Coffee - plant cell culture composition

Abstract

The invention provides a coffee - plant cell culture composition comprising a) roasted and ground coffee and b) a composition comprising plant cell culture grown in vitro, optionally in a form of a powder. In some embodiments, the coffee - plant cell culture composition is in a pod or container and may be used in a coffee machine. In some embodiments, the plant cell culture grown in vitro is grape berry cell culture grown in vitro (RGC).

Description

Coffee - plant cell culture compositionField of the invention

[0001] The invention relates to a composition for making a coffee beverage, the composition comprising coffee which may be ground coffee in combination with plant cell culture grown in vitro in a large scale. The plant cell culture grown in vitro in a large scale may be derived from any plant and any part of the plant, such as a fruit including but not limited to grape, pomegranate, olive, and the like. The composition may be inserted into a pod and be used in a coffee machine or inserted into a coffee bag or a container.Background of the invention

[0002] Caffeine is a stimulant and an adenosine receptor antagonist, reducing the desire to sleep and inducing wakefulness. The effects of caffeine are for short periods and are rapidly degraded by rapidity of ingestion increasing serum concentration spikes and reducing total caffeine with time.

[0003] On the other hand, caffeine toxicity and overstimulation create neuronal excitotoxicity, and can lead to neuronal, synapse cerebral metabolic “burn out”, producing lethargy, mental and physical fatigue, even prolonged insomnia exacerbated by high quickly absorbed caffeine bolus doses typical of caffeinated beverages; even states of transient psychosis with high caffeine bolus that could be attributed to brain cell induced toxicity gradients.

[0004] There is a need for a composition of coffee and other agent that provides brain enhancement, awakening, that is long lasting and does not have side effects that are associated with prolonged use of caffeine. Further, there is a need to combine the short-term effects of the coffee with a composition that increases vascular function and dilation of arteries, improves blood flow, physical energy and mental alertness, or oxidation of blood lipids.Summary of the invention

[0005] In some embodiments, there is provided a coffee - plant cell culture composition comprising a) roasted and ground coffee and b) a composition comprising plant cell culture grown in vitro, optionally in a form of a powder. In some embodiments, the coffee - plant cell culture composition is being added to a pod (capsule) or container.

[0006] In some embodiments, the coffee - plant cell culture composition is used in a coffee machine.

[0007] In some embodiments, there is provided a coffee - plant cell culture composition comprising a blend of soluble coffee and a composition comprising plant cell culture grown in vitro, optionally in a form of a powder.

[0008] In some embodiments, the resulted composition after coffee beverage preparation comprises grape berry polyphenols and piceid resveratrol.

[0009] In some embodiments, the resulted composition after coffee beverage preparation comprises resveratrol in an amount of between 1 -10 mg.

[0010] In some embodiments, wherein the smell and taste of the coffee are maintained, i.e. the addition of the comprising plant cell culture grown in vitro does not change the taste and smell of the coffee in the coffee - plant cell culture compositionDetailed description of the embodiments of the invention

[0011] In some embodiments, the invention relates to a coffee beverage composition, also termed here coffee - plant cell culture composition containing roast and ground coffee and a composition comprising plant cell culture grown in vitro, which may be in a form of a powder. In particular, the disclosure relates to a composition for use in a coffee capsule or pod, which gives a good tasting and appearance of final beverage after the preparation step.EP0756844 describes the various problems which arise from the mixing of roast and ground coffee with other ingredients. In particular, the dissolution of the soluble beverage ingredient, such as different plant extracts, for example , can hinder the extraction of the roast and ground coffee, leading to a poor final flavor.

[0012] The inventors have found that surprisingly, the mixing of a composition comprising plant cell culture grown in vitro with ground and roast ground coffee as described herein, results in a composition in which the coffee taste is not different from a composition having coffee only.

[0013] Moreover, the beneficial effects of the composition comprising plant cell culture grown in vitro remain the same despite the extraction, which may be in high temperature and in a high pressure. As is detailed below the plant cell culture grown in vitro may be fruit cell culture grown in vitro. In some embodiments, the plant cell culture grown in vitro may be grape berry fruit, and any other part of the grapes, pomegranate or olive cell culture grown in vitro.

[0014] In some embodiments, the roast and ground coffee have a particle size distribution of from about 500 to 900pm; and the plant cell culture powder has a particle size distribution of from 60 pm to 150 pm.

[0015] In some embodiments, the coffee - plant cell culture composition contains the ground coffee and the composition of the plant cell grown in vitro in a ratio by weight of from 50:1 to 5:1 . When the ratio falls outside of these ranges, the beneficial effects are not observed. Instead it is found that the coffee is weak, either because of a lack of coffee, or a poor extraction of the large amount of coffee present, presumably due to its coarse size. In some embodiments the ratio by weight of the roast and ground coffee to the plant cell grown in vitro composition is from 40:1 to 10:1 . In some embodimentsthe ratio by weight of the roast and ground coffee to the plant cell grown in vitro composition is from 30:1 to 10:1. According to some embodiments, the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 25:1 to 18:1. According to some embodiments, the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 24:1 to 20:1 . According to some embodiments, the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 22:1 to 20:1.

[0016] In some embodiments, the fruit cell culture grown in vitro is in vitro grown red grape cells (RGC or RGC product). RGC powder contains a matrix of polyphenols including piceid resveratrol, quercetin, anthocyanins, and tannins. In some embodiments, the RGC powder is composition in a form of a powder comprising a cell line culture of grape berry cells grown in vitro in a large scale up process, whereby the cell line culture of grape berry cells is derived from one or more of grape-berry cross section, grape-berry skin, grape-berry flesh, grape seed, grape embryo of seeded or seedless cultivars or grape seed coat; wherein the cell line culture of grape berry cells includes resveratrol in an amount of at least 1000 mg / kg powder. In some embodiments, the RGC is prepared as detailed in WO 2014 / 068557. In some embodiments, the RGC powder is equivalent to VINIA®. However, it is noted that the VINIA® product was not adequate for producing a composition of coffee with RGC product. The inventors have found that in order to combine RGC with coffee particles to a composition suitable for being inserted to a pod and used in a coffee machine it is needed to produce RGC at an average particle size of about 60-150 pm. Further, in some embodiments, the bulk density of the RGC is produced at about 0.26 g / ml. In some embodiments, the bulk density of the RGC is produced at between 0.05 to about 0.5 g / ml. In some embodiments, the bulk density of the RGC is produced at between 0.1 to about 0.4 g / ml. In some embodiments, the bulk density of the RGC is produced at between 0.15 to about 0.3 g / ml. In some embodiments, the bulk density of the RGC is produced at between 0.2 to about 0.3 g / ml. Also, the ratio between the coffee and RGC product amounts is important. VINIA® is made of red grapes (Vitis vinifera) cells grown in vertical bioreactors and comes in a form of fine dry pink-purple powder containing the whole matrix of polyphenols with a high concentration of piceid resveratrol in their natural state that have additive and synergistic effects.

[0017] VINIA® is a complex of red grape cells from the red grape skin / flesh / pulp and seed. Accordingly, VINIA® is a source of the whole matrix of nutrients and polyphenols found throughout the red grape and in red wine in their natural state (physicochemical state) without any genetic manipulation and contains polyphenols (resveratrol, tannins, quercetin, catechins, anthocyanins), vitamins, protein, fiber. Its advantages are in the absence of solvent residuals and the typical taste without bitterness and astringent.

[0018] In some embodiments, the plant cell culture grown in vitro is olive cell culture grown in vitro.

[0019] In some embodiments, the olive cells composition is a composition in a form of a powder comprising olive fruit / leaf cells grown in vitro grown in vitro in a large-scale process. Whereby the olive cells are derived from one or more of section: olive pulp, olive seed, olive petiole or olive leaf, wherein the olive fruit cells include polyphenols such as hydroxy tyrosol, tyrosol, oleuropein, verbascoside and pinoresinol. The composition contains in one embodiment high amounts of polyphenols particularly, the secondary metabolites verbascoside. The unique composition of olive cells (OC), which, as an outcome of scale up process, includes a whole matrix of polyphenols and other healthy ingredients, naturally existing in OC of a plant, with higher concentration, i.e., at least 1%, 2%, 2.5%, 3%, 4%, 5%, 7%, 9%, 10%, 13%, 15%, 17%, 20% or higher verbascoside. As used herein the term "polyphenols" refers to naturally occurring phyto organic compounds having more than one phenol group. Polyphenols may range from simple molecules, such as phenolic acid, to large, highly polymerized, compounds such as hydrolyzed tannins. The phenolic rings of polyphenols are typically conjugated to various sugar molecules, organic acids and / or lipids. Differences in this conjugated chemical structure account for the chemical classification and variation in the modes of action and health properties of the various polyphenol compounds.

[0020] In some embodiments, the olive cell culture is manufactured as described in WO 2017 / 130199.

[0021] In some embodiments, the olive cell culture grown in vitro is a composition in a form of a powder comprising olive fruit / leaf cells grown in vitro grown in vitro in a large-scale process. Whereby the olive cells are derived from one or more of section: olive seed, olive fruit or olive leaf.

[0022] In some embodiments the Olive Cell Product (OCP) is a dry cell line culture of olive cells manufactured in vitro in a large scale comprising verbascoside in an amount of 40 -200 g / kg dry weight

[0023] In some embodiments the Olive Cell Product is a dry cell line culture of olive cells manufactured in vitro in a large scale comprising total polyphenols in an amount of 50-220 g / kg dry weight, wherein the total polyphenols comprises verbascoside in an amount of 40 -200 g / kg dry weight, 1 -O-synapoylglucose and beta-hydroxyverbascoside and the product does not comprise detectable levels of tyrosol, hydroxy tyrosol, and oleuropein.

[0024] In some embodiments, the OCP is manufactured in a large-scale process for the in vitro production of an olive cell culture of olive cells grown comprising: growing olive cells in a flask; inoculating the olive cells from the flask into a first bioreactor; inoculating the olive cells from the first bioreactor into a second bioreactor; optionally inoculating the olive cells from the second bioreactor into a last bioreactor; andharvesting the olive cells from the last bioreactor; wherein the second bioreactor is a last bioreactor or an intermediate bioreactor and wherein the olive cells harvested from the last bioreactor are dried.

[0025] According to some embodiments, the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown.

[0026] According to some embodiments, if the second bioreactor is an intermediate bioreactor, an additional step of inoculating the olive cells to another intermediate bioreactor or to the last bioreactor is performed.

[0027] According to some embodiments, the large scale process of the invention further includes additional steps of inoculating the olive cells from the second bioreactor into any number of sequential intermediate bioreactors.

[0028] In some embodiments, there is provided a process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale comprising: growing olive cells in a flask; inoculating the olive cells from the flask into a first bioreactor; inoculating the olive cells from the first bioreactor into a second bioreactor; optionally inoculating the olive cells from the second bioreactor into a last bioreactor; and harvesting the olive cells from the last bioreactor;

[0029] wherein the second bioreactor is a last bioreactor or an intermediate bioreactor and the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growth medium comprising between about 2-5% sucrose, 9- 20 mg / l, ZnSO4.7H2O, 7-20 mg / l H3BO3, 0.1 -1 mg / l, CuSC 5H2O, 400-900 mg / l MgSC , 100-500 mg / l Ca(NO3)2mg / l and 150-500 mg / l KH2PO4, 350-650 mg / l KCI, 700-1800mg / l KNO3or 200-1500mg / l NH4NO3 between about 0.2-1 mg / l folic acid, between about 2-8 mg / l nicotinic acid, between about 0.2- 1 mg / l thiamine, between about 0.02-0.08 mg / l biotin and pea pepton, wherein the olive fruit / leaf cells grown in vitro comprises verbascoside in the amount of at least between 40 -200 g / kg dry weight and wherein the olive fruit / leaf cells grown in vitro do not comprise detectable levels of tyrosol, hydroxytyrosol, and oleuropein.

[0030] In some embodiments, if the second bioreactor is an intermediate bioreactor, an additional step of inoculating the olive cells to another intermediate bioreactor or to the last bioreactor is performed.

[0031] In some embodiments, the process further including additional steps of inoculating the olive cells from the second bioreactor into any number of sequential intermediate bioreactors.

[0032] In some embodiments, any one of the bioreactors is a 3— 9-liter bioreactor.

[0033] In some embodiments, any one of the bioreactors is a 30— 65-liter bioreactor. In some embodiments, any one of the bioreactors is a 30 -200-1 iter bioreactor. In some embodiments, any one of the bioreactors is a 200— 400-1 iter bioreactor. In some embodiments, any one of the bioreactors is a 200-1100-liter bioreactor. In some embodiments, any one of the bioreactors is a 1000-2000-1 iter bioreactor. In some embodiments, any one of the bioreactors is a 2000— 5000-1 iter bioreactor. In some embodiments, any one of the bioreactors is a 2000-1 OOOO-liter bioreactor. In some embodiments, at least one bioreactor is a disposable bioreactor having air flow of more than 800 liter / h, 1000 liter / h, 1200 liter / h, 1500 liter / h or more.

[0034] In some embodiments, the disposable bioreactor is made from one or more layers of polyethylene. In some embodiments, the disposable bioreactor includes an inner and an outer layer prepared from polyethylene and a middle layer prepared from nylon. In some embodiments, the growth medium further comprises a pea pepton.

[0035] In some embodiments, the pea pepton is in amount of between 50 to 500 g / l. In some embodiments, the pea pepton is in amount of between 100 to 400 g / l. In some embodiments, wherein the pea pepton is in amount of between 200 to 300 g / l. In some embodiments, the growth medium is enriched with one or more of vitamin, cytokine, auxin, glycine, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine, folic acid, 2,4-D, or 2iP. In some embodiments, the amount of 2, 4-D is between 0.3- 0.6 mg / l. In some embodiments, the amount of 2iP is between 0.07-0.2 mg / l. In some embodiments, the amount of glycine is between 0.5-6 mg / l. In some embodiments, the amount of myo inositol is between 50-200 mg / l. In some embodiments, the amount of nicotinic acid is between 2-8 mg / l. In some embodiments, the amount of pyridoxine HCI is between 0.1 -1 mg / l. In some embodiments, the amount of thiamine HCI is between 0.2-0.8 mg / l. In some embodiments, the amount of biotin is between 0.01 - 0.1 mg / l. In some embodiments, the amount of folic acid is between 0.2-0.8 mg / l.

[0036] In some embodiments, there is provided a process for the in vitro production of an olive cell culture of olive leaf, or olive fruit cells grown in vitro in a large scale comprising: growing olive cells in a flask; inoculating the olive cells from the flask into a first bioreactor; inoculating the olive cells from the first bioreactor into a second bioreactor; optionally inoculating the olive cells from the second bioreactor into a last bioreactor; and harvesting the olive cells from the last bioreactor; wherein the second bioreactor is a last bioreactor or an intermediate bioreactor and the size of each bioreactor used in the process is larger than the one in which the olive cells were previously grown; wherein the olive cells harvested from the last bioreactor are dried and wherein the olive cells are grown in bioreactors in a growth medium comprising COCI2.6H2O, CuSO4, FeNaEDTA, H3BO3, KI, MnSO4.H2O, Na2MoO4.2H2O, ZnSO4.7H2O, CaCI2, Ca (NO3)2, KH2PO4, KNO3, Ca(NO3)2, NH4NO3, MgSO4or KCI; Pea pepton; and one or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol,nicotinic acid, pyridoxine, biotin, thiamine and folic acid. In some embodiments, wherein the growth medium comprises:

[0037] Pea pepton; and one or more of vitamin, cytokine, auxin s glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid.In some embodiments, the amount of pea peptone and the one or more of vitamin, auxin, cytokine, glycine, sucrose, myo inositol, nicotinic acid, pyridoxine, biotin, thiamine and folic acid is as follows:

[0038] In some embodiments, the cells are grown in OCR medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose. In some embodiments, the cellsare grown in MS medium at the stage of Erlenmeyers, wherein the medium is supplemented with pea pepton and sucrose.

[0039] In some embodiments, the plant cell culture grown in vitro is pomegranate cell culture grown in vitro. The composition is in a form of a powder comprising a cell culture of pomegranate cell culture (PC) grown in vitro in, whereby the cell culture of PC is derived from one or more of pomegranate sections: pomegranate skin exocarp, mesocarp, pomegranate lamellae and pomegranate seeds. In an embodiment of the invention, the cell culture of PC includes punicalagin and PGG in an amount of at least 1300 mg punicalagin / kg powder and 2600 mg PGG / kg powder.

[0040] Typical pomegranate polyphenols include but not limited to ellagitannins (e.g. punicalagin and punicalin), gallic and ellagic acids and 1 , 2,3,4,6-pentagalloyl glucose (PGG). The pomegranate fruit may be of a wild or cultivated variety.

[0041] In some embodiments, the pomegranate cell culture is manufactured as described in WO 2015 / 102003.

[0042] In an embodiment of the invention, the coffee - plant cell culture composition may include more than one plant cell culture grown in vitro. For example, without being limited, roast ground coffee may be combined with red grape berry cell culture grown in vitro and olive cell culture grown in vitro.

[0043] In some embodiments, the coffee - plant cell culture composition of the invention further comprises one or more flavorings, selected from the group consisting of cream, almond, amaretto, anise, apple, brandy, caramel, cider, cinnamon, cherry, chocolate, mint, cocoa, panna, cream, milk, creme de menthe, French vanilla, grape, hazelnut, soluble coffee, Irish cream, lemon, macadamia nut, orange, peach, peppermint, pistachio, strawberry, vanilla, Wintergreen or a mixture of two or more thereof. The one or more flavorings are present in an amount of less than 5 wt %, 4 wt%, 3 wt%, 2 wt% or less than 1 wt % of the composition.

[0044] In some embodiments, the coffee - plant cell culture composition of the invention does not contain sugar. In some embodiments, the plant cell culture grown in vitro that is added to the coffee contains less than 10%, 8%, 6%, 4%, 2% or less sugar.

[0045] According to another embodiment there is provided a container comprising the composition disclosed herein. The container is suitable for a beverage preparation machine. Such containers are well known in the art and include various pods, pads, sachet, bags .capsules and cartridges, which are optionally made of plastic, paper, polybutylene perephthalate (PBT), food grade plastic with aluminium foil lids and aluminium capsules.

[0046] According to a further aspect there is provided a kit comprising a plurality of the containers disclosed herein.

[0047] According to some embodiments, the coffee - plant cell culture composition includes caffeine.According to some embodiments, the coffee - plant cell culture composition includes a decaffeinated coffee.

[0048] According to some embodiments, if the plant cell culture is grape berry cell culture, the resulted composition in the prepared coffee beverage after extraction comprises resveratrol and / or piceid resveratrol.

[0049] According to some embodiments, if the plant cell culture is grape berry cell culture, the resulted composition after extraction in the prepared coffee beverage comprises resveratrol and / or piceid resveratrol in an amount of between 1 -10 mg.

[0050] According to some embodiments, if the plant cell culture is grape berry cell culture, the resulted composition after extraction comprises resveratrol in an amount of between 3-8 mg.

[0051] According to some embodiments, if the plant cell culture is grape berry cell culture, the resulted composition after extraction comprises resveratrol in an amount of between 5-7 mg.

[0052] According to some embodiments, if the plant cell culture is grape berry cell culture, the resulted composition after extraction comprises resveratrol in an amount of about 6 mg.

[0053] It is noted that the term “resveratrol” herein refers to all resveratrol forms such as: cis piceid resveratrol, trans piceid resveratrol, cis resveratrol and trans resveratrol.Resveratrol Glycoside exp. Resvertrol chemistry structure

[0054] Chemistry structure of the resveratrol and piceid resveratrol of RGC Product, (VINIA®)

[0055] RGC Product, (VINIA®) is Red Grape Cells a natural product consists of ingredients associated with Red Wine such as polyphenols, mainly piceid resveratrol, maintaining the quality and inherent benefits present in nature without any solvent extraction or genetic modification. RGC is a fine pink-purple powder of cells originating from fruit of red grape berry (Vitis Vinifera), which are grown in aqueous media consisting of water and defined nutrients in bioreactors under controlled conditions such as light and temperature. Red grape cells are grown in large scale bioreactors grape cells yield a product that has marked advantages over the current alternative commercial products since it whole complex of polyphenols of grape-cell constituents and a significant higher concentration of graperesveratrol-glycoside (piceid) than in current grape derived products, in their natural state, that have additive and synergistic benefits.

[0056] RGC also contains in addition to piceid resveratrol polyphenols, tannins, quercetin, catechins, anthocyanins, vitamins (B1 , B6, C), protein, fiber, minerals and low level of sugar. The Natural state of piceid resveratrol in RGC permits better absorption and long-lasting ability.

[0057] Many studies have shown that glycosides differ from the parent aglycones molecule (molecules without the sugar / glucose group), not only in their antioxidant and biological activities, but also in their water solubility and bioavailability (Goldberg et al., 1996; Spencer et al., 2001 ; Kuhnle et al., 2000; Orsini et al., 1997; Stojanovic et al., 2002; Stivala et al., 2001 ; Biasutto et al., 2009).

[0058] Resveratrol can be found in a plant as both a glycoside and aglycone. In the production of most resveratrol end-products, the sugar molecule is stripped from the glycoside resveratrol, or it is produced as a synthetic molecule as an aglycone, without the glucose group.

[0059] Indeed, the glycoside form of trans-resveratrol, identified as piceid, is the predominant form of resveratrol found in VINIA® (99% piceid or glycoside resveratrol). The RGC production process is a controlled process favoring the production of piceid while preventing its destruction in the cells. This enables a high and stable VINIA® piceid concentration mirroring nature. VINIA is fast absorbing. The glycosylated structure enables VINIA’s resveratrol to be more soluble in aqueous media such as body fluids. Accordingly, VINIA’s resveratrol presented higher water solubility compared to other sources of resveratrol (Azachi et al., 2014). The high-water solubility of the glycosyl groups in VINIA’s resveratrol explains VINIA’s significantly high gastrointestinal absorption rate, as demonstrated in Azachi et al. 2014, when resveratrol was found in the plasma samples as soon as 20 minutes after ingestion.Red grapes are an important source of resveratrol, one of the most investigated plant polyphenols with a myriad of beneficial health effects. In plants, polyphenols like Resveratrol, are usually found as glycosides, sugar molecules bound to the polyphenols (Alluis et al., 2001 ; Goldberg et al., 1996).

[0060] The inventors have surprisingly found that the coffee - plant cell culture composition of the invention even after the step of heating and / or high pressure to produce coffee beverage into a cup maintains the metabolite structure, composition and function the RGC as described above. More surprisingly, the structure of resveratrol as T-piceid resveratrol is maintained in the coffee beverage also after heating and / or exposing to high pressure.

[0061] According to some embodiments, the particle size of the coffee is between 500-900. According to some embodiments, the particle size of the coffee is between 500-800. According to some embodiments, the particle size of the coffee is between 500-700. According to some embodiments, the particle size of the coffee is between 500-600. According to some embodiments, the particle size of the coffee is between 600-900. According to some embodiments, the particle sizeof the coffee is between 700-900. According to some embodiments, the particle size of the coffee is between 800-900.

[0062] According to some embodiments, the particle size of the plant cell culture grown, such as without limitation RGC powder in vitro is between 60-150 pm. According to some embodiments, the particle size of the plant cell culture grown in vitro is between 70-130 pm. According to some embodiments, the particle size of the plant cell culture grown in vitro is between 60-130 pm. According to some embodiments, the particle size of the plant cell culture grown in vitro is between 70-110 pm. According to some embodiments, the particle size of the plant cell culture grown in vitro is between 50-110 pm.

[0063] According to some embodiments, the particle size of the plant cell culture grown in vitro is between 80-100 pm. According to some embodiments, the particle size of the plant cell culture grown in vitro is about 90 pm.

[0064] According to some embodiments the plant cell culture grown in vitro, such as the RGC powder, is homogenous.

[0065] According to a further aspect there is provided a method for the manufacture of the coffee - plant cell culture composition disclosed herein, the method comprising: grinding roasted coffee beans, and mixing the roast and ground coffee with a plant cell culture grown in vitro powder, the method optionally further comprising filling the composition into a container.As will be appreciated, the grinding will be by any known technique suitable to arrive at the particle size distributions discussed herein. By way of example, a suitable grinding technique would be using roller or disc type grinders.

[0066] According to a further aspect there is provided a method of manufacturing a beverage in a beverage preparation machine, the method comprising: introducing the container described herein into a beverage preparation machine; and passing an aqueous medium through the container to produce a beverage.

[0067] The aqueous medium will typically be water, although another prepared beverage may also be used, such as from another container. The medium is heated during the process, and may be heated to a temperature of from 70 to 95°C.

[0068] The container is suitable for producing a beverage having a volume of 30 to 300 ml, preferably from 30- 250.

[0069] In some embodiments, the invention provides a blend of soluble coffee and a composition comprising plant cell culture grown in vitro, which may be in a form of a powder and may be provided in stick-packs or sachets. The plant cell culture grown in vitro are as described above.

[0070] In some embodiments, the plant cell culture powder has a particle size distribution of from 60 pm to 150 pm.

[0071] In some embodiments, the composition contains the soluble coffee and the composition of the plant cell grown in vitro in a ratio by weight of from 50:1 to 5:1 . In some embodiments the ratio by weight of the soluble coffee to the plant cell grown in vitro composition is from 30:1 to 10:1. According to some embodiments, the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 25:1 to 18:1. According to some embodiments, the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 22:1 to 20:1 .

[0072] In some embodiments, the coffee - plant cell culture composition of the invention enhances wakefulness, concentration, focus, and cognition; with smoother, more gradual onset of enhanced alertness at up to about 400-500 mg of caffeine per serving and 200-500 mg of RGC product. In some embodiments, the coffee - plant cell culture composition of the invention is cold brewed.

[0073] Further, the coffee - plant cell culture composition leads to reduced or eliminated side effects including those of nervousness, shakes, jitteriness, tremors, anxiety, restlessness, insomnia, elevated heart rate or clinically significant blood pressure elevation, urinary frequency, sudden mental fatigue and the like that may appear after drinking coffee without the addition of plant cell culture grown in vitro.

[0074] The surprising and unexpected novel compositions and methods discover a ‘brain metabolism’ optimized, nutrient dense, low glycemic energy blend when added to a blend of coffees becomes a highly potent selective inducer of alertness and mental focus allowing hours of imperceptible smooth onset sustained concentration and feeling of well-being without any of the “jolt”, shakiness, jitters, insomnia, brain fog, energy crash, transient psychosis, induced hypertension, tachycardia, arrhythmias, sugar cravings, or keto-acidotic emergencies associated with prior art caffeinated drinks, beverages, nootropics and or energy drinks.

[0075] In a human clinical trial VINIA® was found to significantly increase vascular function and dilation of arteries as was demonstrated by significant increasing of FMD after consuming 400mg daily over a 90-day period. This promotes the delivery of increased blood flow and oxygen to the body's tissue and organs supporting improved physical energy and mental alertness. Flow-mediated dilation (FMD) as an expression of endothelial dysfunction may serve as an indicator of several diseases.

[0076] Further, as shown in US patent No. 9,867,861 , vascular function was measured by FMD in six subjects that received 200 mg RGC, eight subjects that received 400 mg RGC, and in nine subjects who received a placebo. A statistically significant increase of 2.14±1 .82 mm Hg in FMD wasobserved in the RGC 400 mg group between baseline and the end of treatment (Table 17, p=0.013, therein). FMD was performed by inflating a blood pressure cuff on the upper arm to 200 mm Hg for 5 minutes and then measuring the artery's dilation by ultrasonography after releasing the cuff.

[0077] All eight subjects (100%) from the 400 mg treatment group experienced a positive relative change (>70% in FMD) compared to two out of six subjects (33.3%) from the 200 mg treatment group and two out of nine subjects (22.2%) who received a placebo (FIG. 11 ). The improvement rate observed in the RGC 400 mg group was statistically significantly different from that observed for both the 200 mg group and the placebo group (p=0.015 for 400 mg .vs. 200 mg and p=0.0023 for 400 mg .vs. placebo).

[0078] The results presented in US patent No. 9,867,861 indicate that a daily consumption of RGC powder improves FMD and also improves oxidative stress in mild hypertensive subjects. RGC may also decrease diastolic and systolic blood pressure in subjects with moderate hypertension who are not medically treated.

[0079] In some embodiments, the coffee - plant cell culture composition of the invention improves endothelial and vascular function and dilation of arteries.

[0080] In some embodiments, the coffee - plant cell culture composition of the invention the coffee - plant cell culture composition improves blood flow.

[0081] In some embodiments, the coffee - plant cell culture composition of the invention improves physical energy and mental alertness.

[0082] In some embodiments, the coffee - plant cell culture composition of the invention improves oxidation of blood lipids.

[0083] In some embodiments, the coffee - plant cell culture composition is antioxidative.ExamplesExample 1 :

[0084] Different formulations of roast coffee and RGC were prepared. Roasted coffee in three weights 9.5,10, 10.5 gr were mixed with the following RGC powder (Vinia®) dosages: 400 mg, 450 mg, 500 mg, and the mixture was inserted into pods.

[0085] Coffee beverages of 160 to 280 ml were prepared from these capsules using coffee machine (Keuring machine).

[0086] The coffee beverage was analyzed for piceid resveratrol content using HPLC. Piceid resveratrol peak was identified at 306 nm. Piceid resveratrol peak presented a typical absorbance fingerprint and could be detected above the coffee-polyphenols background.Results:Table 1 : precentage of piceid resveratrol recovery in different formulations of coffee and RGC:Conclusion:

[0087] The amount of the coffee and RGC powder as well as the ratio between them has a significant effect on the resveratrol recovery.

[0088] Since the aim is to provide at least 6 mg piceid resveratrol in the coffee beverages at least 400 to 500 mg of RGC powder should be used. The best formulation is achieved with 400-500 mg RGC and 10 gr coffeeExample 2:Decaffeinated Coffee with RGC Product

[0089] In this experiment, based on the results of example 1 , a formulation of roasted decaffeinated coffee and RGC has been prepared. Roasted decaffeinated coffee of 10 g was mixed with 500 mg RGC powder (Vinia®) and the mixture was inserted into a capsule (pods). Coffee beverages were prepared from these capsules using coffee machine (Keuring machine). The level of piceid resveratrol in the decaffeinated coffee beverage was measured using HPLC analysis.Results:Table 2: precentage of piceid_resveratrol recovery in the coffee beverage after the decaffeinated coffee production in pods containing 10 g decaffeinated coffee and 500 mg RGCConclusion:

[0090] As can be seen, the combination of 10 gr decaffeinated coffee and 500 mg RGC provided a good recovery of piceid resveratrol, and the production was very homogenous. Further, the stage ofthe production has no significant effect on the percentage of recovered piceid resveratrol in the decaffeinated coffee drink.Example 3:

[0091] Piceid resveratrol stability in the coffee beverage was evaluated 8 hours after the preparation. Roasted coffee (10 g) was mixed with 500 mg RGC powder (Vinia®) and the mixture was inserted into pods. Coffee beverages were prepared from these pods using coffee machine (Keuring machine). The level of piceid resveratrol in the coffee beverage was tested immediately after the preparation and after eight hours incubation at 25°C using HPLC analysis.Results:Table 3: precentage of piceid resvertrol recovery in the coffee drink after coffee production with 10 g coffee and 500 mg RGC immediately after production and after 8 hours.Conclusions:

[0092] As can be seen, precentage of piceid resvertrol recovery after 8 hours incubation of the coffee drink with RGC remained like the precentage of piceid resvertrol recovery immediately after the coffee production (decreased from 75.04% to 72.61%).Example 4Improving piceid resveratrol extraction from the brewing coffee drink using pods for Keuring machine

[0093] In this experiment, 10 gram of roasted coffee and 450 or 500 mg of RGC were prepared and the mixtures were inserted into pods. Coffee beverages of 280 ml were prepared from these capsules using coffee machine (Keuring machine).

[0094] The coffee beverage was analyzed for piceid resveratrol content using HPLC. In this experiment the extraction method for measuring the level of piceid resveratrol was improved by using methanol to the measuring extraction process.Table 4: Recovery of piceid resveratrol within the brewing coffee drink utilizing the improved extraction methodResults

[0095] As demonstrated in Table 4 improving the extraction method of the piceid resveratrol from the brewing coffee drink produced from Keuring machine pods increased the recovery of piceid resveratrol to 90-100% compared to the recovery without the extraction by methanol measuring process (Tables 1 ,2,3).Example 5:Product stability of the RGC powderThe stability of the RGC product was established under different conditions: o Freezer -20°C ± 5°C o Refrigerator 5°C ± 3°C o Incubator 25°C ± 2°C with relative humidity of 60% ±5% o Incubator 40°C ± 2°C with relative humidity of 75% ±5%The final product is packed under nitrogen conditions and protected from light.The stability results support 48 months of stability under RT conditions, 48 months (4 years) at 5°C ± 3°C, 48 months at -20°C ± 5°C and 6 months at 40°C. pH treatment

[0096] RGC powder was exposed to low pH (pH 2.5-3) for 24 hours. There was no significant effect on polyphenols composition as well as the amount of piceid resveratrol. It was concluded that low pH (pH 2.5-3) stabilizes polyphenols.Example 6:RGC-enriched coffee powder in bag

[0097] RGC powder was mixed with ground coffee powder (100pg / 5 g) and the mixed powder was inserted into sealed single-use bags (sachets). Preparation of coffee drink (100 ml of water / bag) was done either by mixing the powder with boiling water (mud coffee), or by boiling coffee and water until the boiling point and continuing boiling additional 30 seconds of boiling.Results:Table 5: precentage of piceid resvertrol recovery of coffee samples with RGC powder, in coffee drinks

[0098] The recovery of piceid resveratrol from RGC-enriched coffee after mixing thereofwith boiling water (mud coffee), or by boiling the RGC-enriched coffee and water until the boiling point and continuing boiling additional 30 seconds of boiling was very high (above 95%) and remained stable for at least 10 minutes from the coffee beverage preparation.Example 7:RGC enriched coffee in Nespresso pods

[0099] Resveratrol content in Nespresso pods prepared from coffee powder with RGC was tested as follows:A. Short and Lungo espresso capsules

[0100] 5.3 gr Robusta coffee was mixed with 250 mg RGC powder, and the mix was inserted intoNespresso capsules. The capsules were used to prepare a short espresso beverage of 35-38 ml or lungo espresso coffee beverage ~108 ml. Coffee beverages were prepared from these capsules using coffee machine (Nespresso machine). The piceid resveratrol recovery was analyzed from the beverages prepared also from coffee capsules sampled from the beginning, middle, and end of a production batch using HPLC analysis.Results:

[0101] Nespresso capsules containing a composition of 250 mg RGC and 5.3 gr Robusta coffee were used to prepare short and lungo coffee. As demonstrated in table 5, the recovery of piceid resveratrol was very high 99 -100%. In addition, the production was homogenous and consistent during the different stages of the production. Same recovery was received in the beginning, middle and end.Table 6: Piceid resveratrol recovery in short and lungo espresso beverage‘Average of three experimentsConclusion

[0102] As can be seen in Table 6, short and lungo espresso beverage that was prepared from the combination of 5.3 gr Robusta coffee and 250 mg RGC resulted in a very high recovery of piceid resveratrol and the production was very homogenous. The precentage of recovered piceid resveratrol at different stages of the production (beginning, middle and end) was identicalB. Lungo decaffeinated (decaf) espresso capsules

[0103] 5.6 gr decaf coffee was mixed with 250 mg RGC and the mix was inserted into Nespresso capsules. Decaf coffee pods are premium decaffeinated coffee products that use of chemical-free decaffei nation. This process is known to remove 99.9% of caffeine while preserving the coffee's flavor profile. The capsules were used to prepare lungo espresso coffee beverages —110 ml. Coffee beverages were prepared from these capsules using coffee machine (Nespresso machine). The piceid resveratrol recovery was analyzed also coffee capsules sampled from the beginning, middle, and end of a production batch using HPLC analysis.Table 7: Piceid resveratrol recovery of lungo decaf espresso beverage‘Average of three experimentsResults:

[0104] Nespresso capsules contain a composition of 250 mg RGC and 5.6 gr decaf coffee was used to prepare long coffee in Nespresso capsules. As demonstrated in table 6 the recovery of piceid resveratrol was very high 100%. In addition, the production of the decaf lungo espresso was homogenous and consistent during the different stages of the production and the same recovery was received in the beginning, middle and end.Conclusion

[0105] As can be seen in Table 7, lungo espresso beverage that was prepared from the combination of 5.6 gr Swiss water decaf coffee and 250 mg RGC demonstrated overall average recovery rate for Piceid Resveratrol indicates highly efficient extraction in a very high recovery of piceid resveratrol and the production was very homogenous as at different stages of the production (beginning, middle and end) has no effect on the % of recovered piceid resveratrol.Example 8Particle size and bulk density of RGC

[0106] Roasted coffee and decaffeinated coffee (10 g) were mixed with 500 mg RGC powder or ~5.3 gr coffee mixed with 250 mg RGC for coffee beverages production using Keuring or Nespresso machine at different particle size and bulk density and the mixture were inserted into capsules. The effect of different RGC powder at different particle size and bulk density on the recovery of piceid resveratrol and the capsule pressure was measured.Results:Table 8: Particle size and bulk density of RGCConclusions:Optimal bulk density of 0.26 gr / ml and particle size of 90 pm was essential to receive optimal piceid resveratrol recovery, homogenous coffee beverage and low pressure during the preparation of the coffee pods in the coffee machine.Example 9:

[0107] Taste testing of coffee (decaffeinated and caffeinated) mixed with RGC product and used in a coffee machine was conducted on 60 experimenters (each group).No effect on the organoleptic properties of the coffee, such as taste, and smell was observed by about 70% of the experimenters. Other reported that the taste is richer, great flavor, better mouthfeel and great smell.General conclusions:

[0108] As can be seen from the examples above, optimal bulk density of 0.26 gr / ml and particle size of 90 pm is essential to receive optimal piceid resveratrol recovery, homogenous coffee beverage and low pressure during the preparation of the coffee.

[0109] In addition, the ratio of coffee and RGC powder weights should be kept to ensure reliable and stable recoveries of piceid resveratrol. Achieving consistency and stability in piceid polyphenol recoveries is difficult especially when striving for optimal outcomes in coffee brewing scenarios.

Claims

What is claimed is:

1. A coffee - plant cell culture composition comprising a) roasted and ground coffee and b) a composition comprising plant cell culture grown in vitro, optionally in a form of a powder.

2. The coffee - plant cell culture composition of claim 1 , wherein the coffee - plant cell culture composition is in a pod or container.

3. The coffee - plant cell culture composition of claim 1 , wherein the coffee - plant cell culture composition is used in a coffee machine.

4. The coffee - plant cell culture composition of claim 1 , wherein plant cell culture grown in vitro is grape berry cell culture grown in vitro (RGC).

5. The coffee - plant cell culture composition of claim 1 , wherein the plant cell culture grown in vitro is olive cell culture grown in vitro (OOP).

6. The coffee - plant cell culture composition of claim 1 , wherein the plant cell culture grown in vitro is pomegranate cell culture grown in vitro.

7. The coffee - plant cell culture composition of any one of claims 1 -6, wherein the coffee includes caffeine.

8. The coffee - plant cell culture composition of any one of claims 1 -6 wherein the coffee is decaffeinated.

9. The coffee - plant cell culture composition of any one of claims 1 -8, wherein the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 50:1 to 5:1.

10. The coffee - plant cell culture composition of any one of claims 1 -9, wherein the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 30:1 to 10:1 .

11. The coffee - plant cell culture composition of any one of claims 1 -10, wherein the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 25:1 to 18:1 .

12. The coffee - plant cell culture composition of any one of claims 1 -11 , wherein the weight ratio between the coffee and the composition comprising plant cell culture grown in vitro is between about 22:1 to 20:1.

13. The coffee - plant cell culture composition of any one of claims 4 and 7-12, wherein the resulted composition after extraction comprises grape berry polyphenols and piceid resveratrol.

14. The coffee - plant cell culture composition of claim 13, wherein the resulted composition after extraction comprises resveratrol in an amount of between 1 -10 mg.

15. The coffee - plant cell culture composition of any one of claims 1 -14, wherein the particle size of the coffee is between 500-900 pm.

16. The coffee - plant cell culture composition of any one of claims 1 -15, wherein the particle size of the plant cell culture grown in vitro is between 60-150 pm.

17. The coffee - plant cell culture composition of any one of claims 1 -16, wherein the particle size of the plant cell culture grown in vitro is between 80-100 pm.

18. The coffee - plant cell culture composition of any one of claims 1 -17, wherein the particle size of the plant cell culture is homogenous.

19. A coffee - plant cell culture composition comprising a blend of soluble coffee and a composition comprising plant cell culture grown in vitro, optionally in a form of a powder.

20. The coffee - plant cell culture composition of claim 19, wherein plant cell culture grown in vitro is grape berry cell culture grown in vitro.21 . The coffee - plant cell culture composition of claim 19, wherein the plant cell culture grown in vitro is olive cell culture grown in vitro.

22. The coffee - plant cell culture composition of claim 19, wherein the plant cell culture grown in vitro is pomegranate cell culture grown in vitro.

23. The coffee - plant cell culture composition of any one of claims 19-22, wherein the coffee includes caffeine.

24. The coffee - plant cell culture composition of any one of claims 19-22, wherein the coffee is decaffeinated.

25. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition is cold brewed.

26. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition improves vascular function and dilation of arteries.

27. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition improves blood flow.

28. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition improves physical energy and mental alertness.

29. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition improves oxidation of blood lipids.

30. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the coffee- plant cell culture composition is antioxidative.31 . The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the plant cell culture grown in vitro contains less than 10%, 8%, 6%, 4%, 2% or less sugar.

32. The coffee - plant cell culture composition of any one of claims 1 -19 and 20-24, wherein the smell and taste of the coffee are maintained.