Emulsified compositions comprising yeast oleosomes

Abstract

The present invention relates to emulsified compositions comprising yeast oleosomes, water and exogenous lipids, wherein the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8):1. It further relates to food products comprising such emulsified compositions. It also relates to the use of yeast oleosomes to emulsify exogenous lipids with water, wherein the yeast oleosomes are used in an amount such that the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8):1. It finally relates to a process for the preparation of an emulsified composition comprising yeast oleosomes, water and exogenous lipids comprising admixing yeast oleosomes with the exogenous lipids, such as to obtain a total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio of (at least 0.8):1 and emulsifying the mixture.

Description

[0001] PAT8219EP00

[0002] Emulsified compositions comprising yeast oleosomes

[0003] Technical field

[0004] The present invention relates to emulsified compositions comprising yeast oleosomes, water and exogenous lipids, wherein the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1. It further relates to food products comprising such emulsified compositions. It also relates to the use of yeast oleosomes to emulsify exogenous lipids with water, wherein the yeast oleosomes are used in an amount such that the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1. It finally relates to a process for the preparation of an emulsified composition comprising yeast oleosomes, water and exogenous lipids comprising admixing yeast oleosomes with the exogenous lipids, such as to obtain a total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio of (at least 0.8): 1 and emulsifying the mixture.

[0005] Background of the invention

[0006] Vegetarian and vegan diets are getting more and more frequent, for sustainability, health and ethical reasons. In this framework, all products traditionally produced using ingredients of animal origin tend to be reformulated using only ingredients that do not originate from animals. Emulsified products, such as emulsified acidified sauces, like Ranch sauce, salad sauces, Caesar sauce, dairy alternatives, yoghurt, sauces in the culinary sector, like Hollandaise, Bearnaise and the like, as well as pastry creams, puddings, milk chocolate alternatives, and other vegetarian or vegan desserts, are traditionally emulsified using eggs or lecithin. Eggs, being of animal origin it is highly desirable to find alternative non-animal based solutions. Lecithin can originate from eggs or from plant, such as soy or sunflower. Even though lecithin from plants would be suitable for vegetarian or vegan diets, it would be desirable to provide vegetarian or vegan alternatives to lecithin, which are nutritive while achieving suitable emulsification, preferably while achieving similar or even better emulsification compared to eggs, (in particular egg-yolk) and / or lecithin.

[0007] Yeast oleosomes have been described in the prior art. For example, Nikiforidis et al., RSC Adv., 2014, 4, 25067 discloses natural emulsions based on oil bodies from plants, which have properties close to dairy milk or cream. Oil bodies are extracted from plant materials by using aqueous media. An oil-in-water emulsion, based on intact or partially disrupted oil bodies is obtained and a protein co-extraction takes place. The oil bodies size can be nanometric, up PAT8219EP00 to a few microns. The oil in such emulsions is however originating from the yeast and is present in the structure of oleosome. This document is however silent with respect to the emulsification properties of yeast oleosomes.

[0008] WO98 / 53698A1 discloses emulsion formulations prepared from oil bodies originating from living cells. The content of the document is focused on plant oil bodies, but other sources of oil bodies are mentioned in a very general way, including fungal sources, such as yeast cells. The oil bodies are obtained from a cell, washed and formulated. All disclosed process aspects are focusing on the preparation of formulations from plant seeds. The size of the oil bodies varies between 0.4 and 1.5 pm. This document teaches that rapeseed oleosomes can be used for emulsification purposes, and provides recipes of mayonnaise using such rapeseed oleosomes. This document is silent with respect to emulsification using yeast oleosomes specifically and does not provide any guidance regarding the parameters that impact the emulsification properties of the oleosomes.

[0009] WO2017 / 066569A1 discloses oleosome compositions comprising two different oleosome compositions, preferably originating from two different sources, and having different particles sizes: a first oleosome composition is characterized by a D50 of at least 120 nm and the second oleosome composition is characterized by a D50 of at least 600 nm. Yeast cells are mentioned in very general terms. However, all aspects described in detail are related to plant oleosomes. The use of oleosomes for emulsification purposes is described in general terms, but no example is applicable, so that this document does not provide any guidance regarding the parameters that impact the emulsification properties of the oleosomes.

[0010] Different ingredients from yeast origin have been previously described as having emulsification properties. Several documents disclose the use of whole cells as emulsifiers. For example, CN114009751B discloses inactivated Kluyveromyces yeast cells as emulsifier in sauces and other high fat products. The whole cells are used, even though in inactivated form, and is defined as having a fat content of 1-5%. Similarly, US3995066A describes margarine and low fat spread in the form of a water-in-oil emulsion comprising yeast cells that are not lysed. US2017 / 121671 Al also discloses the use of yeast (or bacteria) cells that are inactivated but not lysed as emulsifiers.

[0011] Other documents relate to yeast extracts as emulsifiers. JP2012 / 231747A describes the use of a yeast extract having a peptide content of 5% by weight or more, a RNA content of 5% by weight or more and a fibre content of 15% by weight of more, based on dry weight. The amount of lipids is not indicated. The emulsifier behaviour of the yeast extract is attributed to the presence of RNA in a sufficient amount in the extract (preferably at least 6.5% by weight, PAT8219EP00 based on dry weight. US2024 / 124831 Al relates to a yeast protein extract, which is said to act as an emulsifier.

[0012] It would be desirable to optimize the emulsification capacity of compositions of yeast origin.

[0013] The prior art also teaches that plant oleosomes can act as emulsifiers. This is the case of US2020 / 237641A1 and US6146645A. It would however be desirable to provide an advantageous alternative to plant oleosomes, such as having improved emulsification capacity than plant oleosomes.

[0014] The present invention aims at solving the above-mentioned problems associated with the emulsification of lipids in products free of ingredients of animal origin, while providing advantageous nutritional value.

[0015] Summary of the invention

[0016] In a first aspect, the invention provides an emulsified composition comprising yeast oleosomes, water and exogenous lipids, wherein the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1.

[0017] In a second aspect, the invention relates to a food product comprising the emulsified composition of the invention.

[0018] In a third aspect, the invention relates to the use of yeast oleosomes to emulsify exogenous lipids with water, wherein the yeast oleosomes are used in an amount such that the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1.

[0019] In a fourth aspect, the invention provides a process for the preparation of an emulsified composition comprising yeast oleosomes, water and exogenous lipids comprising a) preparing a mixture comprising yeast oleosomes, water and exogenous lipids; and b) emulsifying the mixture, wherein the yeast oleosomes are admixed with the exogenous lipids in step a) such as to obtain a total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio of (at least 0.8): l. PAT8219EP00

[0020] Brief description of the drawings

[0021] [Fig. 1] Representation of particle size results of egg yolk, lecithin and CB emulsions without the addition of SDS. Results are shown based on volume-weighted area mean-D(4,3) value and standard deviation bars.

[0022] [Fig. 2] Representation of particle size results of egg yolk, lecithin and CB emulsions with the addition of SDS. Results are shown with D(4,3) value and standard deviation bars.

[0023] [Fig. 3] Viscosity results for emulsions (A to G) created with CB.

[0024] [Fig. 4] Viscosity results for emulsions (Al to Gl) created with egg yolk.

[0025] [Fig. 5] Viscosity results for emulsions (A2 to G2) created with lecithin.

[0026] [Fig. 7] spin test results for emulsions with CB (A to G)

[0027] [Fig. 8] spin test results for emulsions with egg yolk (Al to Gl)

[0028] [Fig. 9] spin test results for emulsions with lecithin (A2 to G2)

[0029] Detailed description of the invention

[0030] General definitions

[0031] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0032] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0033] Reference throughout this specification to "one aspect", "an aspect", "another aspect", "a particular aspect", "combinations thereof' means that a particular feature, structure or characteristic described in connection with the invention aspect is included in at least one aspect of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. PAT8219EP00

[0034] Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0035] The term "comprise(s)", or "comprising" is generally used in the sense of include(s) / including, that is to say, permitting the presence of one or more features or components. The terms "comprise(s)" and "comprising" also encompass the more restricted ones "consist(s)" and "consisting", respectively.

[0036] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0037] The present inventors have used yeast oleosomes to prepare emulsified compositions.

[0038] "Yeast oleosomes" are defined as oil bodies isolated from yeast, which are formed of oil droplets surrounded by a layer (membrane) of proteins, phospholipids and carbohydrates, mainly polysaccharides. Obtaining oleosomes "isolated from yeast" requires the breakage of the yeast cell wall and thus whole yeast cells are excluded from the definition of yeast oleosomes, even though such whole yeast cells contain oleosomes. Also, when yeast oleosomes are provided in the form of a yeast oleosome composition, such as described herein, such composition comprises only a negligible number of remining whole (intact) yeast cells, such as less than 10% by weight of whole yeast cells, based on the total weight of the oleosomes composition.

[0039] Oleosomes also exist in plants. However, plant and yeast oleosomes vary in their composition and properties. Namely, the materials present in the layer separating the oil droplet from the surrounding medium are different between plant oleosomes and yeast oleosomes, which provides yeast oleosomes with a different behaviour compared to plant oleosomes, in diverse applications. As defined herein, yeast oleosomes refer to oleosomes extracted from the yeast cell. Even though the oleosomes can be in the form of a mixture with cell components, such as the yeast cell wall components, such yeast cell wall shall not be intact, so that the oleosomes can freely pass through the yeast cell wall fragments. This excludes oleosomes present inside live yeast cells.

[0040] Yeast oleosomes are preferably present in the form of a mixture (i.e. in the form of a yeast oleosomes composition) of yeast oleosomes with at least one yeast cell wall component, preferably at least one isolated yeast cell wall component (i.e. one or more compound(s) released from the yeast cell wall), such as for example lipids, such as phospholipids, carbohydrates and proteins. Preferably the yeast cell wall component is selected from the group consisting of chitin, P-D-glucan, mannoprotein and combinations thereof. The terms "released" or "isolated" from the yeast cell wall structure herein means that the yeast cell wall component PAT8219EP00 is present in the yeast oleosome composition in free form, i.e. separated from the yeast cell wall structure, and is not part of the structure of a yeast cell wall debris. Thus, in other words, the yeast cell wall component is not fully embedded in the yeast cell wall structure so that it is available for interaction with the surrounding environment. The yeast cell wall component may nevertheless interact or be bound with other components of the yeast oleosome composition, provided it is not embedded in the yeast cell wall structure in a way that it is inaccessible to the yeast oleosomes medium. Indeed, when a yeast cell wall component is present, it is essential that such yeast cell wall component be "released" or "isolated" from the yeast cell wall structure to make the yeast cell wall component available for interaction with other components of the yeast oleosomes composition, with the oral cavity of an individual consuming the yeast oleosomes composition or an emulsified composition comprising it or with other components of the emulsified composition.

[0041] The at least one yeast cell wall component can be present in the yeast oleosome composition either in solubilized or suspended form, or it can be associated with the yeast oleosome membrane or it can be partly solubilized or suspended and partly associated with the yeast oleosome membrane. In a preferred aspect, the at least one yeast cell wall component is associated with the yeast oleosome membrane, preferably through non-covalent bonds, such as electrostatic interactions, hydrogen bonds or hydrophobic interactions. In an embodiment the yeast oleosomes composition comprises at least two yeast cell wall components selected from chitin, P-D-glucan and mannoprotein, one or two of these yeast cell walls components is (are) in solubilized or suspended form, and the other one or two component(s) is (are) associated with the yeast oleosome membrane. Preferably chitin, P-D-glucan and mannoprotein are all associated with the yeast oleosome membrane.

[0042] In a preferred aspect, the at least one isolated yeast cell wall component is present in the yeast oleosomes composition in an amount of at least 1.5 wt%, preferably at least 2 wt%, more preferably at least 2.5 wt%, more preferably at least 3 wt%, more preferably at least 3.5 wt%, more preferably at least 4 wt%, more preferably at least 4.5 wt%, most preferably at least 5 wt%, preferably at least 7.5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt% by weight based on the total weight of the solids in of the yeast oleosomes composition. In a particular aspect, the at least one isolated yeast cell wall component is present in the yeast oleosomes composition in an amount of up to 25 wt%, or up to 30 wt%, or up to 35 wt% or up to 35 wt%, or up to 40 wt% or up to 45 wt% or up to 50 wt%, based on the total weight of the solids in the yeast oleosomes composition. PAT8219EP00

[0043] In a further preferred aspect, the yeast oleosomes composition comprises P-D-glucan in an amount of at least 1.5 wt%, preferably at least 2 wt%, more preferably at least 2.5 wt%, more preferably at least 3 wt%, more preferably at least 3.5 wt%, preferably at least 4 wt%, preferably at least 4.5 wt%, preferably at least 5 wt%, preferably at least 7.5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt% based on the total weight of the solids in the yeast oleosomes composition. In a particular aspect, the at least one isolated yeast cell wall component is present in the yeast oleosomes composition in an amount of up to 25 wt%, or up to 30 wt%, or up to 35 wt% or up to 35 wt%, or up to 40 wt% or up to 45 wt%, based on the total weight of the solids in the yeast oleosomes composition.

[0044] In a particular aspect, the P-D-glucan is in its native form, as obtained after extraction of the oleosomes composition from the yeast cell. In another particular aspect, the P-D-glucan has not been enzymatically degraded in the yeast oleosomes composition.

[0045] In another preferred aspect, the yeast oleosomes composition comprises intact oleosomes in an amount of at least 25wt%, such as 25 to 90wt%, or 25 to 80wt% or 25 to 79wt% or 25 to 60wt% based on the total solids in the yeast oleosomes composition.

[0046] In a preferred aspect, the particle size of the yeast oleosomes composition is characterized by a mean diameter by volume of 0.2 to 7 pm as measured by laser diffraction, preferably using a Anton Paar PSA particle size analyser, preferably with Anton Paar PSA 1190, with a measuring time set to 1 min with 6 repetitions per sample, the reconstruction algorithm being MIE, the refractive index for the material being 1.47 and the absorption index being 0.01, wherein the samples are dissolved in distilled water and the refractive index is set to 1.33. Without wishing to bound by theory. Smaller particle size tends to provide better emulsification due to a higher surface per volume and thus also a higher concentration of emulsifier per volume. Thus, preferably, the mean diameter by volume is of up to 6.5 pm, preferably up to 6 pm, preferably up to 5.5 pm, preferably up to 5 pm, preferably up to 4.5 pm, preferably up to 4 pm, preferably up to 3.5 pm, preferably up to 3 pm, preferably up to 2.5 pm, preferably up to 2 pm, preferably up to 1.5 pm, preferably up to 1 pm.

[0047] In a preferred aspect, the density of the yeast oleosome composition is of 0.8 to 1.1 g / ml.

[0048] In another preferred aspect, the zeta potential at the surface of the oleosomes is in the range of -45 to -5 mV, -45 to -8 mV, -45 to -10 mV, -45 to -20 mV, -40 to -5 mV, -40 to -8 mV, -40 to -10 mV, -40 to -20 mV, or -30 to -20 mV at physiological pH. Such zeta potential is advantageous because within such range, the electrostatic interactions between the oleosomes are sufficiently low to avoid aggregation of the oleosomes and ensure proper PAT8219EP00 physical stability of the emulsified composition. A zeta potential within the present range also contributes to the interaction of the yeast oleosome membrane with the polysaccharides. The zeta potential varies within this range, depending on the proteins present in the oleosome membrane. In a preferred aspect, the zeta potential is measured with a Malvern Zetasizer Nano ZS90 at a sample dilution of 1 : 100 sample:water, with a refractive index of solvent of 1.33, a refractive index of sample of 1.47, an absorption of 0,010, and with a DTS1060 cell.

[0049] In a preferred aspect the pH of the yeast oleosomes composition is of 2.5 to 8, such as 3 to 7, 4 to 7, 5 to 7 or 6 to 7. Such pH range is preferred because it impacts the net charge of the oleosomes and positively impact the physical stability of the yeast oleosomes composition over time.

[0050] In a particular aspect, the yeast oleosomes composition is in liquid form and has a total solids content of 2 to 45 wt%. Higher solids contents are preferred as higher total solids in the yeast oleosomes composition increases the concentration of emulsifier in the emulsified composition of the invention. Thus, the total solids content in the yeast oleosomes composition is preferably of at least 5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt%, preferably at least 25 wt%, preferably at least 30 wt%, preferably at least 35 wt%, preferably at least 40 wt%, based on the total weight of the yeast oleosomes composition.

[0051] In a particular aspect, the yeast oleosomes composition has a fat content of 25 to 60wt%, based on the total solids content in the oleosomes composition.

[0052] Emulsified compositions

[0053] The invention provides an emulsified composition comprising yeast oleosomes, water and exogenous lipids, wherein the total lipids in the emulsified composition to non-lipids solids in the oleosomes ratio is of (at least 0.8): 1.

[0054] By "exogenous lipids", it is referred to herein to lipids that do not form part of the oleosomes. Exogenous lipids are provided in the form of an oil (i.e. a triglyceride composition that is liquid at room temperature) or a fat (i.e. a triglyceride composition that is in solid form at room temperature). Examples of fat or oils include vegetal fat or oils, algal oils, microbial oils, fungal oils and mixtures thereof. In a particular aspect, the exogenous lipids are provided in the form of an oil or fat selected from vegetal fat or oils, algal oils, microbial oils, non-yeast fungal oils and mixtures thereof. In a preferred aspect, the lipids are not animal fat. Accordingly, in an aspect of the invention, the term "lipid" as used herein refers to triglycerides and the terms "total lipids in the emulsified composition" refer to the total amount of PAT8219EP00 triglycerides in the composition, including those originating from the exogenous lipids and those originating from the oleosomes.

[0055] In line with the above definition of lipids, in an aspect of the invention the term "nonlipid solids in the oleosomes" as used herein refers to the total solids in the oleosomes, excluding the triglycerides present in the oleosomes.

[0056] The total lipids in the emulsified composition to non-lipids solids in the oleosomes ratio is calculated according to Equation 1.

[0057] Ratio mtotal lipids in emulsified composition [g] / Ulnon-lipid solids in oleosomes [g] (Equation 1)

[0058] The first member of the ratio ("total lipids in the emulsified composition") refers to the total of the amount of lipids contained in the emulsified composition, including the lipids provided by the oleosomes and the exogenous lipids. The second member of the ratio ("nonlipid solids in oleosomes") refers to the total weight of the oleosomes (or the yeast oleosomes composition when the oleosomes are in such form), minus the water and the lipids content in the oleosomes, thus it refers to the total solids in the oleosomes (or the yeast oleosomes composition), excluding oleosomes lipids. For the avoidance of any doubt, this amount includes any protein, carbohydrate, mineral, vitamin, metabolite, including cell wall components, if any, and all other solid components that are present in the oleosomes composition and which are not lipids, such as defined above.

[0059] The components of the yeast oleosomes membranes, in particular the phospholipids, polysaccharides and proteins, advantageously act as emulsifiers, and stabilise the exogenous lipids in the emulsion, by generating smaller oil droplets, increasing viscosity and / or preventing coalescence. Also, when the oleosomes are provided in the form of a yeast oleosomes composition, it is preferred that this yeast oleosomes composition further comprises additional proteins, polar fatty acids and polysaccharides, in addition to those present in the oleosome membrane, and / or mono-, di- or triacyl glycerides. The yeast cell wall components P-D-glucan, mannoproteins and / or chitin, as defined herein above, when present, act as further emulsifiers or stabiliser.

[0060] In addition, the oil present in the yeast oleosomes increases the total amount of lipids without impairing the emulsion stability, as the lipids in the oleosomes are already emulsified (incorporated in the oleosomes membranes) at the time they are added to the emulsified composition and the soluble fibres stabilize the droplets by creating a protective barrier and herewith decrease droplet coalescence. PAT8219EP00

[0061] Yeast oleosomes are also advantageously characterised by higher nutritional value than lecithin, due to the presence of proteins and fibres. Proteins in the yeast oleosomes further exhibit particularly good nutritional interest.

[0062] In a preferred aspect, the yeast oleosomes are included in the emulsified composition of the invention in the form of liquid or solid yeast oleosomes compositions, such as defined above. Yeast oleosomes in solid form can be either reconstituted with water before admixture with exogenous lipids, alternatively, yeast oleosomes in solid form can be added to a preemulsion containing exogenous lipids and water or in still another alternative, yeast oleosomes in solid form can be added directly to the exogenous lipids, before water is added. When yeast oleosomes are added in the form of a liquid yeast oleosomes composition, the water in the yeast oleosomes composition can either form a sufficient amount of water to form the emulsion. Alternatively, additional water can be added, as desired, depending on the intended final formulation of the emulsified composition of the invention. In a preferred aspect, the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of 0.8: 1 to 95: 1, 0.8:1 to 90:1, 0.8:1 to 85:1, 0.8:1 to 80:1, 0.8:1 to 75:1, 0.8:1 to 70:1, 0.8:1 to 65:1, 0.8:1 to 60:1, 0.8:1 to 55:1, 0.8:1 to 50:1, 08:1 to 45:1, 0.8:1 to 42:1, 0.8:1 to 40:1, 0.8:1 to 35:1, 0.8:1 to 33:1, 0.8:1 to 30:1, 0.8:1 to 28:1, 0.8:127:1, 0.8:1 to 25:1, 0.8:1 to 22:1, 0.8:1 to 20:1, 0.8:1 to 18:1, 0.8:1 to 16:1, 0.8:1 to 14:1, 0.8:1 to 12:1, or 0.8:1 to 11:1. In another aspect the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of 1 : 1 to 95: 1, 1:1 to 90:1, 1:1 to 85:1, 1:1 to 80:1, 1:1 to 75:1, 1:1 to 70:1, 1:1 to 65:1, 1:1 to 60:1, 1:1 to 55:1, 1:1 to 50:1, 1:1 to45:l, 1:1 to 42:1, 1:1 to 40:1, 1:1 to 35:1, 1:1 to 33:1, 1:1 to 30:1, 1:1 to 28:1, 1:1 to 27:1, 1:1 to 25:1, 1:1 to 22:1, 1:1 to 20:1, 1:1 to 18:1, 1:1 to 16:1, 1:1 to 14:1, 1:1 to 12:1, or 1:1 to 11:1.

[0063] In a preferred aspect, the yeast oleosomes are provided in an amount such that the solids of the yeast oleosomes composition (including oleosomes lipids) be present in an amount of at least 0.5 wt%, preferably at least 1 wt%, preferably at least 3 wt%, preferably at least 4 wt%, preferably at least 4.5 wt%, preferably at least 5 wt%, preferably at least 5.5 wt%, preferably at least 6 wt%, preferably at least 6.5 wt%, more preferably at least 7 wt%, even more preferably at least 8 wt%, based on the total weight of the emulsified composition of the invention. In another preferred aspect, the yeast oleosomes are provided in an amount such that the solids of the yeast oleosomes composition (including oleosomes lipids) be present in an amount of up to 30 wt%, or up to 25 wt%, or up to 20 wt%, or up to 18 wt%, or up to 15 wt%, based on the total weight of the emulsified composition of the invention. PAT8219EP00

[0064] The exogenous lipids are preferably present in the emulsified composition in an amount of up to 75 wt%, such as up to 70 wt%, or up to 60 wt%, or up to 50 wt%, or up to 40 wt%, or up to 30 wt%, or up to 25 wt%, or up to 22 wt %, or up to 21 wt%, or up to 20 wt%, or up to 19 wt%, or up to 18 wt%, or up to 16 wt%, or up to 15 wt%, or up to 11 wt%, or up to 10 wt%, based on the total weight of the emulsified composition .

[0065] The emulsified compositions of the invention can further comprise optional ingredients, such as carbohydrates, proteins, micronutrients such as vitamins, minerals or nucleotides, flavours, colorants, preservatives, low calorie sweeteners, spices, taste enhancers, emulsifiers, thickeners, and / or cereals, nuts, legumes, fruits, vegetables, herbs, juice, and the like.

[0066] Examples of carbohydrates include sugars and fibres. Sugars can be used in any amount, based on the desired sweet taste to be imparted to the emulsified composition. For example, sugar can be present in an amount of 1 to 20 wt%, or 3 to 15 wt%, or 5 to 10 wt% based on the weight of the total solids content in the emulsified composition of the invention. Fibres can be present in any desired amount, depending on the desired nutritional value of the emulsified composition. Yeast oleosomes are however particularly advantageous in that they contain fibres, in addition to lipids and proteins, thus reducing the need to add exogenous fibres to the emulsified composition. For example, fibres can be present in an amount of up to 40 wt%, preferably 0.1 to 40 wt%, preferably 0.5 to 40 wt%, preferably 1 to 40 wt%, preferably 5 to 40 wt%, preferably 10 to 40 wt%, preferably 20 to 40 wt%, preferably 20 to 30 wt% based on the weight of the total solids content in the emulsified composition of the invention.

[0067] Examples of proteins include plant proteins, microbial proteins, such as yeast proteins or bacterial proteins, algal proteins, fungal proteins or recombinant proteins, such as precision recombinant animal proteins produced by microbes or functional proteins originating from precision fermentation. Proteins can be present in any suitable amount, depending on the desired application, preferably in an amount of up to 50 wt%, or up to 40 wt%, or up to 30 wt% or up to 20 wt% or up to 10 wt% or up to 5 wt%.

[0068] Micronutrients are also added in the amounts necessary to meet the desired nutritional characteristics of the emulsified composition, depending on its intended use. Functional or medical nutritional compositions for example require fortification with specific amounts of specific micronutrients. Any desired micronutrient can be used in the emulsified composition of the invention, in any desired amount. PAT8219EP00

[0069] Colorants, preservatives and the like are used as appropriate, depending on the desired taste, visual appearance and shelf life of the emulsified composition.

[0070] Any thickener can be used, such as gellan gum, guar gum, P-carrageenan, xanthan, locust bean gum, agar-agar, starch (such as cornstarch, tapioca starch), methylcellulose, P- glucan, gum Arabic, pectin and combinations thereof. The amount of thickener depends on the intended texture of the emulsified composition. For certain purposes a relatively fluid emulsified composition is desired, so that no or little thickener is used, while other applications, such as medical nutrition for individuals having swallowing problems, require thicker emulsified compositions, in which higher amounts of thickeners are used. Any desired amount of thickener can be used in the emulsified composition of the invention.

[0071] The emulsified composition of the invention can further comprise any kind of edible material that is desired, depending on the intended applications. In a particular aspect, the emulsified composition comprises solid particles, preferable vegetal solid particles, such as vegetal flours, in particular cereal or nut flour, for example oat flour, almond flour, rice flour, pea flour, soy flour, coconut flour and mixtures thereof. The solid particle is preferably present in an amount of 0.1 to 20 wt%, preferably 1 to 20 wt%, more preferably 5 to 15 wt%, based on the weight of the total solids content in the emulsified composition of the invention.

[0072] Even though the yeast oleosomes provide good emulsification, the emulsified composition of the invention can optionally comprise further emulsifiers, such as lecithin, preferably vegetal lecithin. In a particular aspect, the emulsified composition of the invention does not comprise lecithin.

[0073] In a particular aspect the composition of the invention does not comprise dairy milk. In another particular aspect the composition of the invention does not comprise dairy ingredients (i.e. components of dairy milk), such as milk fat, milk proteins and / or milk carbohydrates.

[0074] Uses

[0075] The present invention provides for the use of yeast oleosomes to emulsify exogenous lipids with water, wherein the yeast oleosomes are used in an amount such that the total lipids in the emulsified composition to non-lipids solids in the oleosomes ratio is of (at least 0.8): 1.

[0076] In a preferred aspect, the yeast oleosomes are present in the form of a yeast oleosome composition as described above, the exogenous lipids, the amounts and the ratios are as described above. PAT8219EP00

[0077] In another preferred aspect, the yeast oleosomes are used in combination with any of the optional ingredients recited above in the section dedicated to the emulsified composition.

[0078] The use of yeast oleosomes in combination with exogenous lipids in the above- mentioned ratios is advantageous in that such mixture forms a stable emulsion, with small droplet size and suitable sensory attributes, such as mouthfeel.

[0079] Products

[0080] The invention relates to a food product comprising an emulsified composition of the invention. The emulsified composition can constitute by itself a final product. Alternatively, the emulsified composition can be used in combination with other composition(s) and / or ingredient(s) to form a more complex product, wherein the emulsified composition can either be admixed with the other composition(s) and or ingredient(s) or used in combination therewith without mixing.

[0081] Examples of food products and beverages of the invention include any emulsified products or products comprising an emulsion. Examples of such products include emulsified sauces, in particular acidified sauces, like Ranch sauce, salad sauces or Caesar sauce, mayonnaise, and culinary sauces, like Hollandaise, Bearnaise and the like, dairy alternatives, such as milk, cream or yoghurt, emulsified confectionary products such as milk chocolate alternatives, as well as desserts, such as pastry creams or puddings.

[0082] Process

[0083] The invention also relates to a process for the preparation of an emulsified composition comprising yeast oleosomes, water and exogenous lipids comprising a) preparing a mixture comprising yeast oleosomes, water and exogenous lipids; and b) emulsifying the mixture, wherein the yeast oleosomes are admixed with the exogenous lipids in step a) such as to obtain a total lipids in the emulsified composition to non-lipids solids in the oleosomes ratio of (at least 0.8): 1.

[0084] In a preferred aspect, the process comprises before step a) a step of providing a yeast oleosome. Such step of providing a yeast oleosome is preferably performed by isolating oleosomes from yeast in any suitable manner. Preferably, the yeast oleosomes composition is obtained by a process comprising the steps of:

[0085] I. providing oleaginous yeast cells;

[0086] II. lysing the cell wall; and PAT8219EP00

[0087] III. separating the oleosomes from the other yeast cell components.

[0088] The yeast cells can optionally be subjected to pre-treatment, such as: i. washing with water, preferably warm water; ii. washing with an alkaline solution; iii. washing with an acidic solution; and / or iv. enzymatic digestion of the cell wall.

[0089] Step II. can be performed by chemical or mechanical means. Suitable chemical means include: i. alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours; ii. contacting the yeast cells with an organic solvent such as ethanol, methanol or heptane; iii. autolysis; iv. enzymatic treatment, preferably using one or more hydrolase(s), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers; and combinations thereof.

[0090] The one or more hydrolase(s) is preferably selected from one or more protease(s), glucanase(s), mannanase(s), chitinase(s), nuclease(s), beta-glucosidase(s), cellulase(s), xylanase(s), pectinase(s) and combinations thereof. Preferably, it is one or more protease(s) and / or glucanase(s).

[0091] Preferred chemical means for the cell lysis include: i. alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours; ii. autolysis, by heating the cells in the end of the fermentation to a temperature of 50°C or more, reducing the stirring and stopping the aeration until spontaneous lysis of the yeast cell occur (under the action of the endogenous enzymes); iii. enzymatic treatment, preferably using one or more hydrolases (as defined above), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers); and combinations thereof. PAT8219EP00

[0092] Such preferred chemical means are particularly advantageous because they are successful in breaking the cell wall, thus releasing the yeast cell wall components chitin, P-D- glucan and mannoproteins, while preserving the structure of the oleosomes.

[0093] Suitable mechanical means of the cell wall lysis of step II. include high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar, more preferably with 2 to 10 passes at a pressure of 700 to 3000 bar

[0094] When high pressure homogenization is used, the number of passes and the homogenization pressure can advantageously be adapted, when high pressure homogenization is combined with chemical means for lysing the membrane. For example, 1 to 10 passes and a pressure of 500 to 3000 bar is sufficient when the high-pressure homogenization is combined with prior enzymatic treatment, whereas it is preferred to perform the high-pressure homogenization with 2 to 10, preferably 2 to 5 passes at a pressure of 700 to 3000 bar, preferably 700 to 2000 bar, in particular when the high pressure homogenization is performed after alkaline treatment. Preferably high-pressure homogenization is performed in high- pressure homogenizer comprising a nozzle, as it contributes to breaking down the cells.

[0095] High-pressure homogenization as disclosed herein is particularly advantageous over other mechanical means of lysing the yeast cell wall, such as bead milling, ultrasound treatment and microwave treatment, in that it is successful in breaking the cell wall, thus releasing the yeast cell wall components chitin, P-D-glucan and mannoproteins, while preserving the structure of the oleosomes. High-pressure homogenization is also advantageous in terms of yield and allows to control the particle size.

[0096] Chemical and mechanical means for lysing the cell wall can advantageously be combined with each other. Particularly advantageous combinations are: i. alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours, followed by high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar, more preferably with 2 to 10 passes at a pressure of 700 to 3000 bar; ii. enzymatic treatment, preferably using one or more hydrolases (as defined above), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers), followed by high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar, preferably with prior pre-treatment by washing with water or an alkaline solution; PAT8219EP00 iii. autolysis, by heating the cells in the end of the fermentation to a temperature of 50°C or more, reducing the stirring and stopping the aeration until spontaneous lysis of the yeast cell occur (under the action of the endogenous enzymes), followed by high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar, preferably with 2 to 10 passes at a pressure of 700 to 3000 bar;

[0097] In a preferred aspect, cell lysis is performed by one of the following means: i. alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours, followed by high pressure homogenization, preferably with 2 to 10 passes at a pressure of 700 to 3000 bar; ii. autolysis, by heating the cells in the end of the fermentation to a temperature of 50°C or more, reducing the stirring and stopping the aeration until spontaneous lysis of the yeast cell occur (under the action of the endogenous enzymes); and iii. enzymatic treatment, preferably using one or more hydrolases (as defined above), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers), followed by high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar,

[0098] The separation step is preferably performed by i. decantation, preferably using a disk stack at 5000 to 20000 g for 10 seconds to 5 minutes, ii. centrifugation, under any suitable condition, preferably at 3000 to 30000 g, preferably 3000 to 20000 g, preferably 3000 to 10000 g, such as 7000 g in disc centrifuge, such as a continuous disk stack centrifuge, preferably with a residence time of 10 seconds to 10 minutes, preferably 10 seconds to 5 minutes, preferably 30 seconds to 5 minutes for example 1 minute. Preferably, the temperature is of 5 to 85°C, preferably 40 to 65°C. iii. filtration, preferably in cross counter flow, such as by filtration using polymeric or ceramic membranes with pore sizes from 10 kDa to 300 kDa, and from 0.2 pm to 20 pm or iv. Flocculation, preferably by contacting the composition with a flocculant, preferably for 1 to 24 hours at a temperature of 2 to 60°C. Any flocculant known in the art can be used. Preferably the flocculant is selected from inorganic salts such as CaCh, or a natural flocculant such as chitosan, cellulose and the like. The flocculant is preferably used in an amount of 0.1 to 50 mg / L. PAT8219EP00

[0099] Preferably, the separation step is performed by decantation, centrifugation or filtration, as described above. More preferably, it is performed by decantation, as described above.

[0100] In a particularly preferred aspect, the compositions of the present invention are obtained by a process comprising the steps of:

[0101] 1) providing oleaginous yeast cells, wherein the yeast cells are preferably washed and optionally weakening the cell walls by one or more of the following pre-treatments i. alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours; ii. enzymatic treatment, preferably using one or more hydrolases (as defined above), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers); and / or iii. autolysis, by heating the cells at the end of the fermentation to a temperature of 50°C or more, reducing the stirring and stopping the aeration until spontaneous lysis of the yeast cell occurs (under the action of the endogenous enzymes);

[0102] 2) lysing the cell wall by i. Alkaline treatment, preferably by soaking the yeast cells in an aqueous medium at pH 8 to 12 for 1 to 36 hours, preferably 2 to 36 hours; ii. contacting the yeast cells with an organic solvent such as ethanol, methanol or heptane; iii. autolysis, by heating the cells in the end of the fermentation to a temperature of 50°C or more, reducing the stirring and stopping the aeration until spontaneous lysis of the yeast cell occurs (under the action of the endogenous enzymes); iv. enzymatic treatment, preferably using one or more hydrolases (as defined above), applied to the washed biomass and suspended at a concentration of 50 to 200 g / L in water or a suitable pH buffer with an incubation temperature of 30 to 50°C and an incubation time of 0.5 to 8 hours with low stirring or shaking, to allow the degradation of cell wall biopolymers); v. high pressure homogenization, preferably with 1 to 10 passes at a pressure of 500 to 3000 bar, more preferably with 2 to 10 passes at a pressure of 700 to 3000 bar; or vi. any combinations of combinations of any two or more of the means provided in items i. to v.; and

[0103] 3) separating the oleosomes from the other yeast cell components by PAT8219EP00 i. decantation, preferably using a disk stack at 5000 to 20000 g for 10 seconds to 5 minutes ii. centrifugation iii. filtration.

[0104] In an optional further step 4), the composition obtained in the end of step 3) is dried by i. spray drying or ii. lyophilization.

[0105] In such cases, the dry oleosome composition shall be reconstituted with water in step a) of the process of the present invention

[0106] In a preferred aspect of the invention, no washing step is performed after the separation step. This aspect is advantageous in that it avoids removing of the "isolated" yeast cell wall component such as described above that are recovered by the separation step.

[0107] In step a) of the process of the present invention, the yeast oleosomes are admixed with the exogenous lipids and water, if water is added in addition to any water present in the oleosome composition. Any optional ingredient can be added add this stage or later, after the formation of the emulsion, depending on the type of ingredient. The person skilled in the art is able to determine the best moment to add optional ingredients, based on his common general knowledge, for each type of optional ingredient. The yeast oleosomes, exogenous lipids and optional ingredients, as well as their amounts and ratios are as described above in the section dedicated to the composition.

[0108] In step b), the mixture of step a) is homogenized to provide a homogenous composition. Homogenization can be performed by any suitable means known to the person skilled in the art, using any kind of homogeniser or by sonication. Preferably the mixture is subjected to homogenization at pressures of 30 to 500 bar. Preferably, it is homogenized by high-pressure homogenisation, more preferably at a pressure of at least 50 bar, preferably at least 100 bar, more preferably at least 200 bar, more preferably at least 300 bar. In an aspect, the high-pressure homogenization is performed at a pressure of up to 1000 bar, or up to 900 bar, or up to 800 bar or up to 700 bar or up to 600 bar or up to 500 bar. The homogenization can be performed in two steps at different pressures. PAT8219EP00

[0109] Examples

[0110] Example 1: Comparison of the sensory properties of emulsified compositions according to the invention and comparative emulsified compositions

[0111] Materials and Methods:

[0112] The aim of this experiment was to understand the emulsification ability of a yeast oleosomes composition, compared to benchmarks: Egg yolk and Soy lecithin, to understand the rheological characteristics of the obtained emulsions and to understand the stability of the obtained emulsions.

[0113] Sunflower oil was gradually added to all the tested emulsifiers. The mixtures were homogenised with overhead homogeniser (Ultraturrax®) while gradually adding sunflower oil. The emulsion was set for 24h.

[0114] Pure emulsifiers (Total Solids 17 %) were used as control

[0115] Formulations

[0116] For the formulation of the samples the amount of emulsifier and water was matched between samples of the three different emulsifiers tested: yeast oleosomes composition from Yarrowia lipolytica comprising about 5 wt% P-D-glucan, based on (origin: Cultivated Biosciences, Switzerland) (labelled herein as CB cream, samples A, B, C, D, E, F and G, invention), egg yolk (samples Al, Bl, Cl, DI, El, Fl and Gl, comparative) soy lecithin (samples A2, B2, C2, D2, E2, F2 and G2, comparative) contained the same amount of water, the same amount of emulsifier and the same amount of total lipids.

[0117] For yeast oleosomes (CB cream), emulsifier quantity was calculated by subtracting water and oleosomes lipids (i.e. triglycerides (TAG)) from the total quantity of the yeast oleosome composition (i.e. it is the total non-lipid solids of the yeast oleosomes composition). For lecithin, the whole dry matter was considered as emulsifier and for egg yolk as well. Emulsifier to lipids and lipids to emulsifier content for the samples in table 1. PAT8219EP00

[0118] Table 1: Chemical characteristics of emulsifiers:

[0119] Table 2. The ratio between lipids and emulsifier presence

[0120] PAT8219EP00

[0121] Table 3. Emulsified compositions prepared withyeast oleosomes (labelled as CB cream).

[0122] Table 4. Emulsified compositions prepared with egg yolk (comparative). PAT8219EP00

[0123] Table 5. Emulsified compositions prepared with lecithin (comparative).

[0124] Analysis:

[0125] Viscosity: viscosity was measured with Anton Paar rheometer MCR 301, with cylinder geometry (serial number CC27 9342). Shear rate was measured between 0.01 and 100 1 / s. The viscosity was measured at 25°C temperature.

[0126] Particle size analysis (PSD): PSD was measured by laser diffraction with Anton Paar PSA 1190, measuring time was set to 1 min with 6 repetitions per sample. The reconstruction algorithm was MIE, the refractive index for the material was 1.47 and the absorption index was 0.01. Samples were dissolved in distilled water, the refractive index was set to 1.33. The average of the D(4,3) (by volume) of the six measurements was used for the evaluation of the samples.

[0127] Spin test (accelerated instability test): Samples were spined at 10000 rpm for 20 mins using a benchtop 2 ml centrifuge.

[0128] Particle size analysis results:

[0129] Particle size distribution (PSD) was tested both with and without SDS to check for agglomeration or coalescence of the lipid particles. D(4,3) was used to compare the different emulsifiers as it represents the mean diameter of the particle size based on volume-weighted area mean results. The results are provided in [Fig. 1], PAT8219EP00

[0130] Table 6: Raw data particle size CB emulsification (invention):

[0131] Table 7: Raw data particle size egg yolk emulsification (comparative): PAT8219EP00

[0132] Table 8: Raw data particle size lecithin emulsification (comparative):

[0133] For results without addition of SDS: CB emulsion showed smaller particle size up to sample E compared to egg yolk, while for sample F the results were comparable. Sample G from CB emulsion showed a bigger particle size compared to egg yolk. Regarding the comparison between CB and lecithin, CB showed smaller particle size up to sample D.

[0134] SDS was added to the samples to assess the presence of aggregation in the emulsions. CB presented a smaller particle size compared to egg yolk up to sample F, and a smaller particle size compared to lecithin up to sample C, having comparable particle size for samples D.

[0135] Results showed a better emulsifying ability of CB compared to egg yolk up to sample F, where lipids to emulsifier ratio was 27.72:1, and better emulsifying ability compared to lecithin up to sample D, where lipids to emulsifier ratio was 16.63: 1.

[0136] Viscosity results

[0137] Results of the viscosity assessment are provided in [Fig. 3] for samples A to G of the invention, in [Fig. 4] for samples Al to G1 for egg yolk (comparative) and in [Fig. 5] for samples A2 to G2 with soy lecithin (comparative).

[0138] Regarding samples of the invention, viscosity increased for each sample, up to F (i.e. up to a lipids to emulsifier ratio of 27.72: 1), while the emulsion broke in sample G, having a lipids to emulsifier ratio of 33.26:1. High pressure homogenization might lead to a stable emulsion at that lipids to emulsifier ratio. PAT8219EP00

[0139] Emulsions stabilized with CB creams compared to egg yolk emulsions show a higher viscosity for all samples except sample G (lipids to emulsifier ratio 33.26:1), as the emulsion G with CB cream broke.

[0140] CB emulsions compared to lecithin emulsions show a higher viscosity for all samples except samples E, F and G. At lower oil inclusion rates CB shows better emulsification than the benchmark products egg yolk and lecithin. At higher oil inclusion rates the viscosities of egg yolk, lecithin and CB cream emulsions get closer, up to the oil inclusion rate at which CB cream emulsion breaks. High pressure homogenization could be a way to improve emulsification and hence increase the oil inclusion in CB cream emulsions.

[0141] Spin test results

[0142] Spin test showed stability of the emulsion of the invention (CB cream) up to F sample, showing no oil leakage. For G sample, oil leakage was seen, meaning a rupture of the emulsion.

[0143] Spin test showed stability (no oil leakage) for all the samples created with egg yolk.

[0144] Spin test showed stability for all the samples created with lecithin.

[0145] Spin test was performed to accelerate the instability of the emulsions. All the samples were stable except for sample G sample. This indicates that CB emulsion perform similarly to commercially available emulsifiers up to a lipids to emulsifier ratio of 27.72: 1. This might be solved by high pressure homogenization.

[0146] Example 2: Effect of p-D-glucan in yeast oleosomes stability

[0147] The objective of this experiment was to compare the stability of yeast oleosomes having a total solids (TS) content of 20 wt% and comprising about 10 wt% P-D-glucan based on TS, to yeast oleosomes comprising degraded P-D-glucan. To achieve this, samples were prepared by degrading the P-D-glucan contained in a yeast oleosomes composition extracted from Yarrowia lipolytica with a total solids content of 20 wt%, a TAG content of 40wt% and about 5wt% P-D-glucan based on TS (origin: Cultivated Biosciences, Switzerland) (Sample H). One composition was produced by treating the yeast oleosomes composition with endo- glucanase (Sample I) and another one by treating the yeast oleosomes composition with exo- glucanase (Sample J). Endo-glucanase degrades P-D-glucan by cleaving the beta-1,3 bonds, thus cutting the native P-D-glucan into smaller fractions. Exo-glucanase cleaves the nonreducing ends of P-D-glucan, thus releasing alpha-glucose. PAT8219EP00

[0148] Samples H, I and J were kept at the same temperature for the same time as the enzymatic treatment. All samples have also been heated to 90°C (temperature used to inactivate the enzymes).

[0149] The size of the particles in Samples H, I and J was then assessed. For all samples, PSD was measured by laser diffraction with Anton Paar PSA 1190, measuring time was set to 1 min with 6 repetitions per sample. The reconstruction algorithm was MIE, the refractive index for the material was 1.47 and the absorption index was 0.01. Samples were dissolved in distilled water, the refractive index was set to 1.33. The average of the D(4,3) (by volume) of the six measurements was used for the evaluation of the samples. The D4,3 of all samples is provided in Table 9 below.

[0150] Table 9: particle size of Sample H, I and J

[0151] These results show that the particle size of the yeast oleosomes emulsion is very much increased when P-D-glucan is degraded, thus showing a very advantageous role of P-D-glucan on the structure of the emulsion. It also advantageously improves the stability of the emulsion over time, because the smaller the particles, the more stable the emulsion is over time. It is therefore particularly advantageous to use yeast oleosomes comprising P-D-glucan in products such as high protein liquid products, such as protein shakes, in which stability over time is a very important aspect for product quality and acceptance by the consumers.

[0152] Example 3: effect of P-D-glucan on emulsification capacity

[0153] The emulsification capacity of Samples H, I and J used in Example 2 was assessed. An amount of 5 g of Samples H, I or J, respectively, was added to a 40 mL beaker. Another beaker was filled with oil and weighed. Homogenization of the 5 g of sample using a mediumsized disperser (Ika S25N-10G) at medium speed (13500 rpm) was started. Then 1 mL of oil was added with a pipette to the emulsion, until it was emulsified. Additions of 1 mL of oil were repeated until the emulsion broke (i.e. until the viscosity of the emulsion dropped and the emulsion became visibly heterogeneous). The oil beaker was weighed again and the amount of 1

[0154] PAT8219EP00 oil added to the Sample until emulsion broke was calculated as being the difference between the weight of the oil beaker in the beginning of the experiment and the weight of the beaker at the time the emulsion broke.

[0155] The emulsification capacity was calculated by dividing the total amount of TAG (i.e. TAG of oil added (in grams) + TAG in the sample) by the amount of non-TAG TS in the sample placed in the beaker (in grams). Measurements were performed in duplicates. The emulsion viscosity before the breaking point of the emulsion was also assessed on a scale from 1 (very liquid) to 4 (solid-like). The ratio of total fat in the emulsified composition to total nonfat solids in the oleosomes compositions of Samples H, I and J was calculated at the time the emulsion broke. The results are provided in Table 10 below. The Yeast oleosome composition fresh weight (20% TS) was 5g, the yeast oleosome composition dry weight was 1g, the total TAGs in the yeast oleosome composition was 0.46g and the non-TAG solids in the oleosome composition (emulsifier) was 0.54g. PAT8219EP00

[0156] These results demonstrate that degradation of P-D-glucan significantly decreases the emulsification capacity of the yeast oleosome composition. This shows that P-D-glucan in the form of long enough chains, provide improved emulsification capacity to the yeast oleosome composition making it particularly advantageous in high protein product, in particular those in liquid form such as protein shakes, wherein emulsification capacity is a key parameter impacting product stability over time, product appearance, product texture and the possibility to use high amounts of proteins.

[0157] Example 4: comparison of emulsification capacity of yeast oleosomes over plant-based oleosomes

[0158] The emulsification capacity of the yeast oleosome composition of Sample H was compared to that of a 20% TS sunflower oleosomes composition (Sample K).

[0159] The sunflower oleosomes were extracted using the method disclosed in Karfyllakis et al., Soft Matter, 2019, 15, 4639. First, seeds were soaked overnight in an aqueous solution of pH 8, adjusted with 4 M NaOH, and stored at 4 °C. After overnight soaking (16 hours), the excess aqueous solution was drained off using a strainer. The seeds were then ground with demi water in the ratio 1 : 7 (w / v) for 90 seconds in a laboratory blender at maximum speed. The slurry was then passed through a commercial juicer in order to remove the solid leftovers. The crude extract obtained as filtrate was centrifuged at 10000g at 4 °C for 30 min. The cream layer was then carefully removed and subsequently re-suspended in aqueous solution pH 8 at 1 : 4 ratio (w / v) and centrifuged under the same conditions. This washing cycle was repeated once more. The total solids of the obtained composition was of 20%, thus obtaining the sunflower oleosomes composition of Sample K.

[0160] The emulsification capacity of Samples H and K was assessed. An amount of 5 g of Samples H and K, respectively, was added to a 40 mL beaker. Another beaker was filled with oil and weighed. Homogenization of the 5 g of sample using a medium-sized disperser (Ika S25N-10G) at medium speed (13500 rpm) was started. Then 1 mL of oil was added with a pipette to the emulsion, until it was emulsified. Additions of 1 mL of oil were repeated until the emulsion broke (i.e. until the viscosity of the emulsion dropped and the emulsion became visibly heterogeneous). The oil beaker was weighed again and the amount of oil added to the Sample until emulsion broke was calculated as being the difference between the weight of the oil beaker in the beginning of the experiment and the weight of the beaker at the time the emulsion broke. PAT8219EP00

[0161] The emulsification capacity was calculated by dividing the total TAG content (i.e. the TAG amount in the oil added (in grams) + the TAG from the Sample) by the amount of non- TAG TS in the sample placed in the beaker (in grams, here 1 g). Measurements were performed in duplicates. The emulsion viscosity before the breaking point of the emulsion was also assessed on a scale from 1 (very liquid) to 4 (solid-like). The ratio of total fat in the emulsified composition to total non-fat solids in the oleosomes compositions of Samples H and K was calculated at the time the emulsion broke. The results are provided in Table 11 below.

[0162] The Yeast oleosome composition fresh weight (20% TS) was 5g, the yeast oleosome composition dry weight was 1g, the total TAGs in the yeast oleosome composition was 0.46g and the non-TAG solids in the oleosome composition (emulsifier) was 0.54g.

[0163] Table 11: Emulsification capacity (EC) of Samples H and K

[0164] These results demonstrate that yeast oleosomes have a significantly better emulsification capacity compared to plant-based oleosomes such as sunflower oleosomes. This shows that yeast oleosome are particularly advantageous in high protein product, in particular those in liquid form such as protein shakes, wherein emulsification capacity is a key parameter PAT8219EP00 impacting product stability over time, product appearance, product texture and the possibility to use high amounts of proteins.

[0165] Example 5: Comparison of the stability of yeast oleosomes and sunflower oleosomes

[0166] The objective of this experiment was to compare the stability of yeast oleosomes (Sample H) with sunflower oleosomes (Sample K), such as used in Example 4. The size of the particles in Samples H and K was then assessed as described in Example 4. The D4,3 of all samples is provided in Table 12 below.

[0167] Table 12: particle size of Sample H and K

[0168] These results show that the particle size of the yeast oleosomes is smaller than that of plant-based oleosomes, such as sunflower oleosomes, thus showing a very advantageous emulsion structure of yeast oleosomes emulsion. It also advantageously translates in improved stability of yeast oleosomes over plant-based oleosomes, such as sunflower oleosomes, because the smaller the particles, the more stable the emulsion is over time. It is therefore particularly advantageous to use yeast oleosomes in high protein products, particularly in liquid products, such as protein shakes, in which stability over time is a very important aspect for product quality and acceptance by the consumers.

[0169] Example 6: Role of the RNA content in yeast oleosomes on emulsification capacity

[0170] To assess the role of the RNA content in the oleosomes composition, the yeast oleosomes composition of Sample H was treated with a nuclease under conditions prescribed by the supplier to obtain a yeast oleosome sample with reduced RNA content (Sample M). The The yeast oleosome composition of sample H were kept at the same temperature as the enzymatically treated sample L, for the same amount of time as the enzymatic treatment. Both Samples H and L have then been heated to 90°C (temperature required to inactivate the enzymes in Sample L). The samples were then tested for emulsification capacity at 20 % TS and compared. PAT8219EP00

[0171] The emulsification capacity was determined as described in Examples 3 and 4. The ratio of total fat in the emulsified composition to total non-fat solids in the oleosomes compositions of Samples H and M was calculated at the time the emulsion broke. The results are provided in Table 13 below.

[0172] The Yeast oleosome composition fresh weight (20% TS) was 5g, the yeast oleosome composition dry weight was 1g, the total TAGs in the yeast oleosome composition was 0.46g and the non-TAG solids in the oleosome composition (emulsifier) was 0.54g.

[0173] Table 13: Emulsification capacity (EC) of Samples H and M

[0174] Degrading the RNA of Sample H used in the present invention did not significantly impact its emulsification capacity. Hence it can be concluded that the RNA is not playing a key role in the emulsification capacity of yeast oleosomes. PAT8219EP00

[0175] Example 7: Comparison of the emulsification capacity of yeast oleosomes and whole yeast cells

[0176] The emulsification capacity of the yeast oleosome composition of Sample H was compared to that of the intact yeast cells from which such oleosomes are extracted, i.e. intact Yarrowia lipolytica cells (Sample N).

[0177] The emulsification capacity was determined as described in Examples 3 and 4. The ratio of total fat in the emulsified composition to total non-fat solids in the oleosomes compositions of Samples H and N was calculated at the time the emulsion broke. The results are provided in Table 14 below.

[0178] The Yeast oleosome composition fresh weight (20% TS) was 5g, the yeast oleosome composition dry weight was 1g, the total TAGs in the yeast oleosome composition was 0.46g and the non-TAG solids in the oleosome composition (emulsifier) was 0.54g.

[0179] Table 14: Emulsification capacity (EC) of Samples H and N

[0180] The yeast oleosome composition of Sample H (such as used in the present invention) showed a significantly higher emulsification capacity than whole cells of Sample N. Hence the microstructure of the yeast oleosomes composition is important for its functionality and cannot be replaced by isolated whole yeast cells.

Claims

PAT8219EP00Claims1. An emulsified composition comprising yeast oleosomes, water and exogenous lipids, wherein the total lipids in the composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1.

2. The emulsified composition according to claim 1, wherein the yeast oleosomes are provided in the form of a yeast oleosomes composition comprising at least one isolated yeast cell wall component, wherein the yeast cell wall component is selected from lipids, carbohydrates, proteins and combinations thereof, preferably selected from chitin, P-D- glucan, mannoprotein and combinations thereof.

3. The emulsified composition according to claim 2, wherein the at least one isolated yeast cell wall component is present in the yeast oleosomes composition in an amount of at least 1.5 wt%, preferably at least 0.42 wt%, more preferably at least 2.5 wt%, more preferably at least 3 wt%, more preferably at least 3.5 wt%, more preferably at least 4 wt%, more preferably at least 4.5 wt%, most preferably at least 5 wt%, preferably at least 7.5 wt%, preferably at least 10 wt%, preferably at least 15 wt%, preferably at least 20 wt% by weight based on the total weight of the solids in of the yeast oleosomes composition.

4. The emulsified composition according to claim 2 or 3, wherein the particle size of the yeast oleosomes composition is characterized by a mean diameter by volume of 0.2 to 7 pm as measured by laser diffraction, preferably using a Anton Paar PSA particle size analyser.

5. The emulsified composition according to any one of the preceding claims, wherein the oleosomes composition is provided in an amount such that the solids of the oleosome composition be present in an amount of more than 3 wt%, preferably at least 4 wt%, preferably at least 4.5 wt%, preferably at least 5 wt%, preferably at least 5.5 wt%, preferably at least 6 wt%, preferably at least 6.5 wt%, more preferably at least 7 wt%, even more preferably at least 8 wt%, based on the total weight of the emulsified composition.

6. The emulsified composition according to any one of the preceding claims, wherein the yeast oleosomes are provided in an amount such that the solids of the oleosome composition be present in an amount of up to 30 wt%, based on the total weight of the emulsified composition.

7. The emulsified composition according to any one of the preceding claims, wherein the total lipids in the emulsified composition to non-lipid solids in the oleosomes ratio is of 0.8: 1 to 50: 1 preferably 0.8: 1 to 45: 1, preferably 0.8: 1 to 42: 1, preferably 0.8: 1 to 40: 1.PAT8219EP008. The emulsified composition according to any one of the preceding claims, wherein exogenous lipids are present in the emulsified composition in an amount of up to 75 wt%, such as up to 70 wt%, or up to 60 wt%, or up to 50 wt%, or up to 40 wt%, or up to 30 wt%, or up to 25 wt%, or up to 22 wt %, or up to 21 wt%, or up to 20 wt%, or up to 19 wt%, or up to 18 wt%, or up to 16 wt%, or up to 15 wt%, or up to 11 wt%, or up to 10 wt%, based on the total weight of the emulsified composition.

9. The emulsified composition according to any one of the preceding claims, wherein the exogenous lipids are provided in the form of vegetal fat or oils, algal oils, microbial oils, fungal oils or a mixture thereof, preferably in the form of vegetal fat or oils, algal oils, microbial oils, non-yeast fungal oils or a mixture thereof.

10. The emulsified composition according to any one of the preceding claims, wherein the zeta potential at the surface of the oleosomes is in the range of -40 to -5 mV, preferably - 40 to -8 mV, more preferably -40 to -10 mV, even more preferably -40 to -20 mV, most preferably -30 to -20 mV at physiological pH, when measured with a Malvern Zetasizer Nano ZS90 at a sample dilution of 1 : 100 sample:water, with a refractive index of solvent of 1.33, a refractive index of sample of 1.47, an absorption of 0,010, and with a DTS1060 cell.

11. A food product comprising the emulsified composition according to any one of claims 1 to 1012. The food product according to claim 11, wherein the food product is selected from emulsified sauces, dairy alternatives, emulsified confectionary products, and desserts.

13. Use of yeast oleosomes to emulsify lipids with water, wherein the yeast oleosomes are used in an amount such that the total lipids in the composition to non-lipid solids in the oleosomes ratio is of (at least 0.8): 1.

14. A process for the preparation of an emulsified composition comprising yeast oleosomes, water and exogenous lipids comprising a) preparing a mixture comprising yeast oleosomes, water and exogenous lipids; and b) emulsifying the mixture, wherein the yeast oleosomes are admixed with the lipids in step a) such as to obtain a total lipids in the composition to non-lipid solids in the oleosomes ratio of (at least 0.8): 1.

15. The process according to claim 14, wherein the emulsification in step b) is performed by homogenization at pressures of 30 to 500 bar.

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