Cell culture-derived cocoa butter and method of producing the same

Abstract

Disclosed are cell culture-derived cocoa butter (CCCB), cell culture-derived cocoa liquor (CCCL), and cell culture-derived cocoa powder (CCCP) having natural-like fatty acid and triglyceride composition, and methods of producing the CCCB, CCCL and CCCP of the invention from cacao cells suspensions under specific culturing conditions.

Description

[0001] CELL CULTURE-DERIVED COCOA BUTTER AND METHOD OF PRODUCING THE SAME FIELD OF INVENTION

[0002] The present disclosure generally pertains to the field of biotechnological production of cocoa Butter, more specifically the disclosure relates to in vitro production of cocoa butter from cultured cacao cells.

[0003] BACKGROUND OF INVENTION

[0004] Traditionally, cocoa butter (CB) is extracted from seeds (cacao beans) of the Theobroma cacao plant which is native to tropical regions of Central and South America.

[0005] Extraction of natural cocoa butter involves obtaining cocoa nibs by removing the shell of the beans and griding the nibs into a thick paste called cocoa mass or chocolate liquor, which naturally contains cocoa butter. Cocoa mass is pressed to separate the cocoa butter from the solid components, referred to as cocoa powder.

[0006] Cocoa nibs and cocoa mass have 50%-57% w / w total fat content. Natural cocoa butter is essentially made of fat, including 97-98% w / w fatty acids, both saturated and unsaturated. Depending on the cacao variety, three major fatty acids: palmitic (Cl 6:0) 27%-31%, stearic (C18:0) 32%-38%, and oleic (C18: 1) 30%-34% acids, constitute more than 90% of the total fat content of natural cocoa butter, and linoleic acid (C18:2) adds another 1 ,5%-3.5%.

[0007] The fatty acid composition and the balance between the fatty acids in cocoa butter is a major determinant of its physicochemical properties, such as hardness and melting properties, contributing to the smooth texture and mild chocolate flavor of cocoa butter, and to its ability to stay solid at room temperature while melting near body temperature.

[0008] These desirable qualities make traditionally produced cocoa butter perfect for producing chocolate and other food products, as well as for a variety of applications related to cosmetics and skincare due to its moisturizing and emollient properties.

[0009] However, the chocolate industry is facing multiple challenges, including environmental degradation, ethical concerns, and a sharp increase in prices.

[0010] Cocoa farmers often clear tropical forests to plant new cocoa trees rather than reusing the same land, which has spurred massive deforestation in West Africa, particularly in Ivory Coast. Deforestation also leads to a loss of biodiversity and contributes to climate change.

[0011] Climate change has in turn caused a decrease in cocoa yield and increase in pests and diseases impacting the crop. This has resulted in misuse and overuse of pesticides and chemicalfertilizers which causing reduced quality of water resources, contaminated soils, and further reduced biodiversity.

[0012] Child labor is another big problem associated with cocoa farming and is a widespread phenomenon in many West African cocoa farms. Work done by children involves hazardous tasks such as spraying pesticides, carrying heavy loads, climbing cocoa trees for harvesting, or opening cocoa pods with sharp tools like machetes. With limited school attendance, development and education are hindered, making it harder to break the cycle of poverty As climate change and environmental factors reduce the availability of high-quality cocoa, the price of chocolate continues to rise, negatively affecting both consumers and, maybe counter intuitively, exacerbates the poverty and exploitation associated with cocoa farming.

[0013] In vitro production of cocoa butter and cocoa powder from cultured cacao cells offers a promising alternative to traditional production methods by addressing current challenges associated with traditional cocoa farming and providing benefits and advantages.

[0014] Inter alia, it is resilient to climate changes reduces the need of land and water needed for cultivation of cacao plants, as well as the use of harmful agrochemicals.

[0015] Moreover, controlled in vitro growth of cultured cacao cells could potentially enhance the efficiency of production of both cocoa butter and cocoa powder by reducing waste and increasing yield, reducing variability and promoting standardization of quality.

[0016] However, producing a biomass of cacao cells having a desired natural-like fatty acid profile remains a challenge, let alone at an amount sufficient for large scale production, mainly due to technical difficulties in achieving the requirement for balanced fatty acid composition.

[0017] Therefore, there is an unmet need for developing cultured cacao plant-derived cells having a biomass with an improved lipid composition, closely imitating natural cocoa butter.

[0018] SUMMARY OF INVENTION

[0019] The following embodiments are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0020] In some embodiments, there is provided cell culture-derived cocoa butter (CCCB) characterized by a fatty acid profile including 20%-35% w / w palmitic acid, 25%-40% w / wstearic acid, 25%-35% w / w oleic acid, and less than 6% w / w linoleic acid, wherein the culture is a suspension culture derived from a cacao plant.

[0021] In some embodiments, the fatty acid profile includes 27%-33% w / w palmitic acid, 30%-38% w / w stearic acid, 27-34% w / w oleic acid, and 1.5-4.5% w / w linoleic acid. In some embodiments, the fatty acid profile includes less than 0.2% w / w linolenic acid. In some embodiments, the fatty acid profile includes less than 5% w / w docosenoic acid. In some embodiments, the fatty acid profile includes less than 1% w / w ginkgolic acid. In some embodiments, the palmitic acid, stearic acid, and oleic acid, together constitute at least about 90% w / w of the total fatty acid content of the CCCB.

[0022] In some embodiments, the fatty acid profile is further characterized by a triglyceride (TAG) profile including about 16-21% w / w l,3-palmitoyl-2-oleoyl-glycerol (POP), about 39%-41% w / w l-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS), and about 24%-28% w / w 1,3-stearoyl-2-oleoyl-glycerol (SOS). In some embodiments, the TAG profile includes C52 and C50 TAGs at a ratio of about 2.1: 1-2.4: 1 C52% / C50% w / w. In some embodiments, the TAG profile includes C52 and C54 TAGs at a ratio of about 1.4: 1-1.8: 1 C52% / C54% w / w. In some embodiments, the TAG profile includes less than about 4% w / w of any one of l,3-oleyl-2-palmitoyl-glycerol (OPO), 1,2,3-oleyl-glycerol (OOO), l-stearyl-2-palmitoyl-3-oleyl-glycerol (SPO), l-stearyl-l,3-oleyl-glycerol (SOO), 1,2,3- palmitoyl -glycerol (PPP), l-stearyl-2-palmitoyl-3- myristyl-glycerol (SPM), and 1- palmitoyl-2-linoleyl-3- stearyl-glycerol (PLiS). In some embodiments, the TAG profile includes less than about 1% w / w C48 TAGs.

[0023] In some embodiments, the CCCB has a melting temperature of about 20oC-40oC. In some embodiments, the CCCB is derived from a cultured cacao cells biomass having a total fat content of at least 20% w / w.

[0024] In some embodiments, the CCCB is derived from a cacao cell culture which contained about 3%- 10%, or about 4%-8%, sugar upon transition of the cultured cells from logarithmic to stationary phase.

[0025] In some embodiments, the CCCB is derived from a cacao cell culture which contained amolarC / N ratio of about 20: 1-55:1 and / oramass C / N ratio of about 18:1-48:1 upon transition of the cultured cells from logarithmic to stationary phase.

[0026] In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.In some embodiments, the suspension culture is derived from a cacao plant callus. In some embodiments, there is provided a method of producing cell culture-derived cocoa butter (CCCB), cell culture-derived cocoa liquor (CCCL) and / or cell culture-derived cocoa powder (CCCP), the method including culturing cacao cells obtained from a cacao plant in a suspension medium, wherein upon transition of the cultured cells from logarithmic to stationary phase, the suspension medium includes sugar at a concentration of about 3%-10%, or about 4%-8% w / v, thereby obtaining a cacao cell suspension characterized by a fatty acid profile including 20-35% w / w palmitic acid, 25-40% w / w stearic acid, 25-35% w / w oleic acid, less than 6% linoleic acid.

[0027] In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

[0028] In some embodiments, the method includes at least one microscopic observation of the culture for evaluating uniformity in size and shape of the cultured cacao cells, which indicates cell cycle synchronization of the cells.

[0029] In some embodiments, the cacao cell suspension obtained by the method is further characterized by a triglyceride (TAG) profile including about 16-21% w / w l,3-palmitoyl-2-oleoyl-glycerol (POP), about 39%-41% w / w l-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS), and about 24%-28% w / w l,3-stearoyl-2-oleoyl-glycerol (SOS).

[0030] In some embodiments, when the suspension medium does not include sugar at a concentration of about 3%-l 0%, or about 4%-8% w / v, it includes no more than 3% sugar.

[0031] In some embodiments, the sugar is selected from sucrose, fructose, lactose, galactose, maltose, and any combination thereof. In some embodiments, the sugar is not glucose.

[0032] In some embodiments, the suspension medium does not include a plant hormone or a plant regulator. In some embodiments, the suspension medium does not include an agent capable of inducing somatic embryogenesis. In some embodiments, the method does not include a step of fermentation or simulated fermentation. In some embodiments, the method does not include a step of adding a microorganism or an enzyme selected from a protease, a glycosidase, or a lipase.

[0033] In some embodiments, the method further includes a step of harvesting the cacao cells to obtain a cell culture-derived cocoa liquor (CCCL), and extracting the CCCL to obtain CCCB. In some embodiments, the CCCL has a total fat content of at least 20% w / w.In some embodiments, the extracting of the CCCB from the CCCL includes separating fat from remaining biomass by organic solvent or cold pressing, and wherein the remaining biomass constitutes CCCP.

[0034] In some embodiments, the cacao cells are obtained from a cacao plant callus.

[0035] In some embodiments, the method includes the steps of:

[0036] a) culturing the cacao cells in a first suspension medium optionally including an initial sugar concentration of about 4-9% w / w;

[0037] b) replacing the first suspension medium with a second suspension medium including a sugar concentration higher than 2% w / v upon transition of the cells from logarithmic phase to stationary phase, when the first suspension medium did not include an initial sugar concentration of about 4-9% w / w;

[0038] c) growing the cells until liposome formation is observed; and

[0039] d) harvesting the cells to obtain cell culture-derived cocoa liquor (CCCL), and extracting the CCCL to obtain CCCB having a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

[0040] In some embodiments, there is provided a cacao cell culture obtained by the method disclosed herein.

[0041] In some embodiments, there is provided a cacao cell culture obtained by a method including culturing cacao cells obtained from a cacao plant in a suspension medium, wherein upon transition of the cultured cells from logarithmic to stationary phase, the suspension medium includes sugar at a concentration of about 3%-10%, or about 4%-8% w / v.

[0042] In some embodiments, there is provided a cell culture-derived cocoa butter (CCCB) produced by the method disclosed herein.

[0043] In some embodiments, there is provided a cell culture-derived cocoa power (CCCP) produced by the method disclosed herein.

[0044] In some embodiments, there is provided a cell culture-derived cocoa liquor (CCCL) produced by the method disclosed herein.

[0045] In some embodiments, there is provided a cacao-containing product including the CCCB disclosed herein, the CCCP disclosed herein, and / or the CCCL disclosed herein.

[0046] In some embodiments, the cacao-containing product is a chocolate, a chocolatecontaining product, a chocolate-like product, or a chocolate replacement product.In some embodiments, the cacao-containing product is a cosmetic product such as a moisturizer, a balm, a body lotion, a cream, a lip balm, a body butter, a soap, a body wash, a hair conditioner, a mask, and a sun protection cream, spray or balm.

[0047] In addition to the exemplary embodiments described above, further embodiments will become apparent by study of the following detailed descriptions.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Fig- 1 shows non-embryogenic, synchronized cell growth in suspension culture. Representative brightfield (transmitted-light) micrograph acquired using a 10x objective showing elongated, tubular / vacuolated cells with similar morphology and size distribution, consistent with a well -synchronized, non-embryogenic stage. No compact isodiametric cell clumps or embryo-like structures (e.g., globular / heart-stage bodies) are visible, supporting the absence of embryogenic development under these conditions.

[0050] Fig- 2 shows high triglyceride accumulation in synchronized, non-embryogenic suspension cells visualized by BODIPY staining. Representative fluorescence micrograph of BODIPY-stained culture showing abundant punctate green fluorescence corresponding to intracellular neutral lipid droplets (TAG). Cells exhibit a broadly uniform, non-embryogenic morphology (large, vacuolated / parenchyma-like cells with irregular, expanded outlines and no compact, densely cytoplasmic embryo-like aggregates), supporting a non-embryonic developmental stage at the time of imaging. The relatively consistent cell shape and the widespread presence of similarly sized BODIPY-positive droplets across most cells indicate a well-synchronized population with coordinated lipid accumulation. Inset shows a magnified view highlighting discrete lipid bodies. The round insert shows the cells including the lipid droplets at a higher magnification.

[0051] DETAILED DESCRIPTION

[0052] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.Chocolate contains solid particles dispersed throughout a fat matrix, predominantly cocoa butter, thus emphasizing the importance of cocoa butter as a chocolate component. In fact, a composition that does not contain cocoa butter cannot be called “chocolate” in the United States under the Food and Drug Administration Guidelines.

[0053] Natural cocoa nibs, or natural cocoa mass, generally contain about 50%-57% w / w total fat. In terms of fatty acid composition, natural cocoa butter contains about 27%-31% palmitic acid (C16:0), about 32%-38% stearic acid (C18:0), about 30%-34% oleic acid (C18:l), and about 1.5%-3.5% linoleic acid (C18:2), w / w out of the total amount of fatty acids.

[0054] This fatty acid composition is important for the taste of cocoa butter and products made from it, as well as to other physicochemical properties such as hardness, texture, and melting properties.

[0055] However, while growing cacao cells in culture and deriving cocoa powder therefrom is known in the art, production of cell culture-derived cocoa butter (CCCB) has proven far more challenging, resulting in either no production of cocoa butter at all, cocoa butter with olive oil like consistency, or cocoa butter that otherwise fails to provide the desired mouthfeel, taste and / or texture. As it turns out, producing cocoa butter derived from cultured cells which has a fatty acid profile similar to that of cocoa butter produced from cacao beans is very difficult and has so far not been successful. In fact, Steve Stearns, head of strategy and business development of California Cultured, stated in an interview to AgFunderNews (published on September 16, 2025, on the AgFunderNews website): “For years, companies have been trying to replace cocoa butter, and they have used things like hydrolyzed palm fats and shea butter, but they don’t have the same melting profile as cocoa butter and they can have a greasy feel.” Up until now, he said, “Every group working in cacao cell culture has relied on callus cells. The challenge with callus cells is fundamental: they can make excellent cocoa powder, but they simply don’t produce fat. Without fat, you can’t replicate the qualities and the melting properties that make real chocolate.” The solution, as stated in the publication, is producing cocoa butter from somatic embryos instead of from a callus.

[0056] The present invention is based on the surprising finding that when growing callus-derived cacao cells in an in vitro culture and ensuring a certain concentration of sugar at the transition from logarithmic growth phase to stationary phase, the cocoa butter obtained from the cultured cacao cells advantageously has a fatty acid and triglyceride profile which is very similar to that of natural cocoa butter. While sugar (e.g., at about 4%) may be added at the time of the transition from log to stationary phase, it was even more surprisingly found that addinga higher amount of sugar (e.g., at about 8%) when starting the culture leads to the same result and maintains a high sugar concentration at the transition.

[0057] As a further advantage, the herein-disclosed controlled culturing of cacao cells provides a high CCCB yield, i.e. total fat content derived from the cell culture of above 20% and even above 30% w / w. Furthermore, the controlled culturing advantageously ensures low undesired variability in the fatty acid profile of the CCCB, as well as the aroma profile of the cell culture-derived cocoa powder (CCCP) thus ensuring a persistent high-quality produce.

[0058] As shown in Tables 1-3, the CCCB of the invention was found to have a fatty acid profile similar to that of bean-derived cocoa butter, with about 27-31% palmitic acid, 32.5-35.5% stearic acid, 28-33% oleic acid, and low levels of linoleic acid and linolenic acid of about 2.3% and about 0.1%, respectively.

[0059] Palmitic acid (C16:0) is a 16-carbon saturated fatty acid that serves as a critical structural component of the primary triacylglycerols (TAGs) in cocoa butter, specifically POP (l,3-palmitoyl-2-oleoyl-glycerol) and POS (l-palmitoyl-2-oleoyl-3-stearoyl-glycerol). Its primary importance to chocolate features lies in providing hardness and thermal stability. Due to its being a saturated fat with a straight carbon chain, it allows for tight molecular packing that ensures chocolate remains solid and brittle at room temperature with a characteristic "snap." In the context of triglycerides in cocoa butter, palmitic acid typically occupies the outer positions of the glycerol backbone, flanking an unsaturated oleic acid to create a symmetrical molecule. This symmetry is essential for the fat to crystallize into the stable form during tempering, which gives chocolate its glossy finish, prevents fat bloom, and facilitates the sharp, clean melting sensation as the temperature approaches 34°C in the mouth.

[0060] Stearic acid is a long-chain fatty acid abundant in animal fat (up to 30%) as compared to the typically below 5% stearic acid found in vegetable fat, an important exception being cocoa butter. Unlike other fats, the unique chemical structure of stearic acid gives it a relatively high melting point (~70°C) and is thus stable at high temperatures and does not easily become rancid, features important for the chocolate industry. Moreover, in terms of health, unlike other long-chain saturated fatty acids, stearic acid has been found to have no effect on lipoprotein cholesterol concentration.

[0061] Oleic acid provides the smoothness and melt-in-the-mouth feel of chocolate. Moreover, CCCB which contains sufficient amounts of oleic acid has a unique crystalline structure that melts just below body temperature, giving chocolate its signature creamy texture. Oleic acidalso helps prevent chocolate from becoming too brittle or too soft thereby contributing to a stable and desirable consistency.

[0062] The low level of linoleic acid and linolenic acid ensure that the butter is not too oily. Moreover, higher linoleic acid content may interfere with proper tempering, leading to a less stable chocolate with a higher tendency to develop fat bloom, (whitish discoloration due to fat migration). Linolenic acid is even more prone to oxidation, and cocoa butter with high linolenic acid levels compromises the stability of the cocoa butter. Moreover, increased linolenic acid levels may lead to faster rancidity and off-flavors for chocolates as well as lack of snap and greasier mouth-feel.

[0063] Moreover, as opposed to past attempts at producing cocoa butter from cell culture, the CCCB of the invention contains only trace levels of docosenoic acid and ginkgolic acid. Docosenoic acid in chocolate can significantly affect its texture due to its chemical and physical properties. Docosenoic acid is a long-chain monounsaturated, with a lower melting point than cocoa butter’s main fatty acids. Accordingly, high amounts of docosenoic acid can soften the chocolate and make it less firm. The characteristic "snap" of tempered chocolate may also be weakened due to high docosenoic acid concentrations. Moreover, docosenoic acid can interfere with proper crystallization, leading to softer, unstable chocolate, increase the risk of fat bloom as it slows solidification and give a oily and waxy feel to the chocolate, rather than the smooth, creamy texture that well-tempered cocoa butter provides.

[0064] Ginkgolic acid is a fatty acid having cytotoxic and allergenic properties. In fact, many food safety authorities restrict or prohibit ginkgolic acid in consumable products. Ginkgolic acid also has bitter, astringent, and slightly soapy sensory properties. Moreover, due to the amphiphilic nature of ginkgolic acid it can affect how fats and water-based ingredients mix in chocolate.

[0065] In addition, an analysis of triglycerides (triacylglycerols, TAGs) further demonstrates the resemblance of the CCCB to cocoa butter derived from cacao beans such as cocoa nibs, chocolate, and commercial cocoa butter. As shown in Table 4, the levels of different TAGs in the CCCB is very similar to their levels in the bean-derived products, thereby implying a functional similarity including desirable characteristics such as a similar melting temperature (of about 30-35°C) with rapid melt-sharpening at oral temperature, compatible crystallization kinetics, and the ability to form the desirable PV polymorph. Furthermore, this implies that the cultured cacao cells reproduce the key lipid biosynthesis steps (elongation, desaturation, and regioselective acylation) necessary for generating authentic cocoa butter functionality.Cell culture-derived cocoa butter

[0066] CCCB fatty acid composition

[0067] In some embodiments, the present invention provides a cultured-derived cocoa butter (CCCB), characterized by a fatty acid profile including at least palmitic acid, stearic acid, oleic acid, and linoleic acid at levels resembling (with no more than 5%, 2% or 1% deviation) respective levels in cocoa butter extracted from cacao beans.

[0068] The CCCB described herein is defined by being obtained from a cacao cell culture initially derived from cacao plant (Theobroma cacao) cells, by harvesting the cultured cells and extracting the lipid fraction of the cell biomass.

[0069] In some embodiments, the CCCB contains about 20-35% w / w palmitic acid (Cl 6:0), about 25-40% w / w stearic acid (C18:0), about 25-40% w / w oleic acid (C18:ln9c), and less than 6% w / w linoleic acid (C18:2n6c), out of the total fatty acid content. In some embodiments, the CCCB contains about 27-33% w / w palmitic acid, about 30-38% w / w stearic acid, about 28-35% w / w oleic acid, and about 1.5-5% w / w linoleic acid, out of the total fatty acid content.

[0070] Unless indicated otherwise, the % of specific fatty acids presented herein are weight % (w / w) out of the total fatty acid content of the CCCB. Similarly, the % of specific TAGs presented below are w / w out of the total content of TAGs in the CCCB. The total mass of fatty acids is about 90-92% of the total mass of the cocoa butter (the rest corresponds to glycerol and trace elements). The analysis of % fatty acids and TAGs may be carried out by hydrolyzing (saponifying) the triglycerides in the cocoa butter to release fatty acids, and quantifying the individual fatty acids or the TAGs by any suitable method, e.g., by gas chromatography or mass chromatography such as GC-MS (Gas Chromatography -Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry).

[0071] In some embodiments, the CCCB contains about 27-33%, 27-31%, or 30-33% w / w palmitic acid. In some embodiments, the CCCB contains about 30% palmitic acid.

[0072] In some embodiments, the CCCB contains about 32-36% w / w stearic acid. In some embodiments, the CCCB contains about 32.5-35.5% w / w stearic acid. In some embodiments, the CCCB contains about 35% w / w stearic acid.

[0073] In some embodiments, the CCCB contains about 30-35% w / w oleic acid. In some embodiments, the CCCB contains about 33% w / w oleic acid.

[0074] In some embodiments, the CCCB contains less than 5%, 4.5%, 4% or 3% w / w linoleic acid. In some embodiments, the CCCB contains about 1-6%, 2-6%, 2-5%, 2-4%, or 3.5-5.5% w / w linoleic acid. In some embodiments, the CCCB contains about 2% w / w linoleic acid.In some embodiments, the CCCB contains less than 0.25%, 0.2%, 0.15%, or 0.1% w / w linolenic acid (C18:3n3) out of the total fatty acid content.

[0075] In some embodiments, the CCCB contains less than 12%, 10%, 8%, 5%, 1%, 0.5%, or 0.1 % w / w docosenoic acid (C22:ln9) out of the total fatty acid content.

[0076] In some embodiments, the CCCB contains less than 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% w / w ginkgolic acid (15:1) out of the total fatty acid content.

[0077] It is appreciated that the CCCB or the fatty acid profile containing less than a certain % of a certain component (e.g. fatty acid or triglyceride) encompasses that the CCCB or the fatty acid profile contain 0% of that component, i.e., not including that component at all, or including traces which may be less than detection level.

[0078] In some embodiments, palmitic acid, stearic acid, and oleic acid, together constitute at least about 90% w / w of the total fatty acid content in the CCCB. In some embodiments, palmitic acid, stearic acid, and oleic acid, together constitute at least about 93% w / w of the total fatty acid content in the CCCB.

[0079] In some embodiments, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitoleic acid, arachidic acid and behenic acid together constitute at least about 98.5%, 99%, 99.2%, or 99.5% w / w of the total fatty acid content of the CCCB.

[0080] CCCB triglyceride (TAG) composition

[0081] In cocoa butter, fatty acids are mostly esterified as TAGs, which contain three fatty acids esterified to a glycerol backbone. The TAG composition of cocoa butter is a primary determinant of its polymorphism, melting behavior, and functional performance in chocolate manufacturing. The main TAGs which determine chocolate properties are POP (C50, 1,3-palmitoyl-2-oleoyl-glycerol), POS (C52, l-palmitoyl-2-oleoyl-3-stearoyl-glycerol), and SOS (C54, l,3-stearoyl-2-oleoyl-glycerol). Together, these three TAGs typically constitute ~80-90% of the lipid fraction and confer the hallmark melting profile of cocoa butter. TAGs mentioned herein use the following key for the identity of fatty acids: O stands for oleyl, P stands for palmitoyl, S stands for stearyl, M stands for myristyl, and Li stands for linoleyl. In terms of number of carbons: C48 TAGs include SPM, PPP, and PMO; C50 TAGs include PPS, POP, PPO, andPPLi; C52 TAGs include PSS, POS, SPO, PSO, OPO, POO, PLiS, PLiO, POLi, LiPO, LiPLi, and PLiLi; and C54 TAGs include SSS, SOS, SSO, OSO, SOO, OOO, SLiS, SLiO, OLiO, and SLiLi.The ratio between POP, SOP, and SOS determines the hardness of the cocoa butter in that SOS is the "hardest" triglyceride with the highest melting point, while SOS is the "softest" and most prone to melting at lower temperatures. Additionally, having these three TAGs as the predominant TAGs results in the narrow melting range of chocolate, which is due to their similar symmetrical structure (the unsaturated oleic acid at sn-2 position and saturated fatty acids at positions sn-1 and sn-3), which causes them to crystalize tightly together, and to be packed into the highly stable P(Form V) crystal. As a result, the chocolate is brittle at room temperature (solid at 27.5°C) while melting at mouth temperature (34°C). Furthermore, the concentration of POS is particularly important to the stability of the chocolate, preventing fat bloom.

[0082] As shown in Table 4, the distribution of TAGs in the CCCB is similar to that of cocoa butter extracted from chocolate product made from cacao beans, with about 20% POP, about 41% POS, and about 24% SOS.

[0083] In some embodiments, the CCCB contains about 15-25%, 16-21%, or about 20% w / w POP.

[0084] In some embodiments, the CCCB contains about 35-45%, 38-42%, 39-41%, or about 41% w / w POS.

[0085] In some embodiments, the CCCB contains about 20-30%, 24-28%, or about 24% w / w SOS.

[0086] In some embodiments, the CCCB contains about 16-21% w / w POP, about 39-41% POS, and about 24-28% SOS. In some embodiments, the CCCB contains about 20% POP, about 41% POS, and about 24% SOS.

[0087] The ratio between C52 and C54 TAGs indicates chocolate-like (bean-derived) crystallization kinetics and ability to form the desirable PV polymorph. This polymorph is the specific crystal structure of cocoa butter that defines high-quality, professional chocolate. It has a melting Point of 33.8°C-35°C, which is just below human body temperature, thereby providing that signature "melt-in-your-mouth" sensation while remaining solid at room temperature; it creates a rigid, dense crystal lattice that gives chocolate its brittle "snap" when breaking it; and provides a high-gloss, shiny finish.

[0088] In some embodiments, the ratio of C52% / C50% TAGs is about 2:1-2.5:1. In some embodiments, the ratio of C52% / C50% TAGs is about 2.1 : 1-2.4: 1.

[0089] In some embodiments, the ratio of C52% / C54% TAGs is about 1.4: 1-1.8:1. In some embodiments, the ratio of C52% / C54% TAGs is about 1.4:1-1.7:1.In some embodiments, the ratio of POS% / POP% is about 2:1-2.5:1. In some embodiments, the ratio of POS% / POP% is about 2.1 : 1-2.4: 1.

[0090] In some embodiments, the ratio of POS% / SOS% is about 1.4: 1-1.8:1. In some embodiments, the ratio of POS% / SOS% is about 1.4:1-1.7:1.

[0091] In some embodiments, the ratio of C50% / C52% / C54% TAGs is about 22 / 48 / 30. In some embodiments, the ratio of POP% / POS% / SOS% is about 22 / 48 / 30.

[0092] In some embodiments, the CCCB contains less than about 4%, 3%, 2%, or 1% w / w of any one of OPO, OOO, SPO, SOO, PPP, SPM, and PLiS. In some embodiments, the CCCB is essentially devoid of OPO, OOO, SPO, SOO, PPP, SPM, and PLiS. In some embodiments, the CCCB is essentially devoid of OPO. In some embodiments, the CCCB is essentially devoid of OOO. In some embodiments, the CCCB is essentially devoid of SPO. In some embodiments, the CCCB is essentially devoid of SOO. In some embodiments, the CCCB is essentially devoid of PLiS. In some embodiments, the CCCB is essentially devoid of PPP. In some embodiments, the CCCB is essentially devoid of SPM.

[0093] Very low levels (or absence) of C48 TAGs indicates that the CCCB does not exhibit undesired properties such as excessive softness, poor tempering behavior, or oil migration, which are often observed in non-cocoa fat replacements.

[0094] In some embodiments, the CCCB contains less than 1% C48 TAGs. In some embodiments, the CCCB contains less than 0.5% C48 TAGs. In some embodiments, the CCCB is essentially devoid of C48 TAGs.

[0095] The term “essentially devoid of’, with reference to a product being essentially devoid of a certain ingredient, as used herein, means that the product either does not contain that ingredient, or the ingredient is present in the product at a trace amount, e.g., below detection level. In any case, such an ingredient, if present, is present at a level at which the ingredient cannot confer the product with any of the effects associated with the ingredient. In some embodiments, “essentially devoid of’ means that the compound is present at less than 1%, 0.5%, or 0.1%.

[0096] The above TAG profile also falls within the natural compositional envelope defined by ICCO and Codex cocoa butter specifications, indicating that the cell-derived material should be fully compatible with standard chocolate tempering and crystallization protocols without requiring formulation changes.CCCB physical characteristics

[0097] In some embodiments, the CCCB has a melting temperature of about 20-40°C. In some embodiments, the CCCB has a melting temperature of about 25-40°C. In some embodiments, the CCCB has a melting temperature of about 25-35°C. In some embodiments, the CCCB has a melting temperature of about 30-35°C. In some embodiments, the CCCB has a melting temperature of about 33-35°C.

[0098] In some embodiments, the CCCB is solid at a temperature of 27.5°C or lower. In some embodiments, the CCCB is liquid at a temperature of 34°C or higher.

[0099] CCCB produced from cell culture

[0100] In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of more than 2%, 2.5%, 3%, or 3.5% w / v sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of at least 3% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of at least 4% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of 2.5%-9%, 3%-10%, 3%-9%, 3%-8%, 4%-10%, 4%-9%, 4%-8%, 3%-7%, 3%-6%, 3%-5%, 4%-7%, or 4%-6% w / v sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-10% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-8% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-10% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-9% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-8% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-6% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-7% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-6% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of 3%-5% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time inthe presence of about 4% sugar. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in the presence of about 8% sugar.

[0101] In some embodiments, the sugar is selected from sucrose, fructose, lactose, galactose, maltose, and any combination thereof. In some embodiments, the sugar is not glucose.

[0102] In some embodiments, the cacao cells are selected from cacao bean cells (e.g., cotyledon / seed tissue), cacao leaf cells, cacao flower cells, cacao stem cells, and combinations thereof. In some embodiments, the cacao cells are derived from a callus. In some embodiments, the callus is derived from an explant. The explant may originate from any plant part, such as beans (seeds), leaves, flowers, etc.

[0103] In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of at least about 12:1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of at least about 15:1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of at least about 18:1.

[0104] In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of about 12:1-58:1, 12:1-55:1, 15:1-58:1, 15:1-55:1, 18:1-58:1, 18:1-55:1, 20:1-58:1, 20:1-55:1, 15:1-50:1, or 15:1-48:1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of about 15:1-52:1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio or a mass C / N ratio of about 15:1-48:1.

[0105] In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio of about 20:1-55:1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a mass C / N ratio of about 18:1-48:1.

[0106] In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a molar C / N ratio of about 23 : 1 or about 54: 1. In some embodiments, the CCCB is derived from cacao cells cultured at least a portion of the time in a culture medium having a mass C / N ratio of about 20: 1 or about 46: 1.A carbon / nitrogen (C / N) ratio reflects the balance between carbon, providing energy, and nitrogen, providing building blocks for proteins. The carbon in this case may be sucrose and the nitrogen may be nitrates and ammonium salts. A low C / N ratio causes cells to grow faster by synthesizing proteins, amino acids, and nucleic acids. A high C / N ratio causes cells to enter into a metabolic stress state, redirecting the excess carbon into building secondary metabolites such as antioxidants, phenolics, alkaloids, flavors, etc. The ratio is typically modified by the addition of a carbon source (sugar, e.g., sucrose) or by reducing nitrogen salts, e.g. by using “half-strength Murashige and Skoog (MS) medium. A C / N ratio may be defined as a molar ratio, or as a mass (w / w) ratio.

[0107] In some embodiments, the portion of the time is at least about 24h, at least 36h, at least 48h, or at least 96h.

[0108] In some embodiments, the portion of time is from transition from logarithmic phase to stationary phase.

[0109] In some embodiments, the CCCB is derived from a cacao cell culture which contained about 3%- 10%, or about 4%-8%, sugar upon transition of the cultured cells from logarithmic to stationary phase.

[0110] In some embodiments, the CCCB is derived from a cacao cell culture which contained amolar C / N ratio of about 20: 1-55:1 and / oramass C / N ratio of about 18:1-48:1 upon transition of the cultured cells from logarithmic to stationary phase.

[0111] Transition from logarithmic phase to stationary phase may be assessed by any method known in the art. Nonlimiting examples include a growth curve, measuring increase in cell mass or density over time (transition is observed when biomass stops increasing rapidly and begins to plateau), counting cells, depletion of key nutrients (e.g., nitrates), microscopic observations such as of doubling time, and measuring sedimentation volume as an indirect measurement of cell biomass.

[0112] For measuring sedimentation volume, a sample is allowed to settle (by gravity or gentle centrifugation), and the volume of the settled cell mass is measured, referred to as sedimentation volume, or settled cell volume (SCV) / packed cell volume (PCV). Upon transition from log to stationary phase, sedimentation volume stops rising sharply and starts to plateau.

[0113] In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about30%-90%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about 40%-70%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

[0114] In some embodiments, the CCCB is derived from cacao cells cultured by a method including at least one microscopic observation of the culture for evaluating uniformity in size and shape of the cultured cacao cells, which indicates cell cycle synchronization of the cells.

[0115] In some embodiments, the CCCB is not derived from somatic embryos.

[0116] Somatic embryos in cell suspension culture are discrete, organized multicellular structures that arise de novo from somatic cells when the medium and growth-regulator regime induces an embryogenic program. In contrast to non-embryogenic suspension cells (typically vacuolated, irregular / parenchyma-like, and loosely aggregated), somatic embryos are characterized by compact cell aggregates of small, dense, isodiametric cells with high nucleus-to-cytoplasm ratio and progressive organization into embryo-like stages commonly globular or heart / torpedo shaped.

[0117] In suspensions, the presence of somatic embryos is most reliably identified by microscopic observation of these distinct, compact, stage-like structures and by their ability to mature and convert into plantlets under appropriate conditions.

[0118] CCCL, and CCCB derived from CCCL

[0119] Cell cultured-derived cocoa liquor (CCCL) is produced from the cultured cacao cells by harvesting the cultured cacao cells. CCCL is also referred to as cocoa biomass. The CCCL may further be filtered and dried.

[0120] In some embodiments, the CCCB is prepared from CCCL. CCCB may be produced from CCCL by any suitable method, such as extraction using an organic solvent or cold pressing, as further detailed hereinbelow.

[0121] In some embodiments, the CCCL has at least 15%, 20%, 25%, or 30% w / w total fat content. In some embodiments, the CCCL has at least about 20% total fat content. In some embodiments, the CCCL has at least about 25% total fat content. In some embodiments, the CCCL has at least about 30% total fat content. In some embodiments, the CCCL has about 10%-60%, 10%-50%, 10%-40%, 15%-40%, 20%-40%, or 30%-40% w / w total fat content. In some embodiments, the CCCL has about 20%-40% w / w total fat content. In some embodiments, the CCCL has about 30%-40% w / w total fat content.In some embodiments, the CCCL contains about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-40% w / w oleic acid, and less than 6% w / w linoleic acid, out of the total fatty acid content. In some embodiments, the CCCL contains about 27-33% w / w palmitic acid, about 30-38% w / w stearic acid, about 28-35% w / w oleic acid, and about 1.5-5% w / w linoleic acid, out of the total fatty acid content. In some embodiments, the CCCL contains about 20-35% w / w palmitic acid, 33-40% w / w stearic acid, 25-35% w / w oleic acid, and 1.0-6.0% w / w linoleic acid of the total fatty acid.

[0122] In some embodiments, the CCCL contains less than 5%, 4.5%, 4%, or 3% w / w linoleic acid. In some embodiments, the CCCL contains 1-6%, 2-6%, 2-5%, or 3.5-5.5% w / w linolenic acid out of the CCCL total fatty acid content.

[0123] In some embodiments, the CCCL contains less than 0.25%, 0.2%, 0.15%, or 0.1% w / w or less linolenic acid out of the CCCL total fatty acid content.

[0124] In some embodiments, the CCCL contains less than 12%, 10%, 8%, 5%, 1%, 0.5%, or 0.1% w / w docosenoic acid out of the total CCCL fatty acid content.

[0125] In some embodiments, the CCCL contains less than less than 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% w / w ginkgolic acid out of the total CCCL fatty acid content.

[0126] In some embodiments, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitoleic acid, arachidic acid and behenic acid together constitute at least 98.5%, 99%, 99.2%, or 99.5% w / w of the total fatty acid content of the CCCL.

[0127] In some embodiments, there is provided a CCCL according to any of the above embodiments.

[0128] In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of more than 2%, 2.5%, 3%, or 3.5% w / v sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of at least 3% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of at least 4% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of 2.5%-9%, 3%-10%, 3%-9%, 3%-8%, 4%-10%, 4%-9%, 4%-8%, 3%-7%, 3%-6%, 3%-5%, 4%-7%, or 4%-6% w / v sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-10% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-8% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-10% sugar. In someembodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-9% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-8% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 4%-6% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-7% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 3%-6% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of 3%-5% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 4% sugar. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in the presence of about 8% sugar.

[0129] In some embodiments, the sugar is selected from sucrose, fructose, lactose, galactose, maltose, and any combination thereof. In some embodiments, the sugar is not glucose.

[0130] In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of at least about 12:1. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of at least about 15:1. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of at least about 18:1.

[0131] In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of about 12:1-58:1, 12:1-55:1, 15:1-58:1, 15:1-55:1, 18:1-58:1, 18:1-55:1, 20:1-58:1, 20:1-55:1, 15:1-50:1, or 15:1-48:1. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of about 15:1-52:1. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio or a mass C / N ratio of about 15:1-48:1.

[0132] In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio of about 20: 1 -55 : 1. In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a mass C / N ratio of about 18:1-48:1.

[0133] In some embodiments, the CCCL is derived from cacao cells cultured at least a portion of the time in a molar C / N ratio of about 23 : 1 or about 54: 1. In some embodiments, the CCCLis derived from cacao cells cultured at least a portion of the time in a mass C / N ratio of about 20:1 or about 46:1.

[0134] In some embodiments, the portion of the time is at least about 24h, at least 36h, at least 48h, or at least 96h.

[0135] In some embodiments, the portion of time is from transition from logarithmic phase to stationary phase and until liposome formation is observable by staining.

[0136] In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about 30%-90%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about 40%-70%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

[0137] In some embodiments, the CCCL is derived from cacao cells cultured by a method including at least one microscopic observation of the culture for evaluating uniformity in size and shape of the cultured cacao cells, which indicates cell cycle synchronization of the cells.

[0138] Methods for preparing cell culture-derived cacao-containing products

[0139] In some embodiments, there is provided a method of producing CCCB, CCCL and / or CCCP, the method including culturing cacao cells in a suspension medium, wherein during at least a portion of the time of the culturing, the suspension medium includes sugar at a concentration of about 3-10%, or about 3-8%, thereby obtaining a cacao cell suspension characterized by a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

[0140] In some embodiments, there is provided a method of producing CCCB, CCCL and / or CCCP, the method including:

[0141] - culturing cacao cells in a first suspension medium optionally including an initial sugar concentration of about 4-9% w / w;

[0142] - replacing the first suspension medium with a second suspension medium including a sugar concentration higher than 2% w / v upon transition of the cells from logarithmic phase to stationary phase, when the first suspension medium did not include an initial sugar concentration of about 4-9% w / w;

[0143] - growing the cells until liposome formation is observed; and- harvesting the cells to obtain CCCL having a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

[0144] In some embodiments, there is provided a method of producing CCCB, CCCL and / or CCCP, the method including:

[0145] - culturing cacao cells in a first suspension medium including sugar at an initial sugar concentration 2% or less until transition of the cells from logarithmic phase to stationary phase;

[0146] - replacing the first suspension medium with a second suspension medium including a sugar concentration higher than 2% w / v,

[0147] - growing the cells until liposome formation is observed, and

[0148] - harvesting the cells to obtain CCCL having a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

[0149] In some embodiments, there is provided a method of producing CCCB, CCCL and / or CCCP, the method including:

[0150] - culturing cacao cells in a first suspension medium including an initial sugar concentration of about 4-9% w / w;

[0151] - growing the cells until liposome formation is observed, and

[0152] - harvesting the cells to obtain CCCL having a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

[0153] Definitions and embodiments mentioned above and which may be relevant to embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0154] It is appreciated that replacing the first suspension medium with the second suspension may alternatively carried out by transferring the cultured cacao cells to the second suspension medium, or by adding sugar to the first suspension medium.

[0155] In some embodiments, the cacao cells are selected from cacao bean cells (e.g., cotyledon / seed tissue), cacao leaf cells, cacao flower cells, cacao stem cells, and combinations thereof. In some embodiments, the cacao cells are derived from a callus. In some embodiments,the callus is derived from an explant. The explant may originate from any plant part, such as beans (seeds), leaves, flowers, etc.

[0156] In some embodiments, the method includes a further step before culturing the cacao cells, including placing small pieces of cacao plant cotyledon in a tissue culture dish containing medium, developing calli from the cacao plant cotyledon, and deriving cacao cell suspensions from the calli.

[0157] In some embodiments, the initial sugar concentration is about 4-8%. In some embodiments, the initial sugar concentration is about 4%, 5%, 6%, 7,%, 8%, or 9%. In some embodiments, the initial sugar concentration is about 4%. In some embodiments, the initial sugar concentration is about 6%. In some embodiments, the initial sugar concentration is about 8%.

[0158] In some embodiments, the sugar in the second suspension medium is at a concentration of about 4-10%, 4-9%, 4-8%, 4-6%, or 6-8% w / v. In some embodiments, the sugar in the second suspension medium is at a concentration of about 4% w / v. In some embodiments, the sugar in the second suspension medium is at a concentration of about 6% w / v. In some embodiments, the sugar in the second suspension medium is at a concentration of about 8% w / v.

[0159] Transition from logarithmic phase to stationary phase may be assessed by any method known in the art, nonlimiting examples are provided hereinabove.

[0160] In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about 30%-90%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of about 40%-70%. In some embodiments, the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

[0161] Synchronization of the cultured cacao cells is a very important factor in butter production by the cacao cells, since when the culture is not synchronized, some of the cells produce a small amount of TAGs which include a high level of linoleic acid, and since the butter extraction is carried out on the entire cell mass, these cells “contaminate” the product with undesired fatty acids (such as linoleic acid) and corresponding TAGs. Synchronization of the cells may be assessed by determining cell size and shape, e.g., by microscopic observation.Accordingly, in some embodiments, the method further includes at least one microscopic observation of the culture for evaluating uniformity in size and shape of the cultured cacao cells, which indicates cell cycle synchronization of the cells. In some embodiments, the microscopic observation is carried out at least once in 7, 6, 5, 4, 3, 2, or 1 days. In some embodiments, the microscopic observation is carried out at least once in three days. In some embodiments, the microscopic observation is carried out at least once in two days. In some embodiments, the microscopic observation is carried out daily.

[0162] In some embodiments, the microscopic observation is carried out only following the transition from logarithmic to stationary phase.

[0163] In some embodiments, if the microscopic observation of a culture shows lack of synchronization of the cultured cacao cells, the culture is discontinued or fresh medium is applied to the culture.

[0164] In some embodiments, the method further includes filtering the CCCL and drying the filtered CCCL at a temperature above 50°C, or about 55°C.

[0165] In some embodiments, the method further includes extracting CCCB from the CCCL. In some embodiments, extracting is done by a method selected from using an organic solvent such as but not limited to hexane, cold pressing, chloroform extraction, petroleum-ether extraction, acetone extraction, or any combination thereof.

[0166] In some embodiments, the extracting further includes collecting the CCCP which remains after the extraction.

[0167] In some embodiments, the sugar is selected from sucrose, fructose, lactose, galactose, maltose, and any combination thereof. In some embodiments, the sugar is not glucose.

[0168] In some embodiments, the first suspension medium contains no more than 2%, 2.5%, 3%, or 3% sugar. In some embodiments, the first suspension medium contains less than 4% sugar. In some embodiments, the first suspension medium contains about 2% sugar.

[0169] In some embodiments, the first suspension medium has a molar C / N ratio or a mass C / N ratio lower than 20. In some embodiments, the first suspension medium has a molar C / N ratio or a mass C / N ratio lower than 15. In some embodiments, the first suspension medium has a molar C / N ratio or a mass C / N ratio lower than 12.

[0170] In some embodiments, the sugar concentration in the second suspension medium is more than 2.5%, 3%, or 3.5% w / v sugar. In some embodiments, the sugar concentration in the second suspension medium is at least 3% sugar. In some embodiments, the sugar concentration in the second suspension medium is at least 4% sugar. In some embodiments, the sugarconcentration in the second suspension medium is 2.5%-9%, 3%-10%, 3%-9%, 3%-8%, 4%-10%, 4%-9%, 4%-8%, 3%-7%, 3%-6%, 3%-5%, 4%-7%, or 4%-6% w / v sugar. In some embodiments, the sugar concentration in the second suspension medium is about 3%-10% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 3%-8% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 4%-10% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 4%-9% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 4%-8% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 4%-6% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 3%-7% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 3%-6% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 3%-5% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 4% sugar. In some embodiments, the sugar concentration in the second suspension medium is about 8% sugar.

[0171] In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of at least about 12:1. In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of at least about 15:1. In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of at least about 18:1.

[0172] In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of about 12:1-58:1, 12:1-55:1, 15:1-58:1, 15:1-55:1, 18:1-58:1, 18:1-55:1, 20:1-58:1, 20:1-55:1, 15:1-50:1, or 15:1-48:1. In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of about 15:1-52:1. In some embodiments, the second suspension medium has a molar C / N ratio or a mass C / N ratio of about 15:1-48:1.

[0173] In some embodiments, the second suspension medium has a molar C / N ratio of about 20:1-55:1. In some embodiments, the second suspension medium has amass C / N ratio of about 18:1-48:1.

[0174] In some embodiments, the second suspension medium has a molar C / N ratio of about 23 : 1 or about 54: 1. In some embodiments, the second suspension medium has a mass C / N ratio of about 20: 1 or about 46: 1.

[0175] The first suspension medium may be any suitable medium for growth of plant cells in suspension. Some nonlimiting examples include MS (Murashige and Skoog) Medium, DKW (Driver and Kuniyuki Walnut) Medium, and WPM (Woody Plant Medium), having a low C / Nratio, which leads to cell division. The second suspension medium may be based on the same nonlimiting examples as the first suspension medium, with the addition of sugar, thereby increasing the C / N ratio and leading the cells to transition into a non-dividing state during which cocoa butter is accumulated.

[0176] In some embodiments, the second suspension medium is identical to the first suspension medium except for having a higher sugar concentration.

[0177] Liposome formation may be monitored by any suitable method, typically by methods that detect the presence, size, structure, or composition of lipid vesicles. Nonlimiting methods include confocal microscopy, fluorescent microscopy including staining with fluorescent dyes, dynamic Light Scattering (DLS), etc. In some embodiments, liposome formation is observed by liposomal staining, e.g., as described in Rua at al. 2017 (Bioprocess and biosystems engineering, 40, 1479-1492).

[0178] In some embodiments, the first and / or the second suspension medium further include a plant hormone or a plant hormone substitute. In some embodiments, the plant hormone is cytokinin or an analogue thereof (such as kinetin), or an auxin or an analogue thereof (such as 2,4-dichlorophenoxyacetic acid (2,4-D)). In some embodiments, the first and / or the second suspension medium further includes kinetin and / or 2,4-D.

[0179] In some embodiments, the plant hormone or plant hormone substitute is present in the first and the second suspension media at the same concentration.

[0180] In some embodiments, the first and the second suspension media do not include a plant hormone or a plant hormone substitute. In some embodiments, the first and the second suspension media are not capable of inducing somatic embryogenesis. In some embodiments, the first and the second suspension media do not include an agent capable of inducing somatic embryogenesis.

[0181] Treatments

[0182] Traditional treatments of cacao beans in the process of chocolate production typically include at least fermentation, roasting, and conching, which, as noted above, are important for flavor and color development.

[0183] Fermentation of cacao beans is done after harvesting, usually by keeping the raw beans under banana leaves or wooden boxes for about 5-7 days. This process increases the temperature to about 50°C and causes microbial activity by yeast, lactic acid bacteria, and acetic acid bacteria, which convert sugar to alcohol and to acids, and initiate breakdown ofproteins into free amino acids. The result is reduced bitterness and formation of flavor precursors for the Maillard reaction (during roasting).

[0184] Roasting of the beans is typically done at 110-160°C for 20-60 minutes, and transforms flavor precursors into chocolate’s signature profile. The heat triggers the Maillard reaction, which is a chemical reaction between amino acids and sugars, producing volatile aroma compounds, as well as additional processes such as Strecker degradation and polyphenol oxidation. The Maillard reaction also contributes to browning of the chocolate.

[0185] Conching is done during grinding of the chocolate liquor, by mixing and aerating at warm temperatures for several hours, causing some volatile flavor compounds develop and others evaporate, contributing to a silky texture and a more rounded flavor.

[0186] An additional treatment often used for improving flavor and / or color is alkalization (Dutching), which involves treating cocoa-based substances with alkaline solutions such as potassium carbonate (K2CO3), sodium carbonate (NaiCCh), or sodium hydroxide (NaOH), which raises the pH, causing a smoother flavor and darker color.

[0187] The above methods may also be applied to cell culture-derived cacao-based substances. For example, CCCL may be roasted, to go through the Maillard reaction. Additionally, conching may also be applied to CCCL. Alkalization may also be applied to post-harvest culture-derived cocoa-based substances.

[0188] The term “cacao-based substances”, as used herein, encompasses substances obtained in the process of the cacao-based product preparation. Such substances especially include CCCL, CCCB, and CCCP.

[0189] However, instead of the traditional fermentation, simulated fermentation and / or enzymatic treatments may be applied to the cultured cells, as well as to the culture-derived cacao-based substances.

[0190] Enzymatic treatments that are typically carried out during fermentation and include using pectinases, proteases, polyphenol oxidase (PPO), invertases, amylases, and lipases, may be applied during cell culture, or to the harvested CCCL.

[0191] Additionally, simulated fermentation may applied, e.g., by adding microorganisms to the cell culture. For example, the cells may be inoculated with microorganisms such as yeast (Saccharomyces. Candida) or bacterial strains (e.g., Lactobacillus or Acetobacter), possibly with addition of substrates such as sugars, acetate, or peptides.

[0192] The term “simulated fermentation”, as used herein, relates to a process of causing metabolic shifts similar to those which happen during fermentation by other means. Suchmeans include adding microbes (microorganisms) to the cell culture, oxygen deprivation, carbon source overload, low pH, and generating metabolic stress, e.g., by light at certain frequencies.

[0193] Some of the treatments may be followed by roasting, to allow for the Maillard reaction to occur.

[0194] Accordingly, in some embodiments, the method further includes a treatment step of the CCCL for improving flavor and / or color.

[0195] In some embodiments, the treatment step is selected from fermentation, roasting, conching, alkalization, enzymatic treatment, and combinations thereof.

[0196] In some embodiments, the method further includes an enzymatic step including adding to the cell culture at least one enzyme selected from a pectinase, a protease, a polyphenol oxidase (PPO), an invertase, an amylase, a lipase, and combinations thereof.

[0197] In some embodiments, the method further includes a simulated fermentation step including adding to the cell culture a microorganism. In some embodiments, the microorganism is selected from yeast (e.g., Saccharomyces. Candida), bacterial strains (e.g., Lactobacillus or Acelobacler), and combinations thereof.

[0198] In some embodiments, the method does not include a step of fermentation. In some embodiments, the method does not include a step of simulated fermentation. In some embodiments, the method does not include a step of roasting. In some embodiments, the method does not include adding a microorganism. In some embodiments, the method does not include adding an enzyme. In some embodiments, the method does not include adding an enzyme selected from a protease, a glycosidase, or a lipase. In some embodiments, the method does not include adding a plant regulator or a hormone. In some embodiments, the method does not include adding a medium or an agent capable of inducing somatic embryogenesis. In some embodiments, the method does not include steps of somatic embryogenesis.

[0199] A cacao cell culture capable of producing cocoa butter

[0200] In some embodiments, there are provided cultured cacao cells capable of producing the CCCB, CCCL, and / or CCCP disclosed herein.

[0201] In some embodiments, the cultured cacao cells are produced by the methods disclosed herein.

[0202] In some embodiments, the cultured cacao cells are produced by a method including culturing cacao cells obtained from a cacao plant in a suspension medium, wherein upontransition of the cultured cells from logarithmic to stationary phase, the suspension medium comprises sugar at a concentration of about 3%-l 0%, or about 4%-8% w / v.

[0203] Definitions and embodiments mentioned above and which may be relevant to embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0204] In some embodiments, the cultured cacao cells have a biomass including the fatty acid profile or the TAG profile disclosed hereinabove.

[0205] In some embodiments, the cultured cacao cells have fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-40% w / w oleic acid, and less than 6% w / w linoleic acid, out of the total fatty acid content.

[0206] In some embodiments, the cultured cacao cells have a biomass containing at least 15%, 20%, 25%, or 30% w / w total fat content.

[0207] In some embodiments, the cultured cacao cells are selected from cacao bean cells (e.g., cotyledon / seed tissue), cacao leaf cells, cacao flower cells, cacao stem cells, and combinations thereof. In some embodiments, the cultured cacao cells are derived from a callus. In some embodiments, the callus is derived from an explant. The explant may originate from any plant part, such as beans (seeds), leaves, flowers, etc.

[0208] In some embodiments, the cultured cacao cells are not derived from somatic embryo cells. In some embodiments, the cultured cacao cells do not include somatic embryos.

[0209] Products including the cell culture-derived cocoa butter

[0210] In some embodiments, there is provided a cacao-containing product including the CCCB disclosed herein (according to any one or more the above listed embodiments).

[0211] Definitions and embodiments mentioned above and which may be relevant to embodiments in this section also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0212] The cacao-containing products of the invention are intended to encompass any cacaocontaining product.In some embodiments, the cacao-containing product further includes cacao bean-derived cocoa ingredients. In some embodiments, the cacao-containing product further includes cacao bean-derived cocoa butter. In some embodiments, the cacao-containing product further includes cacao bean-derived cocoa powder.

[0213] In some embodiments, the cacao-containing product has a melting temperature of about 20-40°C. In some embodiments, the cacao-containing product has a melting temperature of about 25-40°C. In some embodiments, the cacao-containing product has a melting temperature of about 25-35°C. In some embodiments, the cacao-containing product has a melting temperature of about 30-35°C. In some embodiments, the cacao-containing product has a melting temperature of about 33-35°C.

[0214] In some embodiments, the cacao-containing product is a comestible product. In some embodiments, the comestible product is a chocolate, a chocolate containing product, a chocolate-like product, or a chocolate replacement product. Some nonlimiting examples for a comestible cacao-containing product include chocolate (such as dark chocolate, milk chocolate, chocolate chips, chocolate truffles, filled chocolates, etc.), chocolate cakes (brownies, muffins) and cookies, chocolate ice cream and mousse, chocolate spread, chocolate like spread, chocolate topping, chocolate cream, cocoa spread, cocoa-based beverages (such as hot cocoa, hot chocolate, chocolate milk, chocolate shake), cocoa powder and mixes including cocoa powder, chocolate coatings and spreads, chocolate-coated snacks, supplements (such as flavanol-rich cocoa extract capsules, cocoa-based energy bars).

[0215] In some embodiments, the cacao-containing product includes about 1-60%, 5-60%, 10-60%, 20-60%, or 20-50% w / w cocoa butter. In some embodiments, the cacao-containing product includes about 1-60%, 5-60%, 10-60%, 20-60%, or 20-50% w / w CCCB.

[0216] For example, dark chocolate may include 30-50% cocoa butter or CCCB whereas milk and white chocolates typically include about 20-30% cocoa butter or CCCB.

[0217] In some embodiments, at least 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98% w / w of the cocoa butter in the comestible product is the hereindisclosed CCCB. In some embodiments, all the cocoa butter of the comestible product is the hereindisclosed CCCB.

[0218] In some embodiments, the cacao-containing product is a cosmetic cacao-containing product.

[0219] Non-limiting examples of CCCB-containing cosmetics include: moisturizers, balms, body lotions and creams, lip balms, body butters, soap, body washes, hair conditioners, and masks, sun protection creams, sprays and balms, etc.In some embodiments, the cosmetic cacao-containing product includes 1-100%, or 5-100% cocoa butter or CCCB. For example, the moisturizers and balms may be made from pure cocoa butter or CCCB (i.e., 100%), body lotions and creams may include 5-25% cocoa butter or CCCB and lip balms may include 10-50% cocoa butter or CCCB, body butters may include 15-50% cocoa butter or CCCB, soap and body washes may include 2-10% cocoa butter or CCCB, and hair conditioners and masks may include 5-20% cocoa butter or CCCB.

[0220] In some embodiments, at least 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98% w / w of the cocoa butter in the cosmetic cacao-containing product is the hereindisclosed CCCB. In some embodiments, all the cocoa butter of the cosmetic cacao-containing product is the hereindisclosed CCCB.

[0221] Product by process

[0222] In some embodiments, there is provided the CCCB produced by the hereindisclosed method (according to any one or more the above listed embodiments).

[0223] In some embodiments, there is provided the CCCL produced by the hereindisclosed method (according to any one or more the above listed embodiments).

[0224] In some embodiments, there is provided the CCCP produced by the hereindisclosed method (according to any one or more the above listed embodiments).

[0225] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs.

[0226] As used herein, the terms “cocoa butter” is pale-yellow, edible fat traditionally extracted from cacao beans (also referred to as “Theobroma oil”). It is used to make chocolate, as well as some ointments, toiletries, and pharmaceuticals. It typically has a cocoa flavor and aroma. For use in chocolate manufacture, fermented, dried and roasted cacao beans are separated from their hulls to produce cocoa nibs. Typically, cocoa nibs contain 54-58% of cocoa butter. The cocoa nibs are ground to form “cocoa mass”, also known as “cocoa liquor”.

[0227] The liquor is pressed to separate the cocoa butter from the non-fat cocoa solids, also referred to as “cocoa powder”.

[0228] The term “fat”, as used herein with respect to total fat content, refer to triglycerides, diglycerides, monoglycerides, and free fatty acids, that can normally be used in chocolates and other cacao-containing products. Cocoa butter encompasses pressed cocoa butter, expellercocoa butter, solvent extracted cocoa butter, refined cocoa butter, butterfat, and fractionated butterfat.

[0229] As used herein, the term “CCCB-containing product” may refer to any cacaocontaining product, edible cosmetic or other, including the herein disclosed CCCB.

[0230] As used herein, the term “chocolate” is intended to refer to all types of chocolates or chocolate-like compositions. The term is intended to include all chocolate and chocolate-like compositions that contain at least cocoa butter. The term is intended, for example, to include standardized and non-standardized chocolates, i.e., including chocolates with compositions conforming to the U.S. Standards Of Identity (SOI) and compositions not conforming to the U.S. Standards Of Identity, respectively, including dark chocolate, baking chocolate, milk chocolate, sweet chocolate, semi-sweet chocolate, buttermilk chocolate, skim-milk chocolate, mixed dairy product chocolate, low fat chocolate, white chocolate, non-standardized chocolates and chocolate-like and chocolate-containing / cocoa containing compositions, unless specifically identified otherwise.

[0231] As used therein, the term “chocolate-like product” may be any cacao-containing product having a chocolate taste and include there herein disclosed CCCB, CCCL or / and CCCP.

[0232] As used therein, the term “chocolate replacement product” may be any chocolate alternative that does not include cacao-based ingredients obtained from cacao beans. Nonlimiting examples for a cacao-free alternative include carob (Ceratonia siliqua), roasted mesquite (Prosopis spp. Lucuma (Pouteria hicuma . coconut, sweet potato, beetroot, fermented grains / legumes, and synthetic combinations of artificial chocolate flavor (e.g., from vanillin, ethyl maltol) combined with vegetable fats and sweeteners.

[0233] As used therein, the term “chocolate-containing product” refers to any product including chocolate, which includes the hereindisclosed CCCB, CCCL and / or CCCP.

[0234] As used therein, the term “cacao-containing products” refers to any product including cacao which includes the hereindisclosed CCCB, CCCL and / or CCCP.

[0235] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a cell” means one or more cell(s).The term "preferably" is used to suggest that a certain feature or embodiment of an invention is favored, but it is not strictly necessary for the invention to function.

[0236] As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass deviations / variations of ±20% or in some embodiments ±10%, or in some embodiments ±5%, or in some embodiments ±1%, or in some embodiments ±0.1% from the specified value, as such deviations are appropriate to perform the disclosed methods.

[0237] As used herein, the term “comprising” is synonymous with the terms "including" "containing" or "characterized by" and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. In some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0238] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0239] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0240] EXAMPLES

[0241] Methods

[0242] Cacao Cotyledon cell line establishment from callus

[0243] The procedure for calli development and cell suspensions from cacao cotyledon was based on Tsai & Kinsella, 1981 (Annals of Botany, 48(4), 549-558). Briefly, cacao fruits were picked from Theobroma cacao plants and surface sterilized. The beans were then extractedfrom the fruits, peeled and surface sterilized. The cotyledon was cut into small pieces and plated in petri dishes containing media. Calli were then developed and cell suspensions were derived from the calli.

[0244] Cacao Cotyledon cell line and maintenance

[0245] The Theobroma cacao cotyledon-derived cell line was maintained in suspension in liquid basal medium (BM), and subcultured routinely under aseptic conditions. Prior to experimental treatments, actively growing cells were propagated for 7 days in BM to standardize physiological state before inoculation into the test media. All cultivations were performed in 500 mL Erlenmeyer flasks containing 100 mL working volume of liquid medium. Each treatment was carried out in three biological replicates.

[0246] The BM was either MS (Murashige and Skoog) medium including 4.4 g / L MS basal mixture, 0.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg / L kinetin; or DKW (Driver and Kuniyuki Walnut) medium including 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D).

[0247] Inoculum and transfer procedure

[0248] After 7 days of growth in BM, cells were transferred to a fresh medium (same as above) as follows: cells were allowed to settle for 15 minutes and the volume of the cell was estimated. Each experimental flask was inoculated at 10% v / v cells / medium.

[0249] Media preparation and pH adjustment

[0250] Media were prepared by dissolving MS basal mixture and carbohydrates in deionized water, followed by addition of 2,4-D and kinetin from sterile stock solutions. The initial pH was adjusted to 5.8 for all treatments (e.g., using 1 N KOH or 1 N HC1) prior to sterilization. Media were sterilized by autoclaving, and heat-labile components (if any) were sterile-filtered and added after cooling.

[0251] Culture conditions

[0252] Inoculated flasks were incubated in a dark culture room at 28°C on an orbital shaker at 110 rpm. Cultures were grown for 20 days unless otherwise stated.Biomass determination (dry weight, DW)

[0253] At day 20, culture biomass was determined as dry weight (DW) per liter, as follows: The entire culture (or a certain aliquot) was harvested by vacuum filtration through a preweighed membrane to yield cell culture-derived cocoa liquor (CCCL); cells were rinsed briefly with deionized water to remove residual medium solids; filters containing biomass were dried in a lyophilizer until completely dry; and dry biomass concentration was calculated.

[0254] Triglyceride (TAG) quantification

[0255] Triglyceride content was quantified from harvested biomass collected at day 20 and normalized to DW as follows: The entire dried biomass was transferred to extraction tubes; total lipids were extracted using hexane; the organic phase was collected, and solvent was evaporated to obtain a crude lipid fraction, corresponding to cocoa butter (the cell culture-derived cocoa butter (CCCB) described herein). Triglycerides were analyzed in the CCCB, and quantified by mass chromatography such as GC-MS (Gas Chromatography-Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry).

[0256] Example 1: Improved fatty acid profile of cacao cell suspensions and cocoa butter Cacao-derived cell lines were prepared from cacao plant cotyledon as described in the methods, and cacao cell suspensions were grown and maintained as described above. Sugar was added according to one of two options: Option 1: Adding sugar at a high concentration (about 8% w / v) when starting the culture; or Option 2: Starting with a low (2%) concentration of sugar and adding about 4% w / v sugar upon transition of cells from logarithmic growth to stationary growth.

[0257] Accordingly, the cells were grown in a BM medium, either including about 8% sugar and having a high mass C / N ratio of about 46:1 (Option 1), or including about 2% sugar and having low mass C / N ratio of about 10:1 (Option 2). It appears that even though the C / N ratio was high in Option 1, the available nitrogen still drives growth of the cells until nitrogen is depleted. It turned out that there is still about 6% sugar in the culture at transition between log phase and stationary phase.

[0258] According to Option 2, cell growth was monitored daily and when cells transitioned from logarithmic to stationary growth, sugar was added (or cells were transferred to a fresh MS medium including sugar) to about 4% w / v sugar (glucose or sucrose), elevating the mass C / Nratio to about 20: 1. The depletion of nitrogen and the higher sugar concentrations and C / N ratio causes the cells to transition into a non-dividing state during which cocoa butter is accumulated, until liposomes are formed (tested by liposome staining as disclosed in Rua at al. 2017 (Bioprocess and biosystems engineering, 40, 1479-1492)).

[0259] Transition from logarithmic to stationary growth was determined by daily following cell growth by measurement of sedimentation volume, until growth has slowed down (sedimentation volume stopped increasing and reached a plateau), typically at sedimentation volumes of above 30%.

[0260] Cells were observed daily under a light microscope. The culture maintained a parenchyma-like morphology, with predominantly large, elongated, highly vacuolated cells and relatively low cytoplasmic density. Cells were present mainly as single cells or loose, friable aggregates rather than compact nodular clusters (typical of somatic embryos). Cell proliferation was evident, with an estimated doubling time of <72 hours, but no pro-embryogenic masses or embryo-like structures (e.g., globular bodies) were observed during the observation period. The uniform size and shape of the cells was indicative of synchronization of cell stage, which is very important for butter production. In culture of un synchronized cells butter production was inefficient, yielding with very little TAGs having a high, about 16%-50% linoleic acid. Accordingly, cultures in which the cells did not exhibit a uniform size and shape were discarded. Doubling time was checked also to assess the time of transition from log to stationary phase.

[0261] Fig- 1 shows an example for microscopy showing shape and size of cells in a synchronized suspension culture that was started with 8% sucrose (as per Option 1), and the lack of embryonic cells. Fig. 2 shows accumulation of lipid droplets of TAGs in the synchronized cells.

[0262] Finally, a cocoa biomass (also referred to herein as CCCL) was obtained by harvesting the cells, filtering the biomass and drying at 55°C.

[0263] Cultured cocoa butter (CCCB) was derived from the CCCL by hexane extraction as disclosed in Rua at al. 2017, supra. The remaining biomass (non-fat solid) constitutes the cell culture-derived cocoa powder (CCCP).

[0264] To assess the fatty acid profile resulting from the tested conditions, the fatty acid profile of the obtained cocoa butter was determined based on the standardized analytical methods developed by the Association of Official Analytical Collaboration (AO AC 996.06-1996(2010), Fat (total, Saturated, and Unsaturated) in Foods, Hydrolytic Extraction Gas ChromatographicMethod,” 18th Edition, AO AC Official Method 996.06, AO AC International, Arlington, 2001).

[0265] Table 1 shows the fatty acids profile of five cell culture-derived cocoa butter samples including an initial 2% sugar and either not adding further sugar at transition (CCCB 1-CCCB4, a mass C / N ratio of 10:1) or adding 4% sucrose at transition (CCCB5, a mass C / N ratio of 20: 1), in comparison with a fatty acids profile of cocoa butter derived from cocoa nibs.

[0266] Table 1: Fatty acid profiles of cultured cocoa butter vs. cocoa butter from nibs

[0267]

[0268] As can be seen, the lipid profiles of CCCB 1 to CCCB4 showed a high content of linoleic acid and relatively low content of stearic and oleic acid compared to cocoa nibs.

[0269] However, advantageously and unexpectedly, the lipid profile of CCCB5, to which 4% sugar was added upon transition of cells from log to stationary phase, exhibited an improved fatty acid profile that is very similar to that of naturally produced cocoa butter derived from cocoa nibs, including 30.5% palmitic acid, 32.7% stearic acid, 33.0% oleic acid, and, most importantly, 2.3% linoleic acid, 0.1 linolenic acid.

[0270] It is further noted that additional fatty acids showed a level more similar to cocoa nibs in CCCB5 than in CCCB1-CCCB4, including myristic acid, arachidic acid, linolenic acid, and behenic acid.Finally, as seen from Table 1, the quality of the fatty acid profile is likewise remarkable in that the listed fatty acids constitute 99.7% of the total fat contents (similar to the 99.8% of the cocoa butter derived from the cocoa nibs), thus indicating very low levels of “contaminating” fatty acids.

[0271] Accordingly, a higher sugar % was needed at the transition from log to stationary phase in order to obtain the desired fatty acids profile, similar to that of natural cocoa butter.

[0272] Example 2: Determination of fatty acid profile and yield (CCCB out of CCCL)

[0273] Additional culture-derived cocoa butter was prepared as disclosed above, and fatty acid profiles were determined, as explained above. Total fat content (CCCB yield) was obtained by determining the weight-to-weight ratio of CCCB / CCCL. The results are presented in Table 2 below.

[0274] The controls included cocoa butter was extracted from a commercial chocolate control and a commercial cocoa butter, the tests were CCCB7, grown with an initial 2% sucrose and 4% sucrose added at transition, and CCCB 8, grown only with the initial 2% sucrose.

[0275] Table 2: Fatty acid profile of cultured cocoa butter vs. cocoa butter from nibs

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[0277]

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[0281]

[0282] As indicated in Table 2, CCCB7, derived from cacao cells cultured in a medium with added 4% sugar upon transition resulted in a fatty acid profile very similar to that obtained from cocoa butter extracted from cocoa nibs, and the controls. Similarly, the listed fatty acid constituted 95.5% w / w of the total fatty acid again demonstrating the quality a reproducibility of the hereindisclosed CCCB.

[0283] In addition, as seen from the table, CCCB7 exhibited resulted a high CCCB yield (above 20% and above 30% of the CCCL), about 10 fold higher compared to CCCB 8.

[0284] Example 3: Fatty acid and TAG profiles after growth with different carbohydrates at varying concentrations

[0285] Cacao-derived cells were grown in suspension as described in the methods and in Example 1, and their biomass and TAG content were tested after 20 days (Table 3). Glucose or sucrose were used upon transition from log to stationary phase at the indicated concentrations (BM is the original basal medium), as explained in Example 1. Additionally, fatty acid profiles of the CCCB were evaluated, in comparison to cocoa butter extracted from bean-derived chocolate.

[0286] Table 3: Fatty acid profiles and TAG yield after 20 days of culture

[0287]

[0288]

[0289] As can be seen from Table 3, the identity of the carbon source and its concentration had a large effect on the TAG yield and the fatty acid profile of the CCCB. A low sucrose concentration (starting with 2%) resulted in both a low biomass (23.4 g / L) and a low TAG yield (12,288 pg / g DW); a very high sucrose concentration (starting with 2% and adding 8% upon transition, E) resulted in a higher biomass (80.5 g / L) but a low TAG yield (7,027 pg / g DW); 7.5% glucose instead of sucrose (C) resulted in a very high biomass (144.3 g / L) but very low TAG yield (2,815 pg / g DW); while starting with 2% sucrose and adding 4% sucrose (D) upon transition resulted in a medium biomass (55.1 g / L) but an extremely high TAG yield (340,565 pg / g DW). Accordingly, the presence of about 4-6%, or more (initial 2%, some possibly depleted plus added 4%) but less than 10% (initial 2%, some possibly depleted + added 8%, with) sucrose after the transition produced far more TAG per liter than the other carbon conditions.

[0290] Finally, experiment was done according to Option 1, starting with a total concentration of 8% sucrose, and surprisingly, in spite of the high initial sugar concentrations, the fatty acid profile obtained was similar to that obtained with the addition of 4% sucrose at transition (data not shown). As it turned out, the cells first entered a growth phase in spite of the high initial C / N ratio (probably since nitrogen levels were sufficient), and only after nitrogen was depleted the cells transitioned into stationary phase, producing butter. It was further found that when nitrogen became limiting, the sucrose concentration was still above 6% (data not shown), and TAG was accumulating in the cells.

[0291] It therefore appears that the presence of concentrations of about 4-6% of sucrose at the transition from log phase to stationary phase resulted in the highest butter production.

[0292] With respect to fatty acid profile, that was also strongly influenced by the carbon source. A low sucrose level at transition (0-2%, BM) resulted in high linoleic acid (50.1%) and very low stearic acid (2.4%), far from their concentration in chocolate; 7.5% glucose (C) also yielded high linoleic acid (43.4%) and very low stearic acid (2.8%); addition of 8% sucrose at transition (E) similarly yielded high linoleic acid (42.6%) and very low stearic acid (3.1%); while addition of 4% sucrose at transition (D) resulted in a high stearic acid (35.1%) and lowlinoleic acid (4.3%), which are very similar to their concentrations in chocolate. Oleic acid was also relatively high (28.4%), similar to its concentration in chocolate.

[0293] Again, this shows that among the tested conditions, the presence of 4-6% sucrose at transition resulted in a profile having the closest values to those of cacao beans-derived cocoa butter reference, with stearic, palmitic and oleic acid similar to the reference, low linoleic acid (close to the reference), as well as a high total of C16:0 + C18:0 + C18:l. That in addition to the highest TAG yield, noted above.

[0294] TAG profiles for the CCCB of D (4-6% sucrose at transition) were further evaluated and compared to cocoa butter extracted from cocoa nibs and from chocolate, and to commercial cocoa butter (all derived from cacao beans). The results are presented in Table 4.

[0295] Table 4: TAG profiles for CCCB after 20 days of culture with 4-6% sucrose present upon transition from log to stationary phase

[0296]

[0297] As can be seen from Table 4, the main TAGS, namely POP (C50, l,3-palmitoyl-2-oleoyl-glycerol), POS (C52, l-palmitoyl-2-oleoyl-3-stearoyl-glycerol), and SOS (C54, 1,3-stearoyl-2-oleoyl-glycerol), reproduced the ratios characterizing bean-derived cocoa nibs: POP in CCCB (20.4%) was nearly identical to that of cocoa nibs (20.5%), and somewhat higher than commercial cocoa butter (16.4%); POS was the predominant TAG in all samples, with 41% in CCCB, which is within the natural range (39-41%), and slightly enriched relative to commercial cocoa butter (39.1%); and SOS in CCCB (24.7%) closely matched cocoa nibs concentration (24.2%) and fell within the expected natural range (24-28%).

[0298] This distribution demonstrates that the biosynthetic machinery of the cultured cocoa cells retains the canonical acyltransferase selectivity characteristic of cocoa seeds, particularly the stereospecifically numbered sn-1 / 3 positioning preferences that generate the symmetric POP-POS-SOS pattern.

[0299] It is worth noting that minor TAGs (e.g., OPO, OOO, SPO, SOO, PliS, when O = oleyl; P = palmitoyl; S = stearyl; and Li = linoleyl) appeared only at trace levels, consistent with natural cocoa butter. The absence of atypical TAGs — especially those rich in linoleic or linolenic acids — indicates that the fatty-acid desaturation and elongation pathways in the cell culture system closely replicate those of cocoa cotyledons. This is a critical observation because deviations in unsaturated TAGs often lead to undesirable softening, off-melting, or polymorphic instability in cocoa butter analogs.

[0300] Example 3: Thermal behavior of CCCB is similar to that of bean-derived cocoa butter Thermal behavior of the CCCB was characterized by differential scanning calorimetry (DSC). The melting thermogram (melting profile) was recorded and was found to be identical to that of commercially available bean-derived cocoa butter (data not shown).

Claims

CLAIMS1. Cell culture-derived cocoa butter (CCCB) characterized by a fatty acid profile comprising 20%-35% w / w palmitic acid, 25%-40% w / w stearic acid, 25%-35% w / w oleic acid, and less than 6% w / w linoleic acid, wherein the culture is a suspension culture derived from a cacao plant.

2. The CCCB of claim 1, wherein the fatty acid profile comprises 27%-33% w / w palmitic acid, 30%-38% w / w stearic acid, 27-34% w / w oleic acid, and 1.5-4.5% w / w linoleic acid.

3. The CCCB of claim 1 or 2, wherein the fatty acid profile comprises less than 0.2% w / w linolenic acid.

4. The CCCB of any preceding claim, wherein the fatty acid profile comprises less than 5% w / w docosenoic acid.

5. The CCCB of any preceding claim, wherein the fatty acid profile comprises less than 1% w / w ginkgolic acid.

6. The CCCB of any preceding claim, wherein the palmitic acid, stearic acid, and oleic acid, together constitute at least about 90% w / w of the total fatty acid content of the CCCB.

7. The CCCB of any preceding claim, further characterized by a triglyceride (TAG) profile comprising about 16-21% w / w l,3-palmitoyl-2-oleoyl-glycerol (POP), about 39%-41% w / w l-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS), and about 24%-28% w / w 1,3- stearoy 1 -2-ol eoy 1 -gly cerol ( S O S) .

8. The CCCB of any preceding claim, wherein the TAG profile comprises C52 and C50 TAGs at a ratio of about 2.1 : 1-2.4: 1 C52% / C50% w / w.

9. The CCCB of any preceding claim, wherein the TAG profile comprises C52 and C54 TAGs at a ratio of about 1.4: 1-1.8: 1 C52% / C54% w / w.

10. The CCCB of any preceding claim, wherein the TAG profile comprises less than about 4% w / w of any one of l,3-oleyl-2-palmitoyl-glycerol (OPO), 1,2,3-oleyl-glycerol (OOO), 1- stearyl-2-palmitoyl-3-oleyl-gly cerol (SPO), l-stearyl-l,3-oleyl-glycerol (SOO), 1,2,3- palmitoyl -glycerol (PPP), l-stearyl-2-palmitoyl-3- myristyl-glycerol (SPM), and 1- palmitoyl-2-linoleyl-3- stearyl-glycerol (PLiS).

11. The CCCB of any preceding claim, wherein the TAG profile comprises less than about 1% w / w C48 TAGs.

12. The CCCB of any preceding claim, having a melting temperature of about 20°C-40°C.

13. The CCCB of any preceding claim, wherein the CCCB is derived from a cultured cacao cells biomass having a total fat content of at least 20% w / w.

14. The CCCB of any preceding claim, wherein the CCCB is derived from a cacao cell culture which contained about 3%- 10%, or about 4%-8%, sugar upon transition of the cultured cells from logarithmic to stationary phase.

15. The CCCB of any preceding claim, wherein the CCCB is derived from a cacao cell culture which contained a molar C / N ratio of about 20: 1-55: 1 and / or a mass C / N ratio of about 18:1- 48: 1 upon transition of the cultured cells from logarithmic to stationary phase.

16. The CCCB of claim 14 or 15, wherein the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%.

17. The CCCB of claim 14 or 15, wherein the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

18. The CCCB of any preceding claim, wherein the suspension culture is derived from a cacao plant callus.

19. A method of producing cell culture-derived cocoa butter (CCCB), cell culture-derived cocoa liquor (CCCL) and / or cell culture-derived cocoa powder (CCCP), the method comprising culturing cacao cells obtained from a cacao plant in a suspension medium, wherein upon transition of the cultured cells from logarithmic to stationary phase, the suspension medium comprises sugar at a concentration of about 3%-10%, or about 4%-8% w / v, thereby obtaining a cacao cell suspension characterized by a fatty acid profile comprising 20-35% w / w palmitic acid, 25-40% w / w stearic acid, 25-35% w / w oleic acid, less than 6% linoleic acid.

20. The method of claim 19, wherein the transition from logarithmic phase to stationary phase is characterized by a sedimentation volume of above 30%.

21. The method of claim 19, wherein the transition from logarithmic phase to stationary phase is characterized by a change from an increase to a plateau in sedimentation volume over time.

22. The method of any one of claims 19-21, comprising at least one microscopic observation of the culture for evaluating uniformity in size and shape of the cultured cacao cells, which indicates cell cycle synchronization of the cells.

23. The method of any one of claims 19-22, wherein the cacao cell suspension obtained by the method is further characterized by a triglyceride (TAG) profile comprising about 16-21% w / w l,3-palmitoyl-2-oleoyl-glycerol (POP), about 39%-41% w / w l-palmitoyl-2-oleoyl-3- stearoyl-glycerol (POS), and about 24%-28% w / w l,3-stearoyl-2-oleoyl-glycerol (SOS).

24. The method of any one of claims 19-23, wherein the sugar is selected from sucrose, fructose, lactose, galactose, maltose, and any combination thereof.

25. The method of any one of claims 19-24, wherein the sugar is not glucose.

26. The method of any one of claims 19-25, wherein the suspension medium does not comprise a plant hormone or a plant regulator.

27. The method of any one of claims 19-26, wherein the suspension medium does not comprise an agent capable of inducing somatic embryogenesis.

28. The method of any one of claims 19-27, wherein the method does not comprise a step of fermentation or simulated fermentation.

29. The method of any one of claims 19-28, wherein the method does not comprise a step of adding a microorganism or an enzyme selected from a protease, a glycosidase, or a lipase.

30. The method of any one of claims 19-29, further comprising a step of harvesting the cacao cells to obtain a cell culture-derived cocoa liquor (CCCL), and extracting the CCCL to obtain CCCB.

31. The method of claim 30, wherein the CCCL has a total fat content of at least 20% w / w.

32. The method of claim 30 or 31, wherein the extracting of the CCCB from the CCCL comprises separating fat from remaining biomass by organic solvent or cold pressing, and wherein the remaining biomass constitutes CCCP.

33. The method of any one of claims 19-32, wherein the cacao cells are obtained from a cacao plant callus.

34. The method of any one of claims 19-33, wherein the method comprises the steps of:a) culturing the cacao cells in a first suspension medium optionally comprising an initial sugar concentration of about 4-9% w / w;b) replacing the first suspension medium with a second suspension medium comprising a sugar concentration higher than 2% w / v upon transition of the cells from logarithmic phase to stationary phase, when the first suspension medium did not comprise an initial sugar concentration of about 4-9% w / w;c) growing the cells until liposome formation is observed; andd) harvesting the cells to obtain cell culture-derived cocoa liquor (CCCL), and extracting the CCCL to obtain CCCB having a fatty acid profile including about 20-35% w / w palmitic acid, about 25-40% w / w stearic acid, about 25-35% w / w oleic acid, and less than 6% w / w linoleic acid.

35. A cacao cell culture obtained by the method of any one of claims 19-34.

36. A cacao cell culture obtained by a method comprising culturing cacao cells obtained from a cacao plant in a suspension medium, wherein upon transition of the cultured cells from logarithmic to stationary phase, the suspension medium comprises sugar at a concentration of about 3%-l 0%, or about 4%-8% w / v.

37. A cell culture-derived cocoa butter (CCCB) produced by the method of any one of claims 19-34.

38. A cell culture-derived cocoa power (CCCP) produced by the method of any one of claims 19-34.

39. A cell culture-derived cocoa liquor (CCCL) produced by the method of any one of claims40. A cacao-containing product comprising the CCCB of any one of claims 1-18 or 37, the CCCP of claim 38, and / or the CCCL of claim 39.

41. The cacao-containing product of claim 40 wherein the cacao-containing product is a chocolate, a chocolate-containing product, a chocolate-like product, or a chocolate replacement product.

42. The cacao-containing product of claim 40 wherein the cacao-containing product is a cosmetic product such as a moisturizer, a balm, a body lotion, a cream, a lip balm, a body butter, a soap, a body wash, a hair conditioner, a mask, and a sun protection cream, spray or balm.

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