) composition may comprise in the order of 0.5-30 wt.% (relative to the total composition, including water), especially in the range of 0.5-15 wt.%, of the protein. Proteins of interest and sources of proteins of interest are indicated above. The amount of water in the starting composition may be in the range of at least 50 wt.%, such as especially 70-99.5 wt.% (relative to the total composition, including the water), even more especially in the range of 80-99 wt.%. Further, as indicated above the starting composition may include other starting components than water and protein, such as one or more of a salt, a sugar, a fat, a fatty acid, an amino acid, a vitamin, a gelling agent, and a surfactant.

As indicated above, in a first embodiment the water protein composition may comprise a hydrogel. Hence, in such embodiment the method includes the formation of the hydrogel based on the starting materials including the protein and water. When using a hydrogel as starting material, especially the water protein composition comprises at least 4 wt.%, such as at least 8 wt.% globular protein, in order to form a space spanning network.

Such hydrogel may have any dimension, from micrometers to centimeters.

Especially, in an embodiment the hydrogel may have a volume of at least 0.1 μιη 3 , such as especially in the range of 1-1000 μιη 3 . Relatively larger hydrogels may have a volumes of at least 1 mm 3 , such as even at least 1 cm 3 (indicated as a "macro gel"). In yet another embodiment, the hydrogel has a volume in the range of 1000 μιη 3 - 1 mm 3 . Hence, in embodiments the hydrogel has a volume in the range of 1 μιη 3 - 1 mm 3 , though larger may also be possible (see above).

After the solvent exchange process, such (macro) (hydro)gels also lead to (macro) oleogels. Such (macro) oleogel may be used as such, or may optionally first be processed in smaller oleogel particles. Hence, in an embodiment the oleogel may have a volume of at least 1 μιη 3 , such as in the range of 1-1000 mm 3 , or even larger than 1 cm 3 (and subsequently be processed into smaller oleogel particles). In yet another embodiment, the oleogel has a volume in the range of 1000 μιη 3 - 1 mm 3 . Hence, in embodiments the oleogel has a volume in the range of 1 μιη 3 - 1 mm 3 , though larger may also be possible (see above).

The solvent exchange process is further elucidated below.

Further, as indicated above in a second embodiment protein aggregates are used. Hence, in a further embodiment the water protein composition comprises protein aggregates. When using protein aggregates as starting material, especially the water protein composition comprises at least 2 wt.% globular protein. The oleogel is formed after the solvent exchange method. Whereas the macro oleogel is a gel like body, the protein aggregates based oleogel may have a more paste like character.

Protein aggregates may in embodiments e.g. be obtainable by e.g. providing a solution of proteins, such as WPI, in water. Especially, a substantially complete solubilization is ensured. The pH of the solution may be maintained close to the isoelectric point (Pi), such as in the range of Pi+/-3, especially Pi+/-1, even more especially ΡΪ+/-0.2. Thereafter, the solution is heated to a temperature of at least 80°C, especially for at least 10 min., followed by a cooling to room temperature. A (weak) (opaque) gel may thereby obtained. The gel is broken into small gel fragments by shaking and vortexing. In this way, the protein aggregates may be obtained. This method may especially be applied in the absence of a salt. When a salt is applied, other conditions may be applied, such as a lower heating temperature. Further, it may not be necessary that a gel is formed. Especially, the conditions are chosen close to the gelling point (i.e. not yet a gel), or a gel is formed. The protein aggregates may have dimensions in the range of 100-500 nm, such as 150-300 nm, as can be determined by light scattering. Hence, the water protein composition comprises protein aggregates as described herein may be obtainable by solubilizing globular proteins in water, heating the thus obtained protein solution to provide a gel, and breaking the gel by applying shear, such as shaking and/or vortexing. The dimension(s) especially refer to diameter(s). These protein aggregates may be used in the method as described herein to provide an oleogel, wherein a solvent exchange is applied. It also appears that the solvent exchange may be interrupted to provide of a protein aggregate material as intermediate product, which may then (later) be used to disperse in an oil.

Hence, the invention also provides a method for the production of a protein aggregate material, the method comprising: (i) providing a water protein composition, wherein the composition comprises protein and water and 0-10 wt.% of one or more other components, wherein the water protein composition comprises protein aggregates, and wherein the protein comprises a globular protein; (ii) exchanging in a solvent exchange process at least part of the water by a non-polar solvent, wherein optionally an intermediate solvent is applied wherein water is solvable, to provide a protein aggregate dispersion in a non-polar solvent; and (iii) providing a protein aggregate material by separating in a separation stage the protein aggregates from the non-polar solvent.

In further embodiments, the intermediate solvent comprises one or more solvents selected from the group consisting of acetone, tetrahydrofuran, ethanol, propanol, butanol, and DMSO (see also above). Yet further, the non-polar solvent is selected from the group consisting of C6-10 alkanes. Hence, the solvent exchange process may be as described above, until the stage that substantially all water is removed and lipid material might be introduced. Hence, e.g. in the solvent exchange, the solvent exchange may be started with the intermediate solvent which is then replaced by the non- solvent, to provide the dispersion of the protein aggregates in the non- solvent. By providing the protein aggregate dispersion in a non-polar solvent, a material is obtained that may be dried and stored, and may be used for a later generation of the oleogel. The separation stage may especially include one or more of air-drying and freeze-drying.

The aggregation between proteins can be induced by different methods, which are known as pH-induced, solvent-induced and heat-induced aggregation. In yet another embodiment the water protein aggregates are obtainable by the method as defined above for making a hydrogel.

During the solvent exchange process, the water in the hydrogel and/or protein aggregates is exchanged via an intermediate solvent for the oil. Especially, the solvent exchange process is a stepwise exchange process, wherein in exchange steps the water content is gradually decreased and the intermediate solvent is gradually increased, followed by exchange steps wherein the intermediate solvent content is gradually decreased and the oil content is gradually increased. During a stepwise solvent exchange, the material of interest is transferred from one type of solvent to another. When performing such a solvent exchange with a hydrogel or protein aggregates, the gel or protein aggregates are subsequently immersed into mixtures of solvents for a given time. During the solvent exchange, the solvent composition is changed stepwise into solvents with lower dielectric constants and higher hydrophobicity, creating a potential driving force for swelling or shrinking, since with each step the solvent quality will change, as a result changing the swelling equilibrium.

Hence, in a specific embodiment the solvent exchange process comprises at least a first exchange, wherein the protein composition is exposed to a liquid comprising water and the intermediate solvent, and a second exchange, wherein the protein composition is exposed to a liquid comprising the intermediate solvent and oil.

Further, in yet a more specific embodiment of the solvent exchange process, in a first stage of the solvent exchange process a first series of exchanges are executed wherein the protein composition is exposed to a first series of liquids that decrease in water content to a last liquid (in the first series) substantially comprising the intermediate solvent, and subsequently in a second stage of the solvent exchange process, a second series of exchanges are executed wherein the protein composition is exposed to a second series of liquids that decrease in intermediate solvent content to a last liquid (in the second series) substantially comprising the oil.

When using aggregates, each solvent extraction stage, wherein the protein aggregates are mixed with a liquid, may include thorough mixing, including e.g. high shear, such as with a Vortex. Hence, when before a final solvent exchange stage a gel might have been formed, the gel is physically broken down into protein aggregates (for use in a next stage). Further, each stage may include a removal of at least part of the liquid, such as by evaporation and/or a separation method like centrifugation, and optionally a further (re)suspension in a liquid with a higher intermediate solvent content or oil content, respectively.

As intermediate solvent (herein also indicated as "solvent"), any solvent may be used that is at least partly miscible with water and oil. In embodiments, the intermediate solvent comprises one or more solvents selected from the group consisting of acetone, tetrahydrofuran (THF), ethanol, propanol (such as n-propanol), butanol (such as n-butanol), and dimethyl sulfoxide (DMSO). Alternatively or additionally, the solvent may comprise one or more dichloromethane (DCM), ethyl acetate, dimethylformamide (DMF), acetonitrile (MeCN), propylene carbonate, formic acid, isopropanol (IPA), methanol, acetic acid, and nitromethane. Hence, the term intermediate solvent may also refer to a plurality of different intermediate solvents. Note that the composition of the intermediate solvent may optionally differ per exchange step. For instance, in a first exchange the intermediate solvent may only include ethanol and in a next exchange the intermediate solvent may e.g. include a combination of ethanol and acetone.

When the solvent exchange process was used for the hydrogel, good results were obtained with acetone and THF. When the solvent exchange process was used for the protein aggregates, good results were obtained with acetone, THF, ethanol, and 2- butanol. An advantage of using low temperature boiling solvents, such as acetone, is that these solvents may evaporate and may not be present in the final oleogel and/or may be easily removed therefrom.

Hence, the amount of organic intermediate solvent in the final oleogel may be below the detection limit, such as < 0.5 ppm (mg/kg), such as (well) below 0.1 ppm. However, would still some organic solvent be available in the oleogel, the oleogel may comprise the organic intermediate solvent in an amount of 0.5 ppm - 1 wt.%, especially the amount of organic intermediate solvent is less than 0.01 wt.%. Note that these weight percentages relate to the total amount of organic intermediate solvents.

The solvent exchange step using protein aggregates, may also result in a protein aggregate dispersion in a non-polar solvent, such as pentane, especially n-pentane, hexane, especially n-hexane, cyclohexane, heptane, especially n-heptane, octane, especially n-octane, nonane, especially n-nonane, decane, especially n-decane, and toluene. Other possible suitable non-polar solvents may include 1,4-dioxane, chloroform, diethyl ether, dichloromethane, etc. The non-polar solvent may be removed from the protein aggregates by a drying step, with a method such as air-drying, freeze-drying, spray drying, using a rotary evaporator, sub critical C0 2 drying, etc., to obtain a dried powder. The solvent exchange procedure can be continued or the dried protein can be re- dispersed directly in different oils. Best results were found using hexane, decane and heptane as the non-polar solvent. Herein, the term "solvent" may also refer to a combination of different solvents.

Hence, in specific embodiments the intermediate solvent comprises one or more solvents selected from the group consisting of acetone, tetrahydrofuran, ethanol, propanol, butanol, and DMSO, and wherein especially the non-polar solvent is selected from the group consisting of C6-10 alkanes, and wherein the separation stage includes one or more of air-drying and freeze-drying, though other drying techniques (see above), may also be possible. In yet further specific embodiments, the water protein composition comprising protein aggregates is obtainable by solubilizing globular proteins in water, heating the thus obtained protein solution to provide the protein aggregates, especially to provide a gel, and applying shear to provide the protein aggregates, especially breaking the gel by applying shear, to provide the protein aggregates of globular proteins.

After the solvent exchange process, a substantial amount of the water has been replaced with the oil. The percolating structure of the hydrogel may substantially have been maintained, but the continuous phase has been replaced with the oil. The protein aggregates build together with the oil the oleogel. The oleogel has characteristics like a storage modulus G' (elastic response) being (substantially) larger than a loss modulus G" (viscous behavior), such as at least 10 times higher.

As indicated above, the invention also provides an oleogel obtainable with the above described method. The oleogel thus obtained may be used in food applications or non-food applications. The gel may be used as such, or may be processed into smaller gel particles. In an embodiment, the invention also provides a food product comprising an oleogel phase as defined herein.

Examples of food products that can be prepared by using the present oleogel include baked goods such as cookies and cakes; spreads such as margarine and chocolate spreads; chocolate and fillings; and meat products, such as a ground meat products such as hamburger meat, or a meat emulsion product such as bologna, mortadella, frankfurters, or other sausage products. In an embodiment of the food product, the oleogel phase is a continuous phase, such as e.g. in the case of a spread. In another embodiment of the food product, the oleogel phase is a discontinuous phase with oleogel regions having one or more dimensions (like length, width, height, and diameter) in the range of up to 10 mm, such as in the range of 1 μιη - 10 mm, like 10 μιη - 10 mm. For instance, this may be the case in meat products. Further applications of the oleogel as described herein may be in low fat spreads (e.g. mouth feel and/or stabilization of emulsion), in meat and fish products (e.g. for texture and/or gelling), desserts or dairy products (e.g. for texture, thickening, gelling), confectionary (e.g. for gelling, texture, chewability, stabilization, binding, etc.). Hence, in an embodiment the food product is selected from the group consisting of a pudding, a mousse, a butter, a processed food product, a, farinaceous food product, etc. As indicated above, in a further embodiment the invention provides a non-food product comprising said oleogel phase. For instance, the product is selected from the group consisting of an animal feed product, a cosmetic product, and a pharmaceutical product, etc.. For instance, the oleogel phase may be used as delivery system for hydrophobic molecules, which may be of interest in cosmetic and/or pharmaceutical applications.

The term "substantially" herein, will be understood by the person skilled in the art. The term "substantially" may also include embodiments with "entirely", "completely", "all", etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term "substantially" may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term "comprise" includes also embodiments wherein the term "comprises" means "consists of. The term "and/or" especially relates to one or more of the items mentioned before and after "and/or". For instance, a phrase "item 1 and/or item 2" and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species". Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications. EXPERIMENTAL

The oleogels can be prepared with different methods and with different starting materials (different volumes of hydrogels). Figure 1 gives a non-limiting overview of the different oleogels that can be prepared. Fig. 1A gives an example of an oleogel in the range of 1 cm 3 , Fig. IB an oleogel in the range of 1 mm 3 (i.e. a plurality of oleogels in the form of particles is provided), Fig. 1C a gel paste containing oleogel particles of roughly 1-1000 μιη 3 , and Fig. ID an oleogel prepared from WPI aggregates, such as having volume averaged diameters of about 150 nm. The preparation methods will be discussed below in more detail. SEM and CLSM images for the different gels are given in Figure 2. Figure 2A represents an image of a macrogel, 2B represent an image of oleogels with a volume of roughly 1-1000 μιη 3 , and 2C, a CLSM image of an oleogel paste containing WPI aggregates forming a network.

Experimental series 1: protein aggregates

Protein aggregate examples

In the current research, a protein in oil suspension is produced using whey protein isolate (WPI) and sunflower oil as a model system.

Materials and methods

Materials

Whey protein Isolate, BiPRO (93.2% protein) was purchased from Davisco

Foods International, Inc. (le Sueur, MN, USA) and used as received. Vegetable oils, Sunflower oil (Reddy), Rapeseed oil (Hollands goud), Rice oil (King), Corn oil (Albert Hein), Extra virgin olive oil (Monini) were purchased at a local supermarket and used without further purification. Demineralized water was used throughout the experiments. All Chemicals, Hydrogen chloride, Acetone, Ethanol, 2-Butanol, Tetrahydrofuran (THF), Cyclohexane, Petroleum benzene were of analytical grade and purchased from Merck (Damstadt, Germany), Sigma-Aldrich (Steinheim, Germany) or Actu-All chemicals (Oss, The Netherlands).

Methods

Protein in oil suspension

Production of WPI aggregates

The WPI aggregates produced in this research were based on the method described by Schmitt et al, Food HydrocoUoids 25 (201 1) 558-567. A 4% WPI (powder based) solution (stirred for 2 hours and left overnight to ensure complete solubilisation) was adjusted to pH 5.7 with 1M HC1. This solution was left at room temperature for 20 min while continuously stirring on a magnetic stirrer plate. The pH was corrected again to 5.7 with 1M HC1. After the second pH correction, the samples were heated in a water bath for 15 min at 85°C. The solutions were cooled on ice water. The specific sample composition resulted in the formation of a weak opaque gel, which was broken into small gel fragments by shaking and vortexing for 1 minute.

Production of protein in oil suspension

The solvent exchange method can be performed with different types and number of intermediate steps. In this research either 9 steps (long) or 4 steps (short) were used. For the long method, the WPI aggregates were transferred in 9 steps from a protein in water suspension to a protein in oil suspension. First 15 ml of the protein aggregate solution was mixed with 15 ml acetone to obtain a 50% water / 50% acetone solution. This solution was vortexed and subsequently centrifuged (Hermle Z383K, Hermle Labortechnik GmbH, Wehingen, Germany) for 20 min at 6000 rpm (3904xg) at 20°C (all the following centrifugation steps were performed with the same settings). The supernatant was removed, and the pellet was re-suspended in 30 ml 30%> water/70%) acetone with the use of a high speed blender (Ultra-turrax, T25, IKA Werke, Germany) with a speed of 9500 rpm and centrifuged. This procedure was repeated with pure acetone twice. Then, the pellet was re-suspended in 70%> acetone / 30%> sunflower oil upon mixing and again centrifuged. This procedure was repeated for 50%> acetone/50%) sunflower oil, 30%> acetone/70%> sunflower oil and twice for pure sunflower oil. The last pellet (an opaque gel) was left overnight in the fume hood to evaporate the last traces of acetone. For the short method (4 steps), only pure acetone was used as an intermedate solvent (twice), and the pellet was re-suspended twice in pure oil.

Intermediate solvents

Besides acetone, 2-butanol and ethanol were also used as intermediate solvents using the short method. These solvents were not suitable for the long method because of incomplete miscibility with water (2-butanol) or oil (ethanol). Ethanol showed limited solubility in the oil phase, which made it impossible to complete the solvent exchange directly. Instead, the protein in ethanol suspension was mixed with the oil phase with the use of a high speed blender (Ultra-turrax, T25, IKA Werke, Germany) with a speed of 9500 rpm into a suspension. Subsequently, the ethanol was evaporated at room temperature in the fume hood.

Drying step

In an embodiment, before the protein in intermediate solvent suspension was mixed with the oil phase, the suspension was mixed with hexane (as a representative of a non-polar solvent). The hexane was evaporated by air drying and the dry protein powder was further used to create protein oleogels by mixing the powder directly into different oils.

Final solvents

Besides sunflower oil as final solvent, also rapeseed oil, rice oil, corn oil, and extra virgin olive oil were used to produce the protein in oil suspensions. The short solvent exchange was used, with acetone as an intermediate solvent. Characterisation

Protein content: Dumas

To calculate the protein content of the samples, a calibration curve was made using 2, 4, 6, 8 and 10 mg of D-methionine as a reference for the nitrogen content. About 50-100 mg of sample was weighed. The samples were dried overnight in an oven at 60°C. The samples were analysed in the dumas (Flash EA 1112 Series, N Analyser, Thermo Scientific). A nitrogen conversion factor of 6.38 was used for WPI to convert the nitrogen content to the protein concentration.

Oil content: Soxhlet

0.4-1 g sample was weighed into a filter extraction tube (Whatman, grade 603, 33x118mm). The filters were covered with cotton wool to prevent leaking of the samples and placed into the extraction tube. The oil was extracted with petroleum benzene between 40 and 60°C. The extraction time was set to 3 hours, which was an indication of three extraction rounds. After cooling down the sample, the petroleum benzene was evaporated with a rotary evaporator (Bvichi Rota vapor R-215, Bvichi Labortechnik AG, Switzerland) at 60°C with a rotation speed of 125 rpm connected to a vacuum pomp (Bvichi vacuum controller v-800, Bvichi Labortechnik AG, Switzerland) to decrease the pressure to 200 mbar. The extracted oil of the samples was left overnight in the fume hood and weighed to determine the oil content. Water content: Dry weight content

To determine the water content, approximately 1 g of protein in oil suspension was weighed into an aluminium cup, and dried in the oven (Venticell, MMM Medcenter Einrichtungen GmbH, Grafelfing, Germany) for 24 hours at 105°C. After cooling down the samples were weighed. The loss in weight was assumed to be due to evaporation of water.

Particle size determination: Static Light Scattering

To determine the size of the protein aggregates, the samples were diluted with sunflower oil or water to a protein concentration of 7.5 mg/ml. The samples were mixed with a high speed blender (Ultra-turrax, T25, IKA Werke, Germany) with a speed of 9500 rpm. The particle size distribution of WPI aggregates was determined with the use of static light scatting (Mastersizer 2000, Malvern Instruments, Worcestershire, UK) after 10 minutes of ultra-sonication with sunflower oil or demi water as the continuous phase. The refractive index of water was set to 1.33 and for sunflower oil to 1.469. The average particle size was defined as the mean of the main primary aggregate peak.

Structure of protein in oil suspension

Confocal Laser Scanning Microscopy (CLSM)

The difference in structure of protein in oil suspension produced with the short and long solvent exchange method was analysed with the use of confocal laser scanning microscopy (CLSM). A drop of rhodamine (0.2%) in ethanol was mixed with the oleogel to colour the protein aggregates. The samples were analysed with CLSM (Leica tcs sp5, Leica Microsystems, Wetzlar, Germany). Results and discussion

Preparation of the WPI aggregates

Aggregates were prepared with a protein concentration of 4% and a pH of 5.7, which is close to the pi of whey proteins. As a result, a weak opaque gel is formed. Concentrations less than 4% did not lead to large enough aggregates to form a weak gel. For the production of these WPI aggregates, the adjustment of the pH is a critical step. Small differences in the pH resulted in different sizes of the aggregates. If the initial pH prior to heating is around 5.8, a stable WPI aggregate suspension is produced. At an initial pH of 5.7, a weak gel is formed. This weak gel can easily be broken; the small network pieces can form a pellet when centrifuged at 3900xg for 20 minutes. For this reason pH 5.7 is used for the production of the WPI aggregates.

From WPI aggregates in water to protein in oil suspension

Results show that simply suspending commercially available dried protein in oil is not possible. Also not when the commercially available protein is dried from different solvents. The solvent exchange method, in which the proteins always stay in a wet condition, is necessary to obtain the oleogels. A gradual increase of the acetone and oil content was first used, according to the long solvent exchange procedure. To save time, also a short solvent exchange was performed. Both methods seem to result in, visually, similar protein in oil suspensions. A picture of the suspension (the oleogel) can be found in figure ID. Further, the aggregates of the invention allow formation of an oleogel just by adding the aggregates to the oil, whereas, as indicated above, commercially available protein powders are not able to do so.

To investigate the influence of acetone on the process, other solvents were used instead of acetone. 2-Butanol and ethanol were also used to remove the water. For both solvents, the short solvent exchange method was tested. Small differences were found between the different solvents and compared to acetone. Using the solvent exchange procedure, both 2-butanol and ethanol could be used as an intermediate solvent and results in a similar product as when acetone is used as an intermediate solvent. So, it seems that the intermediate solvent is not limited to acetone, and that other solvents also provide the necessary conditions (which could be conformational changes to the protein aggregates or providing sufficient wetting) to be dispersed in an oil continuous phase. In addition to the use of sunflower oil, also other oils (rapeseed oil, rice oil, corn oil, extra virgin olive oil) were suitable to produce a protein in oil suspension. As observed visually, stability and viscosity are comparable to the protein in oil suspension produced with sunflower oil. Therefore, the oleogelation is not specific for a certain type of fatty acid composition, and can be used for different hydrophobic oils and solvents, or other lipid based materials.

When the solvent exchange using the protein aggregate suspension was performed with acetone as intermediate solvent and hexane as final solvent, a protein aggregate suspension can be created. Subsequently, when the material was dried, e.g. by airdrying, a very light and low-density protein powder was obtained. Microscopy images indicates that severe protein aggregation (clustering) was limited, which is often seen when dried from aqueous environments. When dispersed in vegetable oil (such as sunflower oil), the protein aggregates were found to be of similar size as the inital protein aggregates in water. Table 1 gives an overview of the aggregate (or cluster) size of the powder dried from different solvents and the respective packing density of the protein aggregate powder. See also Figs. 3, 14, 15a-15b. Fig. 3 displays light microscope graphs of WPI aggregates/particles dispersed in sunflower oil after acetone drying (3a) and obtained according to the solvent exchange procedure; Figure 14 schematically gives an overview of the dried powder, dried from water, acetone and hexane, including concomittant SEM graphs; and Figs. 15a- 15b show the volume averaged particle size of the aggregates obtained via solvend exchange via the hydrogel-acetone-oil solvent exchange procedure (Fig. 15a), or the aggregates obtained after solvent exchange with water, water/acetone, or water/acetone/hexane, and then dried from the solvent and redispersed in sunflower oil (Fig. 15b).

The drying step from non-polar solvents changed the properties of the protein-protein and protein- solvent interactions to such extent that clustering of the protein aggregates was prevented and a low density powder was obtained. This powder was found to be easily re-dispersable in other solvents.

The thus obtained three different powders have different densities (see table 1) and thus a different particle size distribution in the sunflower oil. The powder re- dispersed after air drying from hexane had a particle size distribution similar to the 'reference' (Fig. 15a), where the WPI aggregates were transferred to the oil phase using the standard solvent exchange procedure.

Tablel. aggregate size of WPI aggregates suspended in oil after drying from different solvents.

Solvent used in drying step Aggregate size Powder packing density (g/cm 3 )

Water ± 200 micron 0.26

Acetone ± 50% 700 nm, 50% ±200 micron 0.14

(bimodal distribution)

Hexane ± 200 nm 0.07

Oleo reference ± 200 nm

(not dried but solvent exchange) Characterisation

To characterize the protein in oil suspensions obtained with different methods and solvents, the composition and particle size are determined.

Composition

The protein content of the different protein in oil suspensions can be found in Table 2. Depending on the length of the solvent exchange method and the type of intermediate solvent, the protein content is between 7 and 10%. In the case of acetone, the protein content is roughly 8-10%. However, when ethanol or 2-butanol is used as an intermediate solvent, a lower protein content of roughly 7% is found. The differences observed may be a result of the inaccuracy of the last supernatant (oil) removal after centrifugation, leading to dilution of the protein in oil suspension.

Table 2. Protein and water content of different protein in oil suspensions.

the standard deviation is based on a triplicate measurement.

The method to detemine the water content did not lead to reproducable and trustworthy results. For most of the water content measurements, the mass of the samples increased after drying instead of an expected decrease, probably caused by the absorbance of water by the protein aggregates after heating. Therefore, it is expected that the water content is limited, but no exact values can be given. Also the method for the oil determination did not always lead to satisfactory results. Only the oil content for the protein in oil suspension produced with the use of acetone as an intermediate solvent with the long solvent exchange resulted in a reliable result of 89.8% ± 1.1%, which is an average of a triplicate measurement. These results at least indicate that most of the water was removed from the sample, and replaced by oil. Overall, the use of an intermediate solvent to produce a viscous protein in oil suspension results in a pellet with around 8%> protein and 90% oil (figure ID). The remaining 2% will probably be residues of intermediate solvents, (bound) water or a result of inaccurate analysis.

Size of the aggregates

The size of the WPI aggregates was measured with the use of light scattering. The aggregate size of the initial aggregates in water was found to be roughy 210 nm. Some extensive secondary aggregation can be observed in the aqueous phase. In oil, the aggregate size is very similar and no secondary aggregation is present. The average size was found to be 210 ± 30 nm. Due to the solvent exchange, the aggregates fall apart into the primary aggregates. Since the size of the primary aggregates in both the aqueous and oil phase is similar, this indicates that the solvent exchange does not influence the aggregate size. For the WPI aggregates in water, still some larger aggregates are present, which are not observed in the oil phase. Probably the attractive hydrophobic interactions between aggregates in an aqueuous environment are no longer present in a hydrophobic environment. The fact that no hydrophobic interactions can be responsible for aggregate formation in an hydrophobic environment (the oil phase), indicates that the aggregate formation must be dominated by covalent bonds or hydrophilic interactions.

Comparing the short method and the long method with acetone a comparable average size distribution is found. Also the use of another intermediate solvent, such as 2-butanol (2 -but) does not influence the aggregate size. To visualize the aggregate formation in oil, the samples were analysed with light microscopy. Figure 3 shows the aggregation for a protein powder (dried from acetone) added to oil (left) and protein in oil suspensions prepared with the solvent exchange (right). The suspension on the left shows larger dense clustering of aggregates. The sample on the right does not show this extensive aggregation. The solvent exchange enables the aggregates to form a more open network. This network is three-dimensional providing the structure to form a gel. Figure 2C shows the network formation in the CLSM image.

Experimental series 2: Oil binding of macroscopic hydrogels

Macro gel

The following Experimental part especially relates to the macro gel.

However, the general principle of solvent exchange also applies to the protein aggregates (see above). Solvent exchange

During a stepwise solvent exchange, the material of interest is transferred from one type of solvent to another. When performing such a solvent exchange with a hydrogel, the gel is subsequently immersed into mixtures of solvents for a given time. This affects swelling behaviour of the hydrogels, as the solvent quality changes with each step. During the solvent exchange, the solvent composition is changed stepwise into solvents with lower dielectric constants and higher hydrophobicity, creating a potential driving force for swelling or shrinking. Table 3 shows solvents with their dielectric constants.

Table 3: Dielectric constants of solvents used

Material and methods

Materials

Whey protein isolate (Bipro, protein content 93.2%) was obtained from

Davisco international incorporated. Sunflower oil (Reddy, Vandemoortele) was bought at a local supermarket. NaCl was purchased from Sigma Aldrich. Acetone was bought from Actu-All chemicals (801010302) purity (>99%). n-hexane was purchased from Actu-All chemicals (808023502), purity (>99%). THF was bought from Biosolve (20220602/4), purity (>99%). All water used in this research was used in the form of demineralized water. Paraffin oil was purchased from Merck (1.07174.1000). D-Methionine (99% purity) was bought from Sigma Aldrich (227210250).

Methods

Gel preparation

A stock solution of 25% WPI (w/w) (on powder basis) was prepared by stirring WPI powder (Bipro, Davisco international incorporated) and water, for at least 2 hours at ambient temperature. To create WPI hydrogels with different microstructure, the ionic strength of the solution was varied by mixing the stock solution with either water or aqueous solutions and 1M NaCl (Sigma Aldrich) to prepare 15% WPI solutions with 0, 15, 30, 50, 100, 135, 170 or 200 mM NaCl. Obtained solutions were transferred into 5 ml syringes (Omnifix®, B. Braun) (0 = 1 cm) inner-coated with paraffin oil (Merck, 1.07174.1000), and were closed with a screw-cap (Becton Dickinson Infusion Therapy AB, Sweden). The solutions were then heated in a water bath at 85 °C for 30 min to induce denaturation and accompanying network formation of the WPI. After heating, the samples were left to cool down to 7 °C in the refrigerator and stored at these conditions for a maximum of 24h until analysis or further treatment. The resulting gels were taken out of the syringes and cut into cylindrical slabs (d = 5 mm, h = 5mm), with a gel slicer. Solvent exchange

The solvent exchange was done using a 10 step exchange method following the procedure as visualised in Figure 4. During the exchange, the gel samples were placed in custom build porous (0 = 1mm) metal holders, with individual chambers (±lcm 3 ), separated by similar porous membranes. This holder was placed in a 600 or 1000 ml beaker and filled to 150 ml with the exchange solution. At this level the holder including the samples was fully submerged into the solvent. The exchange solutions were continuously stirred with a magnetic stirrer, in order to speed-up the exchange procedure. The beakers were closed by a piece of parafilm or a screw cap, except for the final two steps where the beakers were left open, to allow traces of acetone to evaporate.

Duration of each step in the solvent exchange was at least 7 hours to a maximum of total 65 hours. 7 hours was estimated to be sufficient to achieve a proper solvent exchange within the gels. Both the 100% acetone and 100% oil steps were repeated in order to assure proper water and acetone removal respectively. To investigate the influence of a different intermediate solvent, a similar exchange was performed with tetrahydrofuran (THF), in separate 20 ml glass containers with a screw-cap. These were filled with 10 ml of THF and the solution was not stirred. Besides replacing acetone by THF, the remainder of the exchange procedure was kept the same. Before and after the solvent exchange, samples were weighed to determine the efficiency of the exchange. Further analyses was conducted afterwards, samples were stored in oil until they were analysed. Volume of the gels is calculated using composition measurements and using density values of 998 kg/m 3 for water, 918 kg/m 3 for oil and 1400 kg/m 3 for protein. These values are divided by the initial hydrogel volume to obtain the relative volume. Moisture content

The moisture content of the gels was measured according to the method from AOAC, 2000. Samples with known weight were placed on aluminium trays and placed in an oven (Venticell, labo scientific, Ede, The Netherlands) at 105 °C, for three 5 hours. The weight was again recorded and the moisture content was calculated as: W(sample)-W(sample dry)

Moisture content (%) =— —— (1)

W(sample) ' where W(sample) is the weight of the sample before drying, and W(sample dry) is the0 weight of the sample after drying. Weight loss is assumed to be due to water evaporation.

Oil content

The oil content was determined using a custom exchange method, with n- hexane. First the samples were cut into fine pieces (max ~ 1 mm 3 ), to increase the efficiency of the extraction. The samples were placed into a 20 ml glass container, filled5 with hexane. The flask was closed with a screw cap and stored for at least 15 hours. The hexane was replaced once, repeating the 15 hours of storage, to insure a proper fat removal. After the second period the sample was collected by filtration and left for one hour to evaporate hexane, after which the fat content was determined using: n r / ~ W(sample)- W(leftover)

0 Fat content (%) =— — (2)

J W samp e ' where W(sample) is the weight of the sample before extraction, and W(leftover) is the weight of the sample after hexane treatment

Protein content

The protein content was determined using a DUMAS method. Total Nitrogen content was determined with a Flash EA 1112N/protein analyser (Thermo Scientific, Waltham, US). To calculate the protein content (%w/w), these values were multiplied with a conversion factor of 6.38. A calibration curve was made with methionine. Acetone content

In order to see whether the amount of acetone in the samples was below the GRAS level after evaporation, the amount of acetone was measured. Suspensions with 10% protein in oil were made with protein aggregates prepared at pH 5.7. These were left at room temperature for 48 hours while mildly stirring. After 0, 16, 24, 40 and 48 hours a sample of 10 mL was inserted in a 250 mL Schott bottle. The samples were prepared at least 24h before the measurement to ensure an equilibrium between the sample and the headspace. A calibration curve was prepared with 0, 0.5, 5, 10, 15, 50 and 100 ppm acetone in sunflower oil. The amount of acetone in samples was measured with a mass spectrometer (HR PTR/MS, Ionicon, Innsbruck, Austria) by analyzing the peak at mass 59 and corrected with a blanc.

Oil binding capacity

For the different gels, the oil binding capacity was determined by measuring the total weight/volume of the oleogels and comparing this to the weight of the corresponding hydrogel. It is assumed that the oil binding capacity is related to the swelling capacity of the gels, which is dominated by the microstructure of the gels.

Gel strength

The gel strength of the gels was determined by measuring the Young's modulus with the use of a texture analyzer (TA Instruments). The Young's modulus was determined from the linear regime of the stress-strain curve.

Results & Discussion

The microstructure of the hydrogels was changed by setting the gels at different ionic strength. For samples with a low ionic strength, a fine stranded network is formed, whereas samples with high ionic strength form a more coarse stranded network. A SEM image of the structure of the network is given in figure 2A. The gel strength, as measured as the Young's modulus increased as the coarseness of the gel increased.

Oleogel characterization

For the different oleogels, the oil content and the protein content was determined. An example of such an oleogel can be seen in figure 1A. Table 4 shows the results for the different oleogels. From these results, the oil binding capacity (swelling) of the gels is calculated.

Table 4. Characteristics of some of the oleogels

As expected, the oil binding capacity of the gels is related to the microstructure of the gels as shown in Figure 5. For fine stranded gels (low modulus), a large oil binding capacity is found as the network is more flexible. For coarse stranded gels (high modulus), a lower oil binding capacity is found. Acetone and water content characterization

For some of the gels, the amount of acetone and water was determined. The water content for most of the gels was determined to be less than 1% of water. The acetone concentrations as a function of the dryng time is depicted in Figure 6, and shows that roughly 1 ppm acetone remains in the gel. Water stability

To test the potential of the gels to act as a fat replacer in aqueous environments, we have tested the water stability of oleogels with a dimension of roughly 1 mm 3 . The starting hydrogels (as a template for the production of the oleogels) were prepared by gelation of the WPI solution in oil at a temperature of 90°C. The hydrogels were prepared by injecting droplets with a syringe in oil. A fine stranded gel was prepared from a 25%o WPI solution and no salt added, while a coarse stranded gel was prepared from a 25% WPI solution containing 200 mM of salt. The hydrogels were subsequently treated with a solvent exchange to replace the water for oil. The resulting oleogels can be seen in Figure IB. Results show that when the resulting oleogels were placed in aqueous environments, oil is expelled and water is absorbed by the oleogels. This leads to a decrease in the total oil content and an increase in the water content of the gels. Preliminary results show that the ratio water/oil depends on the microstructure of the gel, but in general, the ratio oil/water ranges roughly from 80/20 to 30/70. Table 5 shows results for the two different types of networks. Oil content (based on total weight) was measured using a Soxhlet apparatus, and the water content was estimated. Table 5. Oil, protein and water content of oleogels immersed in water

Hence, a substantial part of the oil remains in the oleogel.

Experimental series 3: protein type

As shown before oleogels can be prepared by using whey protein isolate and a solvent exchange procedure. In order to determine the flexibility of the protein source, also pig blood serum proteins are used. In a first experiment, determination of the aggregation and gelling properties of three different protein samples was performed. Furthermore, their usability as oleogelators was determined via: (i) Formation of a heat- set protein gel and exchange internal water content for vegetable oil; (ii) Formation of heat-set protein aggregates and usability as oleogelators (oleogel formers).

Materials

Three different protein samples were supplied by Darling international and are derived from pigg blood serum. The composition is given in table 6. All three samples contained serum albumin and serum immunoglobulin, but in different ratio's. Sample "standard" contains both proteins in natural ratio and the sample "Albumin" and "immunoglobulin" are enriched, however still contain both types of protein. Table 6: composition of protein samples

Methods

Hydrogel preparation

Protein powders were slowly added to water at 15% w/w. After 2h stirring at room temperature, solutions were left overnight at 4°C to assure complete hydration. To prepare stock solutions, pH was adjusted to 7 or 9 using IM NaOH or IM HCl and diluted with water to a final protein concentration of 10% w/w. The gels were prepared at 10% w/w (which was well above the critical gelling concentration) by heating at 85°C for 30 minutes. To perform the solvent exchange, gels were placed in perforated stainless steel baskets and immersed into a series of water-acetone (30, 50, 70 and 100%) acetone) and acetone-oil (30, 50, 70 and 100% oil) solutions for 10-12 h per step. The 100% acetone and 100%) oil step was performed twice. Demineralized water was used throughout the experiments and protein concentrations were based on true protein content.

Protein aggregate preparation

To prepare protein aggregates, the sample 'standard' was used. Aggregates could be prepared at different concentrations depending on the pH of the sample and the type of protein used. The pH of the solutions was adjusted to 6, 7 or 9 using IM NaOH or IM HCl. In general, the concentration was roughly 3% for low pH (6) and 5% for high pH (9). Aggregation was initiated by applying heat (85°C) for 30 min. Preparation of the aggregates at the different pH values slighlty changed the microstructure of the aggregates. At pH values closer to the iso-electric point, a more coarse network is formed. For further analysis, the standard sample was used at pH 6 and 3% w/w protein concentration. After cooling, the weak gel was broken down by vortexing (1 min) to create a protein aggregate suspension as a starting material for the solvent exchange. First, the aqueous suspension was centrifuged at 3800 x g for 30 min and the supernatant was discarded. To the pellet 100% acetone was added and the pellet was dispersed by using a rotor-stator homogenizer (Ultra Turrax) at 15.200 rpm for 1 min. Afterwards, the sample was centrifuged at 3800 x g for 20 minutes to collect the pellet. This procedure was repeated once more with 100% acetone and twice with sunflower oil. Afterwards, the protein-in-oil suspension was left to evaporate remaining acetone.

Results

Results solvent exchange on macroscopic hydrogels

After the solvent exchange, the resulting oleogels did not collapse. Gels at pH 9 are more transparent than at pH 7. The exact oil and water content in the gels was not measured. The amount of oil loading is given in Fig, 7, where oil loading (Q 0 ii) is defined as weight oleogel/weight hydrogel. All protein samples show ability to bind oil into their protein matrix, where the immunoglobulin sample had the highest oil loading capacity. Results solvent exchange on protein aggregates

After the solvent exchange using protein aggregates the pellet was collected. The pellet was a smooth paste-like oleogel containing the protein aggregates.

The oleogel was self-supporting. An example of the gel can be found in figure ID. SEM images show the network formation of the aggregates in Figure 2C.

Figure 8 shows the storage modulus, loss modulus and loss tangent as a function of frequency. As can be seen, the storage modulus was around 60kPa and the loss tangent <0.1, indicating a quite firm gel.

Application in cookies

The potential of the oleogels as a solid fat replacer were tested in cookies.

A reference recipe for shortbread cookies with 42.2% flour, 32.8% margerine, 21.1% sugar, 3.5%) vanilla sugar, and 0.5%> salt was used.

Materials and methods

As a replacement for butter (solid fat), we have chosen the oleogel paste as a substitute. The oleogel paste can be prepared with the intermediate solvents acetone or ethanol as an intermediate solvent. To compare the differences, we have used both acetone and ethanol as intermediate solvent to prepare the oleogels (these oleogels are herein also shortly indicated as "acetone oleogel" and "ethanol oleogel", respectively).

The oleogels were prepared using the method as described before.

According to the original recipe, the dough is prepared by mixing the margerine, vanilla sugar and salt. Then, the flour is added to the mixture. The dough is wrapped in foil, and left in the fridge for 1 hour to rest. The dough is rolled into separate cookie doughs of 0.5 cm thickness, and then baked in the oven for 15 minutes at 170°C.

For the cookies in this exemplification research, both acetone- and ethanol- based oleogel paste was used as a substitute for margarine, and they were substituted for

100%. To optimize some of the recipes in terms of colour and texture, part of the sugar was replaced by glucose syrup, and the baking temperature was adjusted. The texture of the dough was evaluated by measuring the firmness and the stickiness of the dough.

Firmness was measured by compression tests using a texture analyser (Stable Micro

Systems, TA.XT.Plus texture analyser). Stickiness of the samples was measured by a double compression test. The negative force was regarded as a measure for stickiness.

The firmness of the cookies was measured with a 3 point breaking force test, and the maximum force (F max ), needed to break the cookies, referred to as the breaking force, was recorded.

Three different cookies were used for the sensory test. The standard cookie with margarine was used as a reference, and two cookies containing oleogel (one with acetone and one with ethanol as intermediate solvent) as a substitute for margarine. For the sensory test, the recipe was slightly adjusted. In the recipes, 50% of the sugar was replaced by glucose syrup.

The sensory panel existed out of 10 untrained panellists The cookies were ranked on a line scale, where the extreme left was marked as low, and the extreme right was marked as high. For each of the attributes, the scaling was transferred into a number between 0 (lowest) and 10 (highest), and a mean/average value was calculated.

Results and discussion

The cookies containing oleogel, margarine and sunflower oil as fat sources were prepared as a first trial, and baked for 15 min at 170°C. The cookie with sunflower oil spread completely, and the final cookie is very brittle, confirming that using oil as a fat source is not sufficient (negative reference), and only solid like fats should be used. The cookies with margerine and the oleogel were much firmer. In order to optimize the texture (firmness) and colour formation of the cookie prepared with oleogel paste, some changes were made to the recipe. To ensure oleogel stability, glucose syrup was used instead of sugar, which was first mixed with the sugar and flour before the oleogel was added. The replacement of sugar by glucose syrup leads to an improvement of the overall texture of the dough. However, the increased browning of the cookie led to very dark colour, which was also observed to be inhomogeneous. To decrease the browning and increase the homogeneity, we have replaced only 50% of the initial sugar content by glucose syrup.

Therefore, for the cookies used in the sensory test, we have replaced 50% of sugar by glucose syrup, the syrup was first mixed with the sugar and the flour, and then the dough was baked at 170°C. As a reference, an original cookie with only sugar was used. For the sensory test, we prepared 4 cookies. The composition is described in Table 7. The cookies are visually quite similar. However, the cookie prepared with sunflower oil is too brittle (and soft) to handle, so this cookie was omitted from the sensory test.

Table 7. Cookies used for sensory test

Texture of the different cookie doughs

To investigate the changes in the dough, we have performed compression tests to evaulate the stickiness of the dough (Figure 9). Stickiness is related to the negative force underneath the curve, which can be seen for both the margarine and the oleogel doughs. However, the cookie dough prepared with margarine shows a larger area, and can therefore be regarded as more sticky compared to the dough prepared with oleogel.

Texture of the cookies

The hardness of the cookies was measured by the force needed to break the cookies, and the results for different cookies are presented in table 8. Table 8. Breaking force of different cookies

The standard cookie with margarine needs a force of about 24N to break. The replacement of 50%> of the sugar in this cookie with glucose syrup doubles the hardness of the cookie. The cookies with only oleogel (100% replacement of margarine) are much softer than the reference with margerine (3-4 softer).

Sensory test

The sensory test was performed with a small untrained panel (10 panellists). The panellists were asked to rate: Crunchiness, crumbliness, fattiness, coating, chewiness, buttery taste, sweetness, off- flavour, after taste and overall liking. The results can be found in Figure 10 and Table 9.

Table 9. Results of sensory evaluation

The reference cookie prepared with margerine is most crunchy and more liked compared to the cookies prepared with oleogel. The cookies with oleogels (both acetone or ethanol based) are perceived more crumbly, more fatty and more coating. By some panellists, the control cookie with margarine was described as too hard. The cookie with ethanol oleogel had for some panellists the best texture, other panellists missed some crunchiness. Even though the oleogel cookies did not contain any butter, they still scored relatively high for buttery taste. For the acetone cookies, a negative off-flavour was perceived, probably because of left-over acetone in the oleogels. A lot of panellist thought that only the cookie with acetone oleogel was an oleogel cookie. They did not notice that the cookie with oleogel prepared with ethanol was not a regular cookie. Although the oleogel cookie (prepared with ethanol) scored lower than the cookie with margerine, 4 of the 10 panellist graded the cookie with ethanol-based oleogel higher than a 6. Overall, the results show that margerine can be replaced by oleogels, but that the texture is not optimal. The replacement of the margerine by butter leads to changes in the texture, and optimization of the recipe is needed to optimize the textural properties. The appearance and the hardness of the cookie can easily be changed by changing the ratio of sugar and glucose syrup and the baking time and temperature of the cookie.

Application in sausages

Pre-made meat batter was prepared by mixing 75% pork filet (=low fat meat), 25% ice/water, 2% nitrite salt brine, and 0.5% spices; Indasia nr. 12. Af a "full-fat" reference 15% back fat was added to the meat batter to a total fat content of 25%. For the low- fat meat batter, 15% of another fat source (sunflower oil or oleogel) was added on top of the 10% fat already present. The total fat percentage of all sausages was 25%. As a replacement for pork fat, oleogel and oleogel paste were used as a substitute. The oleogels were prepared using the methods described before. In the case the oleogel was used in the meat batter, smaller oleogel slices were grinded to small pieces in a mortar before they were added to the meat batter.

Different sausages were prepared by mixing the standard meat batter with or without addition of 1.5% WPI powder and the low fat meat batter with sunflower oil, oleogel paste and oleogel. Mixing was done with a thermomix (Vorwerk) for 30 sec at position 2 followed by 30 sec of only slowly stirring. The sausages were cooked in a waterbath for 15 min at 78°C. The sausages were stored in the fridge overnight before removal of the casing. Texture determination

The sausages were evaluated for their cooking loss (loss of fluid during cooking), their loss in fat content, the firmness, and the juiciness.

The firmness of the sausages was measured with a 3 point bending test in duplicate with a texture analyser (Stable Micro Systems, TA. XT. Plus texture analyser). The maximum force needed to break the sausages was recorded as the breaking force. The prepared sausages have a length of about 11 cm and a diameter of 2.4 cm. The two anchor points were set to a distance of 5 cm.

The juiciness of the sausages was measured by collecting the liquid that was squeezed out of the sausages during the 80% compression test with filter paper.

Sensory evaluation

Five different sausages were used for sensory evaluation. A standard sausage with pork fat as a positive reference, a sausage with pork fat and additional WPI powder, a sausage with sunflower oil as a negative reference and two sausages with oleogel, one with the paste and one with pre-grinded gel pieces.

The sensory panel existed out of 10 untrained panellists. The sausages were ranked on a line scale, where the extreme left was marked as low, and the extreme right was marked as high.

Results and discussion

After mixing of the different meat batter, the batter all look rather similar. There is no visible instability of the oleogel paste and oleogel, and the small pieces of oleogel are not clearly visible. Only the batter with the addition of sunflower oil looks fattier.

The loss in weight of the sausage after boiling is given in table 10. The loss in weight is mainly caused by loss of water, but also some loss in protein and potentially also loss in fat. The sausages with pork fat and additional WPI powder have the least cooking loss, of 12.8 and 10.9%, respectively. The lower water loss for the sausage with additional WPI is probably caused by the water binding ability of the added proteins. The sausage with the oleogel paste shows a similar cooking loss (12.4%) as the control sausage with only pork fat. These three samples have no fat loss due to cooking, and the removed liquid only exists of water and proteins. The sausage with oleogel pieces (number 5) has a cooking loss of 15.7%, and in this case the removed liquid also contains fat as a result of the cooking step. The highest cooking loss is found for the sausage with sunflower oil, about 20%, and also (visually) there is the highest oil leakage from the sausage during cooking.

Table 10. Percentage of cooking loss for the different sausag

The firmness (breaking force) of the sausages is measured with a three point bending test; the maximum force needed to break the sausage is given in Fig. 11 and Table 11. The sausage with sunflower oil is much softer compared to the other sausages. Replacement of fat by oleogels does not lead to large changes in the firmness.

Table 11. Breaking force, the maximum force at fracture of the sausages.

Table 12 represents the juiciness of the cooked sausages. The sausage with sunflower oil, which has the largest cooking loss, is most juicy with 13.4% of liquid which is squeezed out of the sausage during compression. Additionally, the sausage with pork fat and additional WPI powder, which has the lowest cooking loss, also has the lowest juiciness (6.9%>). The juiciness of the two sausages with oleogel (paste and pieces) is between the standard sausage with pork fat and the sausage with sunflower oil.

Table 12. The percentage of liquid squeezed out of a sausage slice during 80% compression, defined as juiciness.

Pork fat + Sunflower

Pork fat Oleogel paste Oleogel

WPI Oil

Juiciness 9.2 ± 0.2 6.9 ± 0.3 13.4 ± 0.1 11.1 ± 0.1 12.8 ± 0.5 All 5 sausages were evualated for 6 sensory attributes. Most panellists found it hard to indicate the differences between the sausages, and the results are given in Table 13 and Fig. 12.

Table 13. Sensory evaluation of the different sausag

The clearest result from the sensory test is the different profile found for the sausage with sunflower oil, compared to the other sausages. The sausage with sunflower oil was the least firm, the least dry, the most juicy and fatty, indicating that fat can not be simply replaced by sunflower oil and keeping the same texture. Although the sausages with pork fat was overall best-liked, the sausages with oleogels scored relatively good. 5 of the 10 panellists even liked the sausage with the oleogel paste (prepared from ethanol) the most. The sausages with the oleogel pieces scored high on off-flavor, caused by the presence of acetone.

Overall, substitution of pork fat by oleogel paste does not change the sensorial properties of the sausage, and oleogels could be a good alternative for pork fat.