Edible scaffolds, their preparation and uses

Abstract

The present disclosure provides a method of producing an edible 3D scaffold and such 3D scaffolds, the method comprising introducing into a mold an edible gel forming formulation, the mold comprising a boundary and an internal pattern, the internal pattern constituting a negative of a 3 -dimensional scaffold structure, providing conditions that cause formation of a gel in said internal pattern; and releasing the gel from the mold, said gel having a 3D scaffold structure. The 3D scaffold is defined as an arrangement of elongated channels having a lumen and being interconnected by channel walls, said channel walls having a thickness that is less than half a channel's cross-sectional dimension. Also disclosed is a method of producing an alternative whole muscle meat, the method comprises introducing at least edible protein mass into an edible 3D scaffold gel defined as an arrangement of elongated channels having a lumen and being interconnected by channel walls.

Description

[0001] EDIBLE SCAFFOLDS, THEIR PREPARATION AND USES

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the food industry and more specifically to the food alternative industry.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] - International patent application publication No. WO2022147357

[0007] - European patent application publication No. EP4144225

[0008] - European patent application publication No. EP4166005

[0009] - International patent application publication No. WO2022079717

[0010] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0011] BACKGROUND

[0012] Plant based food products require the mimicking of organoleptic properties in order to meet consumers' needs. When dealing with whole cut meat alternatives, a specific challenge resides in the mimicking of connective tissue.

[0013] International patent application publication No. WO2022147357 describes a method of preparing a connective tissue analog and meat analog comprising the same; the method comprises combining ingredients comprising a hydrocolloid base and a dietary fiber additive to form a substantially homogenous mixture; hydrating the substantially homogenous mixture to form a hydrated gel; and at least partially dehydrating the gel to obtain an at least partially dehydrated gel comprising a non- covalently cross-linked polymer network, thereby forming the connective tissue analog. European patent application publication No. EP4144225 describes a scaffold including a plant protein and in-vitro meat produced by using the scaffold.

[0014] European patent application publication No. EP4166005 describes a method for obtaining a sterilizable macroporous three-dimensional (3D) tissue engineering scaffold which comprising a network of at least an inter-crosslinked biocompatible polymer. The method comprises preparing a dissolution of the at least a biocompatible polymer, pouring out the solution into molds and freeze.

[0015] International patent application publication No. WO2022147357 describes an edible meat analogue and a method of producing the same, the meat analogue comprising a plurality of protein strands and inter- strand sheaths material.

[0016] GENERAL DESCRIPTION

[0017] The present disclosure provides, in accordance with a first of its aspects, a method of producing an edible 3D scaffold, the method comprising introducing into a mold an edible gel forming formulation, the mold comprising a boundary and an internal pattern, the internal pattern constituting a negative of a 3 -dimensional scaffold structure, providing conditions that cause formation of a gel in said internal pattern; and releasing the gel from the mold, said gel having a 3D scaffold structure; the 3D scaffold is defined as an arrangement of elongated channels having a lumen and being interconnected by channel walls, said channel walls having a thickness that is less than half a channel's cross-sectional dimension.

[0018] Also provided, according to a second aspect of the presently disclosed subject matter, is a 3D scaffold comprising a three-dimensional gel structure (referred to herein, at times, by the term "3D scaffold") including an arrangement of elongated channels having a lumen and being interconnected by channel walls; wherein said channel walls comprise an edible gel and said channel walls have a thickness that is less than half a channel's cross-sectional diameter. In accordance with a third of its aspects, the present disclosure provides a method of producing an alternative whole muscle meat, the method comprises introducing edible mass, at least protein mass, into an edible 3D scaffold gel defined as an arrangement of elongated channels having a lumen and being interconnected by channel walls.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0021] Fig- 1 is an image of a silicon mold used for forming a connective tissue scaffold according to some examples of the presently disclosed subject matter.

[0022] Figs. 2A-2C are images of a mold that can be used for forming molds for forming a connective tissue scaffold such as that of Fig. 1; where Fig. 2A is the plastic mold used for forming the silicon mold of Fig. 1, and Figs. 2B-2C are molds with cells created using Voronoi Algorithm, the latter having a contour of a typical meat cut.

[0023] Fig. 3 is an image of a 3D scaffold obtained using the silicon mold of Fig. 1.

[0024] Fig. 4A-4B are images of 3D scaffold having a Honeycomb structure with 0.4mm wall thickness after being glued to T shape jig and put in between Llyod grippers for tensile test (Fig. 4A), and showing the specimen stretched while being held between two gripper (Fig. 4B).

[0025] Fig. 5A-5F are graphs showing the tensile stress-strain curves and elastic moduli calculations of the evaluated specimens with or without post treatment and with the different wall thickness, of respectively, honeycomb type specimens of the samples 1 to 6 as defined in Table 3 of Example 3.

[0026] Fig. 6 is an image of an alternative meat product including the 3D scaffold disclosed herein and holding protein mass and fat mass.

[0027] Fig. 7A-7B are a cross-section view (Fig. 7A) and top isometric view (Fig. 7B) of a schematic illustration of a 3D scaffold having elongated parallel intersecting channels, each including a respective lumen. DETAILED DESCRIPTION

[0028] The present disclosure is based on the development of edible scaffolds having a predesigned arrangement of thin-walled elongated channels having a lumen / elongated cavity, into which edible protein mass and optionally fat mass (the protein mass and fat mass being typically introduced into different channels) is or can be introduced. The combination of scaffold, after being filled with the edible mass, provides a final product having organoleptic properties that resemble those of alternative whole muscle cuts.

[0029] The 3D scaffold disclosed herein, is a standalone product, in the sense that is produced without the mass to be introduced into channels' lumen. In may thus be regarded as an intermediate product for use in the production of a final edible product. The final edible product would be obtained by filling the channels' lumen with the edible mass, be it a protein mass, fat mass or any other edible mass. The filling of the lumen may be designed with a pattern of fillings (i.e. some channels being filled with protein mass, some with fat mass), to provide the final product with an appearance of a corresponding an animal derived product (e.g. slab appearance).

[0030] The 3D scaffold having the elongated channels that are constructed with a thin channel wall and an inner channel lumen is obtained or obtainable using a mold designed as the negative of the desired arrangement of the thin-walled elongated empty channels.

[0031] The formation of the 3D scaffold involves casting of gel forming material in a specifically designed mold and thereafter release of the cast 3D structure from the mold.

[0032] Specifically, and in accordance with a first aspect of the presently disclosed subject matter, there is provided a method for producing a 3D scaffold, the method comprising: introducing into a mold an edible gel forming formulation, the mold comprising a boundary and an internal pattern, the internal pattern constituting a negative of a 3 -dimensional scaffold structure, providing conditions that cause formation of a gel having said 3D scaffold structure within the internal pattern; and releasing the gel from the mold while maintaining said 3D scaffold structure; the 3D scaffold structure being defined as an arrangement of elongated channels having a lumen, and being interconnected by channel walls, the channel walls have a thickness that is less than half a channel's cross-sectional diameter.

[0033] In accordance with a second of its aspects, the presently disclosed subject matter provides a 3D scaffold comprising a three-dimensional gel structure including an arrangement of elongated channels, the channels having a lumen and being interconnected by channel walls; wherein the channel walls comprise a gel and wherein the channel walls have a thickness that is less than half a channel's cross-sectional diameter.

[0034] Preferably, the gel is an edible gel and the 3D scaffold is an edible 3D scaffold.

[0035] In the context of the presently disclosed subject matter, when referring to an edible entity (be it the gel, the scaffold or any other material described herein as being edible) it is intended for use in food or related applications. Specifically, "edible" defines substance or product to safe for consumption by humans or animals, in accordance with recognized standards and regulations governing food safety. This includes materials that are nontoxic, non-harmful, and suitable for ingestion, either as a component of a food product or as a material used in the production or preparation of food, without causing adverse effects to the consumer. The term encompasses materials that comply with applicable food-grade standards and are approved by relevant regulatory authorities for consumption.

[0036] For the sake of simplicity, the following description including the different examples and definitions are applicable to both the presently disclosed method and the presently disclosed 3D scaffold, with the necessary changes according to the specific context. Thus, for example, when referring to the elongated channels, it is to be understood to refer to those of the mold, forming part of the first aspect of the presently disclosed subject matter, as well as to the 3D scaffold of the second aspect of the presently disclosed subject matter.

[0037] In the context of the presently disclosed subject matter, when referring to a "mold", it is to be understood to refer to a tangible structure having distinct boundaries (i.e. a defined physical perimeter) within which resides the internal pattern. This internal pattern embodies the negative impression or, in other words, the exact reverse configuration of the 3D scaffold to be produced therewith.

[0038] More specifically, the internal pattern provides at least the negative of the arrangement of the elongated empty channels and their walls, as defined with respect to the 3D scaffold.

[0039] In the context of the presently disclosed subject matter, when referring to "elongated channels" it is to be understood to mean essentially linear, extended passages suitable to receive and accommodate filling mass, such as protein mass, the channels having a length (longitudinal dimension) greater than their width (cross sectional dimension). In other words, the 3D scaffold is a product that is absent of the filling mass, and the filling mass, e.g. protein mass, can be later introduced.

[0040] A channel that is considered "essentially linear" is one in which the nominal direction of the channel follows a generally linear path, allowing for some level of deviation. This deviation may be up to ±10°, or in more precise cases, up to ±3° or even ±1° from a perfectly straight line. The term "essentially linear" should be understood to accommodate a small percentage of deviation, potentially up to 20% of the channel length, but typically less than 10%, and in some cases, less than 3% or even 1%, where the channel may exhibit slight curvatures or bends.

[0041] In some examples of the presently disclosed subject matter, the elongated channels have a length of at least 5mm. Accordingly, the internal pattern of the mold is constructed so as to provide channels with a length of at least 5mm.

[0042] In some examples of the presently disclosed subject matter, the elongated channels have an inner (net) cross-sectional dimension of between about 1 mm and about 5 mm. This is to be understood to mean also that the mold is constructed with an internal pattern that is configured to provide elongated channels with an inner cross-sectional dimension of between about 1 mm and about 5 mm.

[0043] In the context of the presently disclosed subject matter, the term "inner cross-sectional dimension" is to be understood to refer to the largest dimension perpendicular to the channel's longitudinal axis, and without accounting to the thickness of the channel's wall at the cross section (i.e. net cross section, the dimensions between the inner surface of the channel's wall). The inner cross-sectional dimension is greater than the wall's thickness.

[0044] In some examples of the presently disclosed subject matter, the walls of the channel have a thickness that is less than half the channel's cross-sectional dimension.

[0045] In some examples of the presently disclosed subject matter, the walls of the channel have a thickness that is less than a third the channel's cross-sectional dimension.

[0046] In some examples of the presently disclosed subject matter, the walls of the channel have a thickness that is less than quarter the channel's cross-sectional dimension.

[0047] In some examples of the presently disclosed subject matter, the walls of the channels have a thickness that is less than 0.4mm; at times, less than 0.3mm, when the 3D scaffold is in wet form; or less than 0.2mm; at time, less than 0.1mm when measured on the 3D scaffold that is in dried or dry form (irrespective of the cross-sectional dimension of the channel).

[0048] When referring to dried form or dry form, in the context of the presently disclosed subject matter, it is to be understood to encompass the 3D scaffold including up to about 20wt% water; at times, up to about 15wt% water; or even, at times, up to 10wt% water. Drying can be achieved by any method known in the art, as further described herein below.

[0049] When referring to wet form, in the context of the presently disclosed subject matter, it is to be understood to encompass the 3D scaffold including at least 20wt% water; at times, at least 30wt% water; at times, at least 40wt%; at times, at least 50wt%; at time, at least 60wt%; at times, at last 70wt%; at times, at least 80wt%; at times, at least 90wt%; at times, at least 95wt%; at least 98wt% or even up to about 98wt%.

[0050] In some examples of the presently disclosed subject matter, when the cross- sectional dimension is about 1 mm, the wall's thickness is less than 0.5mm, at times, less than 0.3mm; at times, less than 0.25mm.

[0051] In some examples of the presently disclosed subject matter, when the cross- sectional dimension of the channel is 5mm, the wall's thickness will be less than 2.5mm; at times, less than 0.825mm; at times, less than 0.625mm.

[0052] In some examples of the presently disclosed subject matter, the channel walls have a thickness that is less than 0.2mm, when the 3D scaffold is in dried form. In some examples of the presently disclosed subject matter, the channel walls have a thickness that is less than 0.1mm; when the 3D scaffold is in dried form.

[0053] In some examples of the presently disclosed subject matter, the channel walls have a thickness that is in between about 0.01mm and about 0.2mm; when the 3D scaffold is in dried form.

[0054] In some examples of the presently disclosed subject matter, the channel walls have a thickness that is in between about 0.05mm and about 0.15mm; when the 3D scaffold is in dried form.

[0055] In some examples of the presently disclosed subject matter, the elongated channels within the 3D scaffold have essentially the same cross-sectional dimensions and / or a same average wall thickness.

[0056] In some examples of the presently disclosed subject matter, the elongated channels of the 3D scaffold may vary in their cross-sectional dimensions and / or in their wall thickness. As such, a single, standalone 3D scaffold may comprise a plurality of channels having different cross-sectional dimensions and / or different wall thickness.

[0057] In some examples of the presently disclosed subject matter, the mold (i.e. the internal pattern) is constructed to provide channels with an inner cross-sectional area of between about 1mm2and about 25mm2. As a result, the channels within the 3D scaffold have inner cross-sectional areas of between about 1mm2and about 25mm2.

[0058] In some examples of the presently disclosed subject matter, the channels within the 3D scaffold have inner cross-sectional areas of between about 2mm2and about 20mm2; at times, between about 2mm2and about 15mm2; at times, between about 2mm2and about 10mm2. At times, about 3mm2.

[0059] In the context of the presently disclosed subject matter, the term "inner cross-sectional area" is to be understood to refer to the two-dimensional surface spanned within the internal boundaries of the channel's wall, when viewed perpendicular to the channel's longitudinal axis, and without accounting to the thickness of the channel's wall at the cross section (i.e. net cross section). In some examples of the presently disclosed subject matter, the channels (or their negative impression in the mold) are essentially parallel or essentially aligned to have a same nominal direction.

[0060] The term “nominal direction'’’’ as used herein refers to a direction where significantly more than 50% of the channels (or the negative impression of the channels, in the mold) have a direction of up-to ±45 degrees from that nominal direction, when the channels (or their negative impression in the mold) are viewed from any direction perpendicular to the channel's direction. The term “nominal direction” may also refer to the average of the channels' direction as found using high magnification imaging as described herein. The nominal direction is a solid angle, where its projection on each of the 2 views, is the average direction found at this view.

[0061] In the context of the present disclosure, the term "essentially" is used to denote some level of deviation, such as 1%, 2%, 3%, 10%, or even up to 20%, from a defined parameter.

[0062] In this context, when referring to “essentially parallel channels” or “parallelly oriented channels” or "essentially aligned channels" it is to be understood to refer to the orientation of at least 80% of the channels, at times, at least 85%; at times, at least 90%; at times, at least 95% of the channels one with respect to the other when viewed within a specimen, to be generally parallel. In this context, the essential alignment is within a specimen having a dimension of at least lcm*2cm*2cm.

[0063] The term "essentially parallel" or “generally parallel” should be understood to encompass the nominal direction of the longitudinal axis to be at most ± 10°, at times, at most ± 3°, at most ±1°.

[0064] In some examples, the internal pattern is configured such that at least 50% of the elongated channels have the same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of about 10mm* 20mm* 20mm (4000mm3); the short, 10mm dimension being the dimension aligned with the direction of the channels.

[0065] For the sake of simplicity, in the above and below description and in the appended claims, when referring to a 3D scaffold or to a sample of the 3D scaffold having dimensions of 10mm* 20mm* 20mm it is to be understood to have the short dimension (10mm) in the direction of the longitudinal axis of the elongated channels. Since the other two dimensions in the sample are greater than the longitudinal dimension of the elongated channels, the short dimension can be regarded as the thickness of the sample.

[0066] At times, at least 60% of the elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of about 10mm* 20mm* 20mm; at times, at least 70% of the elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of about 10mm* 20mm* 20mm; at times, at least 80% of the elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of about 10mm* 20mm* 20mm at least 90% of the elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of about 10mm* 20mm* 20mm.

[0067] In some examples of the presently disclosed subject matter, the internal pattern of the mold is configured to allow for at least a portion of the channels to intersect, to form an organized collection of interconnected channels. The dimensions of the intersecting channels need not to be the same as those of the elongated channels.

[0068] In some examples of the presently disclosed subject matter, the intersecting channels form an angle with the longitudinal axis of the elongated channel.

[0069] In some examples of the presently disclosed subject matter, the intersecting channels are essentially perpendicular to the longitudinal axis of the elongated channel.

[0070] A schematic illustration of elongated channels being intersected by other channels (or holes) is provided in Figs 7A-7B.

[0071] Without being bound thereto, it is believed that there is an advantage of having intersecting channels, the point of intersection creating intersecting holes in the standalone (i.e. unfilled) 3D scaffold. During the mass filling inside the channels, the mass will pass through the holes between the channels' columns and create a connection between the channels of the filled mass. Such interactions may affect adhesion of the whole structure and better interlocking of the structure.

[0072] Thus, in the context of the present disclosure, when referring to intersecting channels, it also encompasses holes, and these holes are not necessarily elongated channels. In this connection, it is to be clear that if or when present, the holes have a controlled location and are considered patterned holes and not spontaneously formed holes. In other words, the location of the intersecting holes is determined or determinable by the design of the internal pattern of the mold.

[0073] In some examples of the presently disclosed subject matter, at least a portion of the elongated channels traverses the entire 3D scaffold, spanning from one end point to the opposite end point extend from one end to another end of the 3D scaffold.

[0074] In some examples of the presently disclosed subject matter, elongated channels are open at least at one end thereof, i.e. the channel does not extend along the entire longitudinal direction of the mold or the 3D scaffold.

[0075] In some examples of the presently disclosed subject matter, elongated channels are open from both ends thereof, i.e. the channel extends along an entire longitudinal direction of the mold or the 3D scaffold.

[0076] The cross section of the channel may have different shapes.

[0077] In some examples of the presently disclosed subject matter, at least a portion of the channels have a polygonal contour, e.g. have a polygonal cross section.

[0078] In some examples of the presently disclosed subject matter, the polygonal cross section includes polygonal shapes with at least 3 sides, this includes, without being limited thereto, quadrilaterals (4 sides), pentagons (5 sides), hexagons (6 sides), and heptagon (7 sides).

[0079] In some examples of the presently disclosed subject matter, the internal pattern is configured to provide channels with a hexagon cross section; and accordingly, the 3D scaffold comprises channels with a hexagon cross section.

[0080] In some examples of the presently disclosed subject matter, the internal pattern is configured to provide essentially aligned channels with a hexagon cross section. This may be regarded as an arrangement of essentially aligned pillars having a hexagonal cross section. According to this non-limiting example, the 3D scaffold thus comprises essentially aligned channels with a hexagon cross section.

[0081] In some examples of the presently disclosed subject matter, the internal pattern comprises the negative impression of a 3D honeycomb scaffold structure; and accordingly, the 3D scaffold comprises a 3D honeycomb scaffold structure. Fig. 1 provides an image of an exemplary honeycomb mold that can be used for forming a connective tissue.

[0082] Fig. 2A-2C are images of molds that can be used for forming a mold according to the present invention, Fig. 2A can be used for forming a mold for forming a 3D scaffold having an internal pattern of honeycomb, while Figs. 2B-2C are images of other types of molds (for forming a mold according to the present invention), the first is a rectangular mold with cells created using Voronoi algorithm and 3D printed via conventional FDM 3D printer and the second is similarly formed, but now the overall shape is adjusted to a typical meat cut.

[0083] In some examples of the presently disclosed subject matter, the scaffold walls pattern is defined by applying the Voronoi function, i.e. the channel walls have a cross-section structure defined by Voronoi function, and accordingly, the 3D scaffold comprises a Voronoi scaffold structure. The inputs of the Voronoi function are selected in such a manner that the channels dimensions and cross-sectional area fit the previously defined dimensions for the channel wall, channel cross section and other disclosed dimensions of the 3D scaffold.

[0084] In the context of the presently disclosed subject matter, when referring to a "Voronoi scaffold" or " Voronoi cross section" it is to be understood to refer to a three dimensional architectural or structural framework created using the Voronoi tessellation concept. In this connection, it is to be appreciated that applying Voronoi principles is only an example of possible tessellation and other techniques can be used to construct the 3D pattern, such as mathematical or algorithmic methods or image processing of livestock meat or even manual design of a pattern, e.g. by industrial designer. The design of the 3D scaffold pattern can be, for example, inspired by existing and accessible patterns of meat fibers structures, determined via photography, microscopy and / or histology, bearing in mind that the channels structure and pattern is aimed, inter alia, to imitate the transverse section of muscle fascicles.

[0085] In some examples of the presently disclosed subject matter, at least a portion of the channels are designed to have or have a round or curved contour, e.g. have a round or curved cross section. In some examples of the presently disclosed subject matter, at least a portion of the channels are designed to have or have a hybrid cross section, i.e. at least part of the cross section is defined by straight lines and angles therebetween and at least some other parts of the cross section are curved lines.

[0086] The method comprises introducing into the mold an edible gel forming formulation.

[0087] In the context of the present disclosure, the term "introducing" in connection with preparing the 3D scaffold, is to be understood to encompass any action that results in filling the internal pattern of the mold with edible gel. The introducing aims at filling the open spaces of the internal pattern until reaching an essentially full embedment of edible gel (formed from the edible gel forming formulation) within the internal pattern of the mold.

[0088] In some examples of the presently disclosed subject matter, the gel forming formulation, prior to said introduction, is in the form of semi solid or solid particles, and the introducing of the gel forming formulation into the mold (and eventually the formation of a gel within the internal pattern of the mold) , according to the presently disclosed method, comprises placing the solid or semi-solid edible gel forming formulation, e.g. in a form of a sheet or in a particulate form, over an open top of the mold and heating the mold to a temperature sufficient to cause melting of the solid or semi solid edible gel forming material resulting in a gel forming molten that flows by gravitational forces into the internal pattern of the mold.

[0089] In some examples of the presently disclosed subject matter, the introducing of the gel forming formulation into the internal pattern of the mold comprises pouring molten gel forming formulation into the internal pattern of the mold.

[0090] In some examples of the presently disclosed subject matter, the introducing of the gel forming formulation into the mold comprises injection of molten gel forming formulation into the internal pattern of the mold.

[0091] Heating of the mold, when necessary, can be prior to, concomitant with or after placing the solid or semi-solid edible gel forming material thereon.

[0092] In some examples of the presently disclosed subject matter, the heating of the mold is prior to placing the solid or semi-solid edible gel forming material thereon. In some examples of the presently disclosed subject matter, the mold is heated to a temperature sufficient to maintain the gel forming formulation in molten state, and to allow flow of the gel forming melt into the spaces formed by the internal pattern.

[0093] In some examples of the presently disclosed subject matter, the method also comprises cooling of the mold and the gel forming molten therein. Thus, according to the presently disclosed method, some of the conditions for the formation of the 3D scaffold using the defined mold involve heating of the mold and after the gel forming formulation is embedded within the internal pattern (the internal spaces) of the mold, the conditions comprise cooling the mold and the gel material therein. Without being limited thereto, the cooling causes the hardening of the gel material resulting in the 3D scaffold, and facilitates the extraction of the 3D scaffold from the mold.

[0094] Extraction of the 3D scaffold from the mold can be performed using techniques known in the art. For example, and without being limited thereto, the 3D scaffold can be removed using mechanical ejection tools, such as draft angles, mold extraction devices, lifting surfaces; air ejection techniques, such as compressed air; vacuum assisted ejection, spray release agents (suitable for the food industry) etc.

[0095] In some examples of the presently disclosed subject matter, the method also comprises drying the 3D scaffold gel, after being extracted from the mold.

[0096] When referring to drying, in the context of the presently disclosed subject matter, it is to be understood to encompass any technique of water removal. The drying does not need to reach complete dryness. In some examples of the presently disclosed subject matter, when referring, with respect to the 3D scaffold, to dry or drying or dried 3D scaffold, it is to be understood to mean that the 3D scaffold has up to about 20wt% water; at times, up to about 15wt% water; at times, up to about 10wt% water; at times, up to about 5wt%; at times, up to about 3wt%; at times, up to about 1 wt%. Water content in the scaffold gel can be determined using, for example, Differential Scanning Calorimetry (DSC), or by moisture analyzer as known in the art.

[0097] In some examples of the presently disclosed subject matter, the drying is by any one or combination of freeze drying, heating and air dry of the 3D scaffold gel.

[0098] In some examples of the presently disclosed subject matter, the drying comprises at least freeze drying of the 3D scaffold gel. Accordingly, in some examples of the presently disclosed subject matter, when drying is applied, the 3D scaffold in accordance with the presently disclosed subject matter is in dried or dry form.

[0099] In addition or alternatively to drying, the 3D scaffold, post extraction, may be submerged into an ionic batch (an anti-solvent agent). An example of an anti-solvent agent may be KC1 dissolved in filtered water, to which ethanol is added.

[0100] The post extraction treatment, such as the drying and / or the ionic bath may contribute to preservation of the geometric structure of the extracted 3D scaffold.

[0101] In some examples of the presently disclosed subject matter, the method also comprises applying onto surfaces of the internal pattern of the mold an edible protein binding agent. In this connection, it is to be understood that the edible protein binding material is to an extent that does not cause blocking or filling of the channels' lumen and in fact would not affect that thickness of the walls to extend the dimension requirements of the walls and / or the dimension relationships between the wall and the lumen.

[0102] In the context of the presently disclosed subject matter, the term "edible protein binding agent" denotes any edible agent that facilitates or promotes the binding of the protein mass to be introduced into the elongated channels, to the channel walls.

[0103] Non-limiting examples of edible protein binding agent include gluten, glucomannan, locus bean gum, mastic resin, casein, guar gum, gum Arabic, xanthan gum, a psyllium, a chitin, an inulin, a pectin, a dextrin, a starch, a cellulose, a hemicellulose, a lignin, a citrus fiber extract, dietary enzyme, a transglutaminase analogs or derivatives thereof, or any combination thereof. Preferably, the protein binding agent is a non-animal derived / vegan protein (e.g. artificially produced casein).

[0104] In some examples of the presently disclosed subject matter, the edible protein binding agent comprises gluten.

[0105] In some examples of the presently disclosed subject matter, the edible protein binding agent comprises gluten powder.

[0106] The mold per se can be made from materials known in the art of molding, such as, silicon, polyurethane, latex rubber, metal, aluminum, fiberglass-reinforced plastics (FRP), wood and others. Further, the mold per se can be made by a variety of techniques known in the art for the fabrication of rigid molds, including, for example, binder jetting, material jetting, direct energy deposition (DED), powder bed fusion (PBF), fused deposition molding (FDM), mechanical milling, laser machining and others. For example, the mold of Fig. 1 was prepared using a 3D printing Stereolithography (SLA) using "Smooth SIL 940" silicon model, with Shore 40 food grade. Other techniques may include CNC. Further, for example, Fig. 2A is a 3D printed mold from solid polymer prepared by the same technique as the mold of Fig. 1. Using the mold of Fig. 1, a 3D scaffold was obtained (5% CAR+5% glycerol +l%gellan, See Table 1 below), an image of which is shown in Fig. 3. Fig. 3, being an example only, clearly shows the formation of interconnected channels, having each a lumen that is suitable for accommodating a mass.

[0107] Fig. 2A-2C are molds used for forming a mold according to the present invention.

[0108] As further examples, Fig. 7A-7B show a cross-section view (Fig. 7A) and top isometric view (Fig. 7B) of a schematic illustration of a 3D scaffold having elongated parallel, intersecting channels including the channel walls, each including a respective lumen. This illustration of a mold exemplifies intersecting holes to allow communication of mass from one lumen to another.

[0109] The gel forming formulation can comprise a variety of edible gels.

[0110] In some examples of the presently disclosed subject matter, the gel forming formulation comprises material that forms hydrogel. Thus, in such cases, the gel forming formulation is a hydrogel forming formulation.

[0111] In some examples of the presently disclosed subject matter, the gel forming formulation comprises material that forms a hydrocolloid. Thus, in such cases, the gel forming formulation is a hydrocolloid forming formulation.

[0112] In some examples of the presently disclosed subject matter, the hydrocolloid forming formulation comprises water and at least one hydrocolloid forming agent.

[0113] In some examples of the presently disclosed subject matter, the gel (or at times, hydrocolloid) forming agent is selected from the group consisting of Carrageenan, Agar, Gelatin, Sodium Alginate, Gellan Gum, Pectin, Xanthan Gum, Guar Gum, Methylcellulose, Starch, Konjac, Locust Bean Gum, Tara Gum, Curdlan and Chitosan. In some examples of the presently disclosed subject matter, the hydrocolloid forming agent is Carrageenan (CAR).

[0114] In some examples of the presently disclosed subject matter, the hydrocolloid forming agent is Kappa Carrageenan (K-CAR). In some examples of the presently disclosed subject matter, the gel forming formulation comprises at least 5% CAR (out of total wet weight), preferably at least 5% K-CAR.

[0115] In some examples of the presently disclosed subject matter, the hydrocolloid forming formulation comprises a combination of two or more hydrocolloid forming agents, at least one being selected from the above non-limiting list of hydrocolloid forming agents.

[0116] Non-limiting Examples of gel forming materials that can be used in the context of the presently disclosed subject matter and their physical properties is provided in Table 1 below, each formulation constituting an independent example of the presently disclosed subject matter.

[0117] Table 1: physical properties of exemplary gel forming material

[0118] Thus, without being limited to these examples, it is clear that by variations of the gel forming formulation can allow variability in the physical properties. Further, the above examples show that in some cases there may be an advantage of combining gellan and / or glycerol with CAR, and in some cases, there may be an advantage of adding to the gel forming formulation gellan.

[0119] In some examples of the presently disclosed subject matter, the gel forming formulation comprises other additives, not necessarily gel forming agents.

[0120] In some examples of the presently disclosed subject matter, the gel forming formulation comprises water-soluble polyol(s). In some examples, the polyol is selected from the group consisting of glycerol, Maltitol, sorbitol, xylitol, erythritol, and isomalt and combinations of same.

[0121] In some examples, the polyol is or comprises at least glycerol (also known as glycerin).

[0122] In some examples of the presently disclosed subject matter, the formulation comprises at least 5% CAR with glycerol.

[0123] In some examples of the presently disclosed subject matter, the amount of glycerol in the gel forming formulation is designed to be in the range of about l%w / w and 10%w / w out of the total wet weight.

[0124] In some examples of the presently disclosed subject matter, the formulation comprises Gellan and at least 5% CAR out of the total wet weight.

[0125] In some examples of the presently disclosed subject matter, the amount of Gellan in the gel forming formulation, if present, is designed to be in the range of up to 3%w / w out of the total wet weight.

[0126] In some examples of the presently disclosed subject matter, the formulation comprises glycerin and Gellan with at least 5% CAR out of the total wet weight. The amount of water mixed with the dry components of the gel forming formulation may range based on the selected components.

[0127] In some examples of the presently disclosed subject matter, the gel forming formulation can comprise animal free fat or oil, e.g. plant-based oil(s).

[0128] Without being limited thereto, the gel forming material can comprise any one or combination of the following oils: olive oil, coconut oil, sunflower oil, soybean oil, canola oil, avocado oil, almond oil, grapeseed oil, flaxseed oil, palm oil, hemp seed oil,

[0129] Without being limited thereto, the gel forming material can comprise any one or combination of cacao butter, shea butter, coconut fat.

[0130] Without being limited thereto, the gel forming material can comprise a combination of at least one animal free fat (such as those listed above) and at least one animal free oil (such as those listed above).

[0131] For example, and without being limited thereto, the plant-based oil can include coconut oil and the formulation can include between about l%w / w and 15%w / w plantbased oil.

[0132] In some examples, the gel forming material may comprise also protein, and when including protein, the amount thereof would typically be up to 20%w / w. In some examples, the protein forming the gel material comprises at least gluten, e.g. a liquid gluten formulation.

[0133] In some examples, the gel forming material comprises a combination of hydrogel and protein, in such cases the amount of the protein would typically be less than 20%w / w, at times, up to 10%w / w or up to 5%w / w.

[0134] Based on the above, the presently disclosed subject matter thus provides a 3D scaffold as defined herein above.

[0135] Specifically, there is disclosed herein a 3D scaffold comprising a three- dimensional gel structure including an arrangement of elongated channels having a lumen and being interconnected by channel walls; wherein said channel walls comprise an edible gel and said channel walls have a thickness that is less than half a channel's cross-sectional diameter. Also, provided herein is a 3D scaffold obtained or obtainable by the presently disclosed method.

[0136] The 3D scaffold can be characterized by its physical properties, allowing it to withstand the later filling with protein mass.

[0137] In some examples of the presently disclosed subject matter, the 3D scaffold is one that was subjected to at least one post extraction treatment, and preferably at least one of freeze drying and ionic bath immersion.

[0138] In some examples of the presently disclosed subject matter, the 3D scaffold is characterized by its capability to withstand a maximum load of at least 3N, as determined on a specimen having dimensions of about (90~l 10)*(40~50)*10mm and wall thickness that is equal or less than 400 micron, by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s, is at least 0.05 MPa.

[0139] In some examples of the presently disclosed subject matter, the 3D scaffold specimen is characterized by its capability to withstand a maximum load of at least 5N; at least 10N; at least 15N; at least 20N; at least 25N; at least 30N.

[0140] In some examples of the presently disclosed subject matter, the 3D scaffold is characterized by a tensile strength of at least 0.05 MPa, as determined on a specimen having dimensions of about (90~l 10)*(40~50)*10mm and wall thickness that is equal or less than 400 micron by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s.

[0141] In some examples of the presently disclosed subject matter, the 3D scaffold specimen is characterized by a tensile strength of at least 0.5MPa.

[0142] In some examples of the presently disclosed subject matter, the 3D scaffold is characterized by a maximum strain of at least 0.4 mm / mm, as determined on a specimen having dimensions of about 90-110*40-50* 10mm and wall thickness that is less than 400 micron by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TPA instrument equipped with a 100N load cell at a speed of 5mm / s.

[0143] For illustrative purposes, Figs. 4A-4B show gripping of a 3D scaffold specimen having a Honeycomb structure with 0.4mm wall thickness that was glued at its top and bottom ends to a T shape accessory used as grippers. The specimens were gripped by Llyod gripper for tensile tests (Fig. 4A) and then stretched under specific conditions (Fig. 4B)

[0144] In some examples of the presently disclosed subject matter, the 3D scaffold is characterized by a hyperelastic material properties exhibited as a nonlinear stress-strain curve, with the ability to undergo elastic deformations and return to its original shape upon unloading.

[0145] In some examples, the stress-strain curve is characterized by an initial nonlinear elastic behavior, followed by a high deformation range without plastic deformation, and an ultimate stretch limit defined by the maximum stress point before the material exhibits failure or necking.

[0146] In some examples of the presently disclosed subject matter, the 3D scaffold is one having a wall thickness of about 50 microns and is subjected to freeze-drying treatment. Such 3D scaffold is characterized by a maximum load of equal or at least 44.5N, a tensile strength of equal or at least 2.86 MPa, and a maximum strain of equal or at least 0.49 mm / mm.

[0147] In some examples of the presently disclosed subject matter, the 3D scaffold is one having a wall thickness of about 400 microns and is subjected to ionic bath immersion. Such 3D scaffold is characterized by a maximum load equal or of at least 63.7N, a tensile strength equal or of at least 0.74 MPa, and a maximum strain equal or of at least 1.09 mm / mm.

[0148] In some examples of the presently disclosed subject matter, the 3D scaffold has a honeycomb structure and is formed from a gel forming formulation comprising at least 5wt% CAR, about 5wt% glycerol, and about lwt% gellan F.

[0149] In some examples of the presently disclosed subject matter, the 3D scaffold has a honeycomb structure and is formed from a gel forming formulation comprising at least 5wt% CAR, about 5wt% glycerol, and about lwt% gellan F, and upon extraction from the mold, the extracted 3D structure is subjected to freeze drying.

[0150] In some examples of the presently disclosed subject matter, the 3D scaffold has a honeycomb structure and is formed from a gel forming formulation comprising at least 5wt% CAR, about 5wt% glycerol, and about lwt% gellan F, and upon extraction from the mold, the extracted 3D structure is subjected to post-treatment by immersion in an ionic bath comprising an anti-solvent.

[0151] The presently disclosed subject matter also provides, in accordance with a further aspect, a method of producing an alternative whole muscle meat, the method comprises introducing edible protein mass into an edible 3D scaffold gel defined as an arrangement of elongated channels interconnected by channel walls (the walls comprising the edible gel). In this context, there is also provided the alternative whole muscle meat / cut obtained using the disclosed 3D scaffold and the methods of using it.

[0152] Without being bound by theory, the combination of the presently disclosed 3D scaffold and the protein mass introduced into the channel lumen of the 3D scaffold, provides improved properties to the final edible product, in terms of texture and other organoleptic properties desired for alternative, plant based whole muscle meat. It was envisaged that the 3D gel structure imitates the functionality of the connective tissue surrounding bundles of muscle fibers also known as fascicles, while the protein mass provides the functionality of the muscle fibers.

[0153] Thus, for the sake of simplicity, it is to be understood that the above description and different definitions of features and alternative examples provided in connection with the presently disclosed method of producing the edible 3D scaffold and the presently disclosed edible 3D scaffold per se, are also applicable and define different examples and alternatives to the method of producing the alternative whole muscle cut.

[0154] The protein mass introduced into the edible 3D scaffold is an edible protein mass.

[0155] The term "protein mass" as used herein, denotes a composition comprising one or more edible proteins. In some examples, the protein mass is pliable, with a sufficient degree of flowability (under pressure) to allow its introduction into the elongated channels of the 3D scaffold, without causing any damage to the scaffold structure.

[0156] In some examples of the presently disclosed subject matter, the protein mass is in fluid form.

[0157] In some examples of the presently disclosed subject matter, the protein mass has a physical state that permits its pouring or injection into the channels. In some examples of the presently disclosed subject matter, the protein mass comprises the one or more edible proteins, in combination with other substances, such as hydrogels, fats, coagulation agents etc.

[0158] In some examples of the presently disclosed subject matter, the protein mass comprises at least 10%w / w, at times, at least 20%w / w, at times, at least 30%w / w protein(s).

[0159] In some examples of the presently disclosed subject matter, the protein mass comprises between 10%w / w and 30%w / w protein(s).

[0160] In some examples of the presently disclosed subject matter, the protein mass comprises between 15%w / w and 25%w / w protein(s).

[0161] The protein mass being introduced into the 3D scaffold is to be considered as a wet composition. In some examples, the wet protein mass comprises at least 50%w / w water.

[0162] In some examples of the presently disclosed subject matter, the protein mass comprises between 55% and 80% water; at times, between 60% and 75% water; at times between 65% and 70% water.

[0163] In some examples of the presently disclosed subject matter, the protein mass comprises plant derived (e.g. isolate or concentrate) proteins (including peptides and amino acids). Without being limited thereto, the plant source for the protein mass can be any one or combination of soy, wheat, legume (e.g. pea, chickpea, beans), rapeseed and corn as well as many other plant-based protein sources as known in the food industry.

[0164] In some examples of the presently disclosed subject matter, the protein mass can comprise protein derived from sources other than plants, such as algae, fungi (e.g. yeast), bacteria and microorganisms in general.

[0165] In some examples of the presently disclosed subject matter, the protein mass can comprise texturized vegetable protein (TVP). TVP is known in the art to be used as a meat extender or vegetarian meat and is usually created by extruding protein isolates or concentrates using high shear, pressure and heat, from vegetable sources such as wheat, pea and others. TVP is commercially available in different sizes from large chunks to small flaks. In some examples of the presently disclosed subject matter, the protein mass comprises TVP. In some examples of the presently disclosed subject matter, the minimal amount of TVP within the protein mass is at least 10%w / w of dry weight TVP, out of the total protein in the protein mass. At times, the minimal amount is at least 15%w / w of dry TVP, at times at least 20%w / w of dry TVP; at times at least 25%w / w of dry TVP; at times at least 30%w / w of dry TVP; at times at least 35%w / w of dry TVP; at times at least 40%w / w of dry TVP; at times at least 45%w / w of dry TVP; at times at least 50%w / w of dry TVP; at times at least 55%w / w of dry TVP; at times at least 60%w / w of dry TVP.

[0166] In some examples of the presently disclosed subject matter, the minimal amount of TVP within the protein mass (out of the total protein), as determined before being introduced into the scaffold, is at least 20%w / w of wet TVP out of the total protein; at times, at least 30%w / w of wet TVP out of the total protein; at least 35%w / w of wet TVP out of the total protein. At times, the minimal amount is at least 40%w / w of wet TVP; at times, at least 45%w / w wet TVP; at times, at least 50% w / w TVP; at times, at least 55% w / w TVP; at times, at least 60% w / w TVP; at times, at least 65% w / w TVP; at times, at least 70% w / w TVP; at times, at least 75% w / w TVP.

[0167] The protein mass does not include only TVP. In some examples, the protein containing material contains at least 30% non-TVP protein matter.

[0168] In some examples of the presently disclosed subject matter, the protein mass also comprises fibrous material, i.e. natural fibers.

[0169] The protein mass can be introduced into the elongated channels of the 3D scaffold by any techniques known in the art that is suitable for such protein mass deposition.

[0170] Without being limited thereto, the introduction of the protein mass into the 3D scaffold's elongated channels, can be by the use of fluid filling techniques, such as, piston filling machines (e.g. SGP-250 Liquid Piston Filler Machine), peristaltic filling machines, gravity filling machines, vacuum filling machines, overflow filling machines, timepressure filling machines, net weight filling machines, electrical caulking gun (e.g. Markita DCGI80) equipped with a nozzle (in the mm size, e.g. 4 mm).

[0171] To facilitate the introduction of the protein mass without causing unnecessary pressure on the channel walls, and further to facilitate flow of the protein mass within the channel, the dispensing nozzle is selected to have a cross-sectional dimension that is smaller than that of the cross-sectional dimension of the elongated channel.

[0172] In some examples of the presently disclosed subject matter, the introduction of the protein mass is into a single 3D scaffold.

[0173] In some examples of the presently disclosed subject matter, the introduction of the protein mass is into two or more 3D scaffold, stacked one on top of the other, at times, with the elongated channels of the two or more 3D scaffolds being aligned so as to allow passage of protein mass from channels of one 3D scaffold to its adjacent 3D scaffold.

[0174] In some examples of the presently disclosed subject matter, the method of producing the alternative whole cut comprises stacking one on top of another 3D scaffolds, as disclosed herein, already filled with protein mass.

[0175] In some examples of the presently disclosed subject matter, when the method comprises stacking one on top of another 3D scaffolds already filled with protein mass, the gel forming the 3D scaffold and / or the protein mass, of the different scaffold layers of 3D scaffolds, may be the same or different.

[0176] In some examples of the presently disclosed subject matter, the method of producing the alternative whole cut comprise introducing edible fat into the lumen of at least part of the elongated channels.

[0177] In some examples of the presently disclosed subject matter, the method of producing the alternative whole cut comprise introducing edible fat in between two stacked 3D scaffolds holding the protein mass.

[0178] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0179] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %. The indefinite articles “a” and “an” as used herein in the description and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one". It must be noted that, as used in this description and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0180] The clause “and / or” as used herein in the description and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified.

[0181] As used herein in the description and in the claims “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of or “exactly one of” or, when used in the claims, “consisting of” will refer to the inclusion of exactly one element of a number or list of elements.

[0182] As used herein in the specification and in the claims, the phrase “at least one” should be understood to mean at least one element that is or can be selected from any one or more elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.

[0183] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. Throughout this description (including the Examples) and claims which follow, all transitional phrases such as “ comprising", ^including", ^carrying", ^having", ^containing", “involving”, ^holding", “ composed of and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “ consisting of and “ consisting essentially of shall be closed or semiclosed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises" , "comprising", "including" , “having^ and their conjugates mean "including but not limited to". The term “ consisting of means “ including and limited to" . The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0184] It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0185] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0186] It is appreciated that certain features of the presently disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0187] Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

[0188] Disclosed and described, it is to be understood that the presently disclosed subject matter is not limited to the particular examples, process steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the presently disclosed subject matter will be limited only by the appended claims and equivalents thereof.

[0189] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the presently disclosed subject matter. DESCRIPTION OF NON-LIMITING EXAMPLES

[0190] The following non-limiting Example illustrates formation of a matrix-like connective tissue structure into which edible, non-animal proteins are introduced creating a whole meat alternative muscle cut structure.

[0191] EXAMPLE 1 - Preparation of a Connective Tissue Scaffold

[0192] For forming the edible scaffold that forms the connective tissue of the whole meat alternative muscle cut structure, a silicon mold was constructed to function as a negative of the desired cellular matrix shape and porosity of the connective tissue to be produced. In this non-limiting Example, the mold, shown in Figure 1, had the shape suitable to provide a scaffold with an internal configuration of a honeycomb, defined by hexagon open cells.

[0193] Specifically, the silicon mold was created to provide hexagon cells having a circumcircle diameter of about 2.5mm and a hexagonal cell wall thickness of about 0.2mm.

[0194] Preparation o f a mold

[0195] The mold was prepared from food grade Silicon, with 40 Shore A hardness that was poured into a plastic rigid mold as shown in Figure 2, having hexagonal cells with a wall thickness of about 0.2mm. In fact, the plastic mold of Figure 2, is essentially a mirror structure of the scaffold to be obtained.

[0196] Preparation o f connective tissue using mold

[0197] The connective tissue (CT) forming material was prepared from a mixture comprising 5% Carrageenan, 5% Glycerol, 1% Gellan, 89% Water, which was heated to 90°C and steered in thermomixer until a homogeneous CT forming material was obtained.

[0198] Process A:

[0199] The homogeneous CT forming material was cooled to room temperature and then broken into pieces and placed over the Silicon mold until completely covered. The mold with the CT pieces were placed into an oven, pre-heated to 90°C for 20 minutes until all CT forming material melted and penetrated into the Silicon mold leaving a thin layer of CT forming material over the mold. Onto the thin layer of CT forming material, an additional portion of molten CT forming material is poured and put back into the oven set to a temperature of 115°C, for an additional 10 minutes, allowing any trapped air bubbles to be released from the molded CT forming material.

[0200] Process B:

[0201] Molten CT forming material was poured onto the oven heated mold and then spread and pressed against the mold's open cells. The mold with the pressed CT forming material was then placed back in the open, set at 95°C, for 10 minutes, after which it was allowed to cool outside at room temperature.

[0202] The mold with the scaffold made of the CT forming material was cooled at 4°C for 15 minutes and after removing excess CT forming material (e.g. the layers on top of the mold), the CT scaffold was extracted from the silicon mold.

[0203] The CT scaffold is shown in Figure 3, which has a honeycomb internal structure.

[0204] Post-formation Processing o f the CT Scaffold

[0205] Freeze-drying

[0206] For preserving geometric structure of the CT scaffold, the CT scaffold is subject to freeze-drying so as to remove water from the scaffold's walls. Freeze drying provides improved long term structural stability as compared to CT scaffold prepared by the same procedure, yet, without the freeze drying.

[0207] The conditions of freeze drying are provided in Table 2 including temperature, duration, vacuum.

[0208] Table 2: Freeze-Drying Conditions

[0209] An alternative to freeze drying may involve immersing the CT scaffold within ionic bath (e.g. anti-solvent including 18 g KC1 dissolved in 250ml filtered water, into which 250 ml ethanol is added). Surface treatment

[0210] To increase adhesion of the protein mass introduced into the open cells of the CT scaffold, the cells surface is scattered with edible gluten powder and placed in an oven heated to 50°C for 24 hours.

[0211] EXAMPLE 2 - Connective Tissue Scaffold Characteristics The physical properties of the 3D scaffold samples were determined. Specifically, honeycomb scaffolds were prepared from a formulation comprising 5wt% CAR, 5wt% glycerol and lwt% gellan F, to provide honeycomb specimens (90~l 10*40-50* 10mm) with 20 micron, 50 micron, 200 micron or 400 micron wall. Some of the specimens were subjected to post treatment by either freeze-drying according to Table 2 above, or by immersing within ionic bath (anti-solvent including 18 g KC1 dissolved in 250ml filtered water, into which 250 ml ethanol is added). Table 3 provides the different formulations and post treatments of the characterized specimens. Table 3 - Specimens for characterization

[0212] The tensile strength of combined specimens was analyzed. To this end, the specimens were glued with cyanoacrylate glue at their edges (top and down) to a T shape accessory which are needed for gripping behavior. The specimens were gripped by Llyod gripper for tensile tests (see Figure 4A). Then, at room temperature (23 °C±2 °C), each of the specimens was stretched using LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s. Figure 4B provides an image of a tensile strength measurement of the same 3D scaffold having a Honeycomb structure with 0.4mm wall thickness, showing the specimen stretched while being held between two gripper. The results are presented in Table 4. Presenting the max load [N], tensile strength

[0213] [MPa] and strain at break (mm / mm).

[0214] Table 4: mechanical properties of tested specimens Table 4 shows that the different specimens can withstand a max load of at least 3N. Further, it is clear from these results that post treatment, e.g. freeze drying or ionic bath immersion significantly improved the mechanical properties of the specimens.

[0215] As appreciated, the tensile strength graphs of a tensile test displays stress (G) on the y-axis and strain (a) on the x-axis. Stress is the force applied per unit area, while strain is the proportional change in length of the material.

[0216] In the honeycomb structures of Table 3, the true cross-sectional area was determined by addition of the total walls thickness in the cross-section area, excluding the voids in between.

[0217] Figs. 5A-5F are graphs showing the tensile stress-strain curves and elastic moduli calculations of the evaluated specimens with or without post treatment and with the different wall thickness, defined in Table 3 (Fig. 5A - specimen 1, Figure 5B- specimen 2 etc). Specifically, Figs. 5A-5F show strain-stress curves for tensile experiments performed of scaffold samples. All curves were analyzed for extracting low-strain modulus and high-stress modulus, via manually selecting data points in linear regions of the first portion of the test and of the second portion of the test and making linear fit and extracting the slope value, representing the average elastic moduli of the sample in the respective strain range. This was done to show the non-linear (non-Hookean) elastic behavior of the scaffold specimens, resulting in the scaffold structure that is being shape- deformed in low strain, but then reaching extension and imposing stress on the wall material extension rather than shape deformation (also seen in Figs. 4A- 4B).

[0218] The tested specimens exhibited properties of hyperplastic materials.

[0219] Generally, tensile test graph of a hyperelastic material differs from that of conventional linear materials because hyperelastic materials display nonlinear stressstrain behavior, often associated with significant deformations. Hyperelastic materials are a class of materials that can undergo large elastic deformations while still returning to their original shape upon unloading. They are commonly used to model rubber -like materials, soft tissues, and elastomers.

[0220] Further, generally, a hyperelastic material graph of a tensile test is divided into segments: Initial Behavior:

[0221] In the initial phase of the test, as stress is applied, the hyperelastic material behaves elastically. However, unlike linear materials, the stress-strain curve is nonlinear from the beginning. The stress response increases more rapidly than the strain due to the material's highly deformable nature.

[0222] Nonlinear Elastic Region:

[0223] As the material continues to deform under increasing stress, the stress-strain curve maintains its nonlinear trend. Hyperelastic materials often exhibit strain-stress relationships described by various mathematical models, such as the Neo-Hookean, Mooney-Rivlin, or Ogden models. These models capture the nonlinear behavior of the material under deformation.

[0224] High Deformation Range:

[0225] Hyperelastic materials can withstand significant deformations without undergoing plastic deformation or permanent damage. This behavior allows them to be stretched or compressed to large strains and still return to their original shape when the load is removed.

[0226] Ultimate Stretch Limit:

[0227] The stress-strain curve will eventually reach a point where the material's deformation capabilities are exhausted. This could be represented by the point of maximum stress, often referred to as the ultimate stretch limit. Beyond this point, the material might begin to show signs of failure or exhibit more pronounced nonlinear behavior.

[0228] Failure or Necking (If Applicable) :

[0229] Depending on the specific behavior of the hyperelastic material, it might experience necking or localized deformation before ultimate failure. In some cases, the material might rupture or tear, leading to a rapid decrease in stress. EXAMPLE 3 - Preparation of a Whole Muscle Cut using the Connective Tissue Scaffold

[0230] To obtain the alternative whole muscle cut, the open cells of the CT scaffold of Example 1 were filled with protein mass using a piston filling machine (Makita equipped with a 3mm nozzle). The protein composition included 20% Soy protein isolate, 10% Gluten, 10% Canola oil and 60% Water.

[0231] The resulting alternative whole muscle cut contained the channels formed from the gel material, and constituting the connective tissue (CT) of the 3D scaffold. The lumen of the channels constituting the connective tissue accommodates protein mass or fat mass, as clearly shown in Fig. 6.

Claims

CLAIMS:

1. A method of producing an edible 3D scaffold, the method comprising- introducing into a mold an edible gel forming formulation, the mold comprising a boundary and an internal pattern, the internal pattern constituting a negative of a 3 -dimensional scaffold structure,- providing conditions that cause formation of a gel in said internal pattern; and- releasing the gel from the mold, said gel having a 3D scaffold structure; the 3D scaffold is defined as an arrangement of elongated channels having a lumen and being interconnected by channel walls, said channel walls having a thickness that is less than half a channel's cross-sectional dimension.

2. The method of claim 1, wherein said internal pattern is configured to provide channels with a length of at least 5mm.

3. The method of claim 1 or 2, wherein said internal pattern is configured to provide channels with an inner cross-sectional dimension of between about 1 mm and about 5 mm.

4. The method of any one of claims 1 to 3, wherein said internal pattern is configured to provide channels with an inner cross-sectional area of between about 1mm2and about 25mm2.

5. The method of any one of claims 1 to 4, wherein said internal pattern is configured to provide channel walls with a thickness of less than 0.2mm, when said 3D scaffold is in dried form, or channel walls with a thickness of less than 0.4mm, when said 3D scaffold is in wet form.

6. The method of any one of claims 1 to 5, wherein said internal pattern is configured such that at least 50% of the elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of 10mm*20mm*20mm.

7. The method of any one of claims 1 to 6, wherein said internal pattern is configured such that at least 50% of the elongated channels intersect at least one other elongated channel, when viewed in a segment of the 3D scaffold having dimensions of 10mm*20mm*20mm.

8. The method of any one of claims 1 to 7, wherein said internal pattern is configured to provide channels with a hexagon cross section.

9. The method of any one of claims 1 to 8, wherein said internal pattern is configured to provide a 3D honeycomb scaffold structure.

10. The method of any one of claims 1 to 7, wherein said internal pattern is configured to provide channels with a Voronoi cross section.

11. The method of any one of claims 1 to 10, wherein said gel forming formulation comprises solid or semi-solid edible gel forming particles and said introducing of the gel forming formulation into said mold comprises placing the gel forming formulation including the solid or semi-solid edible gel forming particles over said mold and heating the mold to a temperature sufficient to cause melting of the solid or semi solid edible gel forming particles into a gel forming molten and flow of said gel forming molten into said internal pattern.

12. The method of claim 11, comprising heating the mold prior to placing the solid or semi-solid edible gel forming particles thereon.

13. The method of any one of claims 1 to 10, wherein said introducing of the gel forming formulation into said mold comprises placing molten gel forming formulation onto the mold.

14. The method of claim 13, comprising heating the mold prior to placing said molten gel forming formulation.

15. The method of any one of claims 12 to 14, comprising heating the mold to a temperature sufficient to maintain said gel forming formulation in molten state, and allowing flow of said gel forming molten into said internal pattern.

16. The method of any one of claims 1 to 15, wherein said introducing of the gel forming formulation into said mold comprises injection molding of molten gel forming formulation into the internal pattern of said mold.

17. The method of any one of claims 1 to 16, wherein the conditions that cause formulation of the gel in said internal pattern comprises cooling of said mold and said gel forming molten.

18. The method of any one of claims 1 to 17, comprising drying said 3D scaffold gel by any one or combination of freeze drying, heating, air dry of the 3D scaffold gel.

19. The method of claim 17, wherein said drying comprises freeze drying.

20. The method of claim 19, wherein said freeze drying is of the 3D scaffold gel after being released from the mold.

21. The method of any one of claims 1 to 20, comprising applying onto surfaces of the internal pattern an edible protein binding agent.

22. The method of claim 21, wherein said edible protein binding agent comprises gluten.

23. The method of claim 22, wherein said edible protein binding agent comprises gluten powder.

24. The method of any one of claims 1 to 23, wherein said gel forming formulation is a hydrogel forming formulation.

25. The method of any one of claims 1 to 24, wherein said gel forming formulation is a hydrocolloid forming formulation.

26. The method of any one of claims 1 to 25, wherein gel forming formulation comprises water and at least one hydrocolloid forming agent selected from the group consisting of Carrageenan, Agar, Gelatin, Sodium Alginate, Gellan Gum, Pectin, Xanthan Gum, Guar Gum, Methylcellulose, Starch, Konjac, Locust Bean Gum, Tara Gum, Curdlan and Chitosan and combinations of same.

27. The method of claim 26, wherein said gel forming formulation comprises at least Carrageenan.

28. A scaffold comprising a three-dimensional gel structure including an arrangement of elongated channels having a lumen and being interconnected by channel walls; wherein said channel walls comprise an edible gel and said channel walls have a thickness that is less than half a channel's cross-sectional diameter.

29. The 3D scaffold of claim 28, wherein said elongated channels have a length of at least 5mm.

30. The 3D scaffold of claim 28 or 29, wherein elongated channels have an inner cross-sectional dimension of between about 1 mm and about 5 mm.

31. The 3D scaffold of any one of claims 28 to 30, wherein said elongated channels have an inner cross-sectional area of between about 1mm2and about 25mm2.

32. The 3D scaffold of any one of claims 28 to 31, wherein said elongated channels have a wall a thickness of less than 0.2mm.

33. The 3D scaffold of any one of claims 28 to 32, wherein at least 50% of said elongated channels have a same nominal direction, when viewed in a segment of the 3D scaffold having dimensions of 10mm(width)*20mm*20mm.

34. The 3D scaffold of any one of claims 28 to 32, wherein said elongated channels have a hexagon cross section.

35. The 3D scaffold of any one of claims 28 to 32, wherein said elongated channels provide a 3D honeycomb scaffold structure.

36. The 3D scaffold of any one of claims 28 to 32, wherein said elongated channels have a Voronoi cross section.

37. The 3D scaffold of any one of claims 28 to 36, in wet form.

38. The 3D scaffold of any one of claims 28 to 36, in dried form.

39. The 3D scaffold of claim 38, being in freeze-dried.

40. The 3D scaffold of any one of claims 28 to 39, wherein said edible gel is or comprises a hydrogel.

41. The 3D scaffold of any one of claims 28 to 40, wherein said edible gel is or comprises a hydrocolloid.

42. The 3D scaffold of any one of claims 28 to 41, wherein said edible gel is or comprises water and at least one hydrocolloid forming agent selected from the group consisting of Carrageenan, Agar, Gelatin, Sodium Alginate, Gellan Gum, Pectin, XanthanGum, Guar Gum, Methylcellulose, Starch, Konjac, Locust Bean Gum, Tara Gum, Curdlan and Chitosan and combinations of same.

43. The 3D scaffold of any one of claims 28 to 42, wherein said edible gel is or comprises at least Carrageenan.

44. The 3D scaffold of any one of claims 28 to 43, wherein said edible gel is or comprises at least 5%w / w Carrageenan.

45. The 3D scaffold of any one of claims 28 to 44, wherein said edible gel is or comprises at least Carrageenan and Gellan.

46. The 3D scaffold of any one of claims 28 to 45, wherein said edible gel is or comprises at least Carrageenan and glycerin.

47. The 3D scaffold of any one of claims 28 to 46, wherein said edible gel is or comprises at least Carrageenan, glycerin and Gellan.

48. A 3D scaffold obtained or obtainable by the method of any one of claims 1 to 27.

49. The 3D scaffold of any one of claims 28 to 48, characterized by at least one of the following properties, when measured on a specimen having dimensions of about 90~l 10*40-50* 10mm and wall thickness that is equal or less than 400 micron: capability to withstand a maximum load of at least 3N, as determined by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s, is at least 0.05 MPa. a tensile strength of at least 0.05 MPa, as by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s; a maximum strain of at least 0.4 mm / mm, as determined by a tensile test conducted at room temperature (23 °C ± 2°C) using a LLOYD TP A instrument equipped with a 100N load cell at a speed of 5mm / s a hyperelastic material properties exhibited as a nonlinear stress-strain curve, with the ability to undergo elastic deformations and return to its original shape upon unloading; and / or a stress-strain curve characterized by an initial nonlinear elastic behavior, followed by a high deformation range without plastic deformation, and an ultimate stretch limit defined by the maximum stress point before the material exhibits failure or necking.

50. A method of producing an alternative whole muscle meat, the method comprises introducing protein mass into a 3D scaffold gel defined as an arrangement of elongated channels having a lumen for accommodating said protein mass, the elongated channels being interconnected by channel walls.

51. The method of claim 50, wherein at least one of the following conditions is fulfilled: said channel walls have a thickness that is less than half a channel's cross-sectional diameter; said channel walls have a thickness that is less than 0.2mm.

52. The method of claim 50 or 51, wherein said elongated channels have a length of at least 5mm.

53. The method of any one of claims 50 to 52, wherein said elongated channels have an inner cross-sectional diameter of between about 1 mm and about 5 mm.

54. The method of any one of claims 50 to 53, wherein said elongated channels have an inner cross-sectional area of between about 1mm2and about 25mm2.

55. The method of any one of claims 50 to 54, wherein said channel walls have a thickness of less than 0.2mm, when said 3D scaffold is in dried form, or less than 0.4mm when said 3D is in wet form.

56. The method of any one of claims 50 to 55, wherein at least 50% of the elongated channels have a same nominal direction when viewed in a segment of the 3D scaffold having dimensions of 10mm*20mm*20mm.

57. The method of any one of claims 50 to 56, wherein at least 50% of the elongated channels intersect at least one other elongated channel when viewed in a segment of the 3D scaffold having dimensions of 10mm*20mm*20mm.

58. The method of any one of claims 50 to 57, wherein said 3D scaffold gel has channels have with a hexagon cross section.

59. The method of any one of claims 50 to 58, wherein said 3D scaffold gel has 3D honeycomb scaffold structure.

60. The method of any one of claims 50 to 59, wherein said 3D scaffold gel has channels with a Voronoi cross section.

61. The method of any one of claims 50 to 60, wherein said gel is an edible hydrogel.

62. The method of any one of claims 50 to 61, wherein said gel is a hydrocolloid.

63. The method of any one of claims 50 to 62, wherein said gel comprises at least one hydrocolloid forming agent selected from the group consisting of Carrageenan, Agar, Gelatin, Sodium Alginate, Gellan Gum, Pectin, Xanthan Gum, Guar Gum, Methylcellulose, Starch, Konjac, Locust Bean Gum, Tara Gum, Curdlan and Chitosan and combinations of same.

64. The method of any one of claims 50 to 63, wherein said protein mass is an edible protein mass.

65. The method of any one of claims 50 to 64, wherein said introducing of the protein mass is by pouring or injection of the protein mass into the 3D scaffold channels.

66. The method of any one of claims 50 to 65, wherein said protein mass is a pliable protein mass.

67. The method of any one of claims 50 to 66, wherein said protein mass comprises at least 10% (w / w) protein and at least 50%(w / w) water.

68. The method of any one of claims 50 to 67, wherein said protein mass comprises plant protein.

69. The method of any one of claims 50 to 68, wherein said protein mass comprises texturized vegetable protein (TVP).

70. The method of any one of claims 50 to 69, wherein said introducing of protein mass is into two or more 3D scaffolds, stacked one on top of the other, with the elongated channels of the two or more 3D scaffolds being aligned.

71. The method of any one of claims 50 to 69, comprising stacking at least one 3D scaffold accommodating the protein mass, on top of at least one other 3D scaffold, accommodating the protein mass.

72. The method of claim 71, wherein each of the stacked 3D scaffolds can comprise the same or different gel and / or the same or different protein mass.

73. The method of any one of claims 50 to 72, comprising introducing edible fat into the lumen of at least part of the elongated channels and / or in between two stacked 3D scaffolds.

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