Methods of producing coffee drinks

Abstract

The present invention relates to methods of producing a coffee drink based on in vitro coffee plant cell biomass. The methods include cultivating an in vitro coffee plant cell biomass optionally in the presence of at least one microorganism, wherein the biomass comprises somatic embryos, as well as drying, roasting and brewing the biomass to create the coffee drink. Coffee drinks based on in vitro coffee plant cell biomass comprising somatic embryos are also provided.

Description

METHODS OF PRODUCING COFFEE DRINKSCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 710,939 filed on October 23, 2024, which is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to methods of producing a coffee drink based on in vitro coffee plant cell biomass. The methods include cultivating an in vitro coffee plant cell biomass, optionally in the presence of at least one microorganism, wherein the biomass may comprise somatic embryos, as well as drying, roasting and brewing the biomass to create the coffee drink. Coffee drinks based on in vitro coffee plant cell biomass comprising somatic embryos are also provided.BACKGROUND OF THE INVENTION

[0003] Coffee production faces several challenges, including economic, environmental, and social issues. Some of the problems associated with coffee production include child labor and exploitation, deforestation, use of pesticides and fertilizers in coffee cultivation, climate change, crop disease, and significant water usage. Coffee farm productivity is impacted by these issues.

[0004] Propagation of coffee plants has been traditionally carried out using rooted cuttings or by grafting, but these techniques require time and labor investments to obtain material with the desired genetic traits and health. In the search for solutions to this problem, plant cell culture has been investigated for the propagation and regeneration of coffee plants. Somatic embryogenesis (SE) is one of the techniques employed for the clonal propagation of coffee plants. It involves collecting somatic (non-reproductive) explant materials, such as staminodes or epicotyls, and placing them in culture medium to form somatic embryos. With the right combination of nutrients and growth regulators, these tissues can develop into plantlets in vitro.

[0005] Several articles describe the regeneration of plants using SE, including Avila- Victor, et al., Plants 2023, 12, 1237; Awada, et al., Int. J. Mol. Sci. 2019, 20, 4665; Boxtel, et al., Plant Cell, Tissue and Organ Culture 44: 7-17, 1996; Etienne, et al., 2013, Tree Physiology 33, 640-653; Etienne, et al., Front. Plant Sci., 2018, Volume 9.

[0006] In 1974, Townsley, et al., Can. Inst. Food Sc. Technol. J. 1974, 7 (1), 79-81, explored the feasibility of generating coffee cells in a laboratory, and first introduced the concept of substitutingtraditional green coffee beans with cultured coffee cells. In the Materials and Methods section, the article describes a coffee tissue culture process. Succulent new growth stem tissue sections were excised from Coffea arabica L. plants and used to initiate cell cultures. The tissue sections were incubated in the dark at 28°C to induce callus formation. Among the resulting calluses, the most vigorous and friable was selected for the preparation of single-cell suspension cultures. Solid media were prepared by adding 10 g / L of agar to these liquid media. The media are designed for callus induction, establishment, and maintenance; they are not designed for embryogenic differentiation. The cells generated are callus-derived, more precisely nondifferentiated, totipotent somatic cells; they are not embryogenic cells.

[0007] More recently, Aisala, et al., Journal of Agricultural and Food Chemistry 2023, 71 (47), 18478-18488, describes bioreactor-grown coffee cells that are dried and roasted to create coffee -like aroma and flavor, with chemical and sensory evaluations. The coffee cell culture described in this article involves callus culture and cell suspension culture. The media are designed for callus induction, establishment, and maintenance; they are not for designed for embryogenic differentiation. The coffee cell culture (CC) of Aisala, et al., is then compared with traditional coffee (TC) in the article of Khushvakov, et al., ACS Food Science & Technology 2024, 4 (8), 1890-1903 and cell-cultured coffees (CC), focusing on precursors in unroasted coffee, aroma in roasted coffee, and sensory profiles in brewed coffee. The findings reveal that unroasted CC contains higher monosaccharides, lower amino acids, and different organic acids, with low or absent chlorogenic acids, caffeine, and trigonelline. In roasted CC, there is a lower intensity of certain aroma compounds, but an abundance of furfurals, aldehydes, and hydrocarbons. In fact, the article demonstrates that coffee drinks made from cell-cultured material do not align with consumer and market expectations.

[0008] FI 130781 Bl (Teknologian tutkimuskeskus VTT Oy) describes a method for producing a cell-cultured ingestion product. The method comprises the steps of providing parts of a plant, pre-processing them to obtain a cell culture by cultivating the cells as a cell suspension in a nutrient medium, separating the cells from the medium and drying them to obtain a powder. The powder is then pressed into tablets and roasted. The document does disclose details of the cell culture medium and does not describe somatic embryos.

[0009] An object of the present invention is to provide coffee drinks based on cell-cultured coffee plant material that deliver a sensory experience and flavor profile comparable to traditional coffee.BRIEF STATEMENT OF THE INVENTION

[0010] The methods of the invention allow the production of coffee drinks using cell-cultured coffee plant material that offer a sensory and flavor profile similar to that of traditional coffee.

[0011] The present invention provides a method of producing an in vitro coffee plant cell biomass for preparing a coffee drink, the method comprising:a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, wherein the cultivation is optionally performed in the presence of at least one microorganism; b) drying the biomass from step a) at least partially; andc) optionally, roasting the biomass from step b).

[0012] The present invention also provides a method of producing a coffee drink based on in vitro coffee plant cell biomass, the method comprising:a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, wherein the cultivation is optionally performed in the presence of at least one microorganism; b) drying the biomass from step a) at least partially;c) roasting the biomass from step b); andd) brewing the biomass from step c) with water to create the coffee drink.

[0013] The present invention provides an in vitro coffee plant cell biomass comprising somatic embryos obtained from the methods of the invention.

[0014] In the invention, the above biomass materials are used as intermediates in coffee drinks product manufacture.

[0015] The present invention provides a coffee drink based on in vitro coffee plant cell biomass obtained from the method of the invention. The coffee drink may be selected among espresso, americano, cappuccino, latte, flat white, macchiato, cortado, mocha, affogato, cafe au lait, cold brew, iced coffee, nitro cold brew, Turkish coffee, Vietnamese coffee, cafe Cubano, frappe, and instant coffee.

[0016] The present invention also generally relates to the use of an in vitro coffee plant cell biomass comprising somatic embryos to produce a coffee drink.

[0017] In the present invention, the biomass may:- have a caffeine content of at least 2.0 mg / g, based on the total weight of the dry biomass, preferably at least 2.3 mg / g, at least 2.6 mg / g or at least 3.0 mg / g;- have an amino acid content of at least 4 wt.%, based on the total weight of the dry biomass, preferably at least 5 wt.% or at least 6 wt.%;- have a sucrose content of at least 2 wt.%, based on the total weight of the dry biomass, preferably at least 3 wt.%, at least 4 wt.% or at least 5 wt.%;- may produce at least one secondary metabolite among trigonelline and chlorogenic acids; preferably both of these secondary metabolites;- have a trigonelline content of at least 0.3 mg / g, based on the total weight of the dry biomass, preferably at least 0.32 mg / g, at least 0.35 mg / g or at least 0.5 mg / g;- have a chlorogenic acids content of at least 5.0 mg / g, based on the total weight of the dry biomass, preferably at least 6.0 mg / g, at least 7.0 mg / g or at least 9.0 mg / g; and / or- be characterized in that the content in Cd and / or Pb in the biomass is less than 10 ppb, less than 5 ppb, less than 1 ppb or even less than 0.5 ppb, based on the total weight of the dry biomass.DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention is based on the utilization of cell cultures for the production of agricultural commodities rather than production of farmed crops. The term is defined in Heiko Rischer, et al., “Cellular agriculture — industrial biotechnology for food and materials”, Current Opinion in Biotechnology, 61, 2020, Pages 128-134. The present invention aims at providing methods for the production of coffee drinks based on cellular biomass and extracts. In other words, the present invention is not based on cell cultures for plant propagation or regeneration.

[0019] The present invention generally relates to methods for producing a coffee drink based on in vitro coffee plant cell biomass, wherein such biomass comprises somatic embryos. In particular, these somatic embryos may be obtained by methods involving the in vitro multiplication, development and maturation of somatic embryos using direct somatic embryogenesis (DSE) and / or indirect somatic embryogenesis (ISE). The patent application also claims coffee drinks which can be obtained from such methods, as well as the use of an in vitro coffee plant cell biomass comprising somatic embryos to produce a coffee drink.

[0020] This invention specifically concerns methods for producing a coffee drink based on in vitro coffee plant cell biomass wherein the biomass presents a high-volume accumulation of caffeine suited for use in the food industry. Biomass and plant cell extract can be produced at industrial scale by methods involving somatic embryogenesis (SE). More specifically, an object of the present invention is to provide coffee drinks based on cell material produced in vitro, wherein the coffee drinks are comparable to coffee bean products, and which can be produced at an industrial scale in order to replace the use of coffee beans to produce coffee and coffee -like products. The present invention solves many of the issues associated with coffee production. The production processes of the present invention contribute to a more sustainable, controlled, and consistent coffee production process.

[0021] One key element of the method of the present invention is that the coffee cell biomass comprises, or consists essentially of, somatic embryos. In particular, these embryos preferably produce a high caffeine content. In fact, producing coffee drinks based on somatic embryos cell lines with a high caffeine content creates enhanced flavor body, and aroma development.

[0022] The somatic embryos of the present invention also produce a distinctive sugar profile that differs from that of callus plant material described in the prior art or traditional coffee. In parlicular. the somatic embryos of the invention have a high sucrose content, more precisely a sugar mixture comprisingglucose and sucrose, with a high sucrose content. Without being bound by theory, this is believed to contribute to a staged release that enhances the complexity in the flavor and aroma development. Glucose and fructose are the primary sugars driving the Maillard reaction, leading to the formation of pyrazines and Strecker aldehydes responsible for malty, nutty, and cocoa-like notes characteristic of coffee. Sucrose itself does not directly participate in the Maillard reaction; however, during roasting, it gradually hydrolyzes into glucose and fructose, thereby serving as a reservoir that feeds these reactions over time. The high sucrose content is thus advantageous for creating flavor and aroma, as it serves as a source of reducing sugars that promote Maillard reaction-driven development of flavor and aroma.

[0023] These embryos also preferably produce a high-amino acids (AAs) content. This is another key differentiator over callus plant material described in the prior art or traditional coffee. Higher levels of branched chain amino acids (valine, leucine, isoleucine) and phenylalanine have been observed, contributing to roasted, nutty, and coffee-like notes. Increases in sulfur-containing precursors such as methionine and cysteine also influence sulfur-impact odorants (e.g., methional and 2-furfurylthiol), which contribute to the characteristic fresh coffee or herbal aroma.

[0024] These embryos also produce a higher chlorogenic acids (CGAs) content, compared to callus plant material described in the prior art. In fact, producing coffee drinks based on somatic embryos cell lines with high CGAs content creates enhanced bitter taste and astringency, which is characteristic of coffee.

[0025] In fact, the present invention is concerned with producing coffee drinks based on somatic embryos biomass. For this purpose, the method of the present invention involves a biomass comprising somatic embryos. This biomass may notably be obtained through a process involving somatic embryogenesis. In some embodiments, the method of the present invention involves a biomass obtained through at least one step of somatic embryogenesis (SE).

[0026] More specifically, the present invention is based on the use of a biomass comprising somatic embryos, which can be produced at an industrial scale. According to the present invention, somatic embryos, or extracts from such somatic embryos, may be used in coffee drinks. Preferably, the somatic embryos biomass utilized in the process of the present invention produces an increased amount of caffeine as compared to immature embryos which can be extracted from coffee plant material.

[0027] As used herein, the terms “in vitro” or “in vitro somatic embryogenesis” mean a process constructed in a controlled environment, such as a petri dish, a bioreactor or other artificial conditions.

[0028] As used herein, the term “caffeine-rich” means containing at least 2.0 mg / g. For example, a caffeine -rich biomass or cell extract means respectively a biomass or a cell extract containing at least at least 2.0 mg / g of caffeine, based on the total weight of the biomass. The caffeine content of a material may be measured according to the method described in the article of Wale, et al., “Level of caffeine,trigonelline and chlorogenic acids in green coffee (Coffea arabica L.) beans from Amhara region, Ethiopia”, Journal of Agriculture and Food Research, Volume 16, 2024, 101082, on biomass samples which are preferably dried until their moisture content reaches 10 to 11 wt.%. In particular, High-Performance Liquid Chromatography with Variable Wavelength Detection (HPLC-VWD) may be used and involve separating compounds in biomass samples using HPLC chromatography and then detecting and quantifying them based on their absorbance at various wavelengths using a variable wavelength detector.

[0029] As used herein, the term “caffeine” typically refers to 1,3,7-trimethylxanthine, but also includes, in the context of the present invention, any caffeine derivatives, for example theobromine, theophylline, paraxanthine, and 7-methylxanthine.

[0030] As used herein, the term “sucrose -rich” means containing at least 4 wt.% of sucrose (or 4 % by weight). For example, a sucrose-rich biomass or sucrose -rich cell extract means respectively a biomass or a cell extract containing at least 4 wt.% of sucrose, based on the total weight of the dry biomass of the cell extract. The sucrose content of a material is measured on dry materials (e.g., dry intact biomass), i.e., by weighing the extracted sucrose after the sample has been dried using ASTM E1755-01(202). The sucrose content may also be measured on wet samples, in case the moisture content of the sample is known (e.g., moisture content of 50 wt.% or 80 wt.%).|0311 As used herein, the term “amino-acid-rich” or “AA-rich” means containing at least 4 wt.% of amino acids (or 4 % by weight). The amino acid profile comprises both essential and non-essential amino acids, including alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (altogether A As), reflecting a rich and balanced composition favorable for Maillard- type reactions during roasting. The amino acids in the coffee plant tissue may be analyzed using automated precolumn derivatization followed by high-performance liquid chromatography. Reference is made to Schuster, R. (1988). Determination of Amino Acids in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn Derivatization and HPLC. Journal of Chromatography, 431, 271-284. For example, an amino-acid-rich biomass or amino-acid-rich cell extract means respectively a biomass or a cell extract containing at least 4 wt.% of amino acids, based on the total weight of the dry biomass of the cell extract. The amino acid content of a material is measured on dry materials (e.g., dry intact biomass), i.e., by weighing the extracted amino acids after the sample has been dried. The amino acid content may also be measured on wet samples, in case the moisture content of the sample is known (e.g., moisture content of 50 wt.% or 80 wt.%).

[0032] As used herein, the term “chlorogcnic-acid-rich” or “CGA-rich” means containing at least 0.5 wt.% of chlorogenic acids (or 0.5 % by weight). The CGA profile includes 3-O-caffeoylquinic acid,4-0-caffeoylquinic acid, 5-0-caffeoylquinic acid, as well as feruloylquinic and dicaffeoylquinic acids. The chlorogenic acid (CGA) content includes mono- and di-caffeoylquinic acids and feruloylquinic acid derivatives. For example, a CGA-rich biomass or CGA-rich cell extract means respectively a biomass or a cell extract containing at least 0.5 wt.% of CGAs, based on the total weight of the dry biomass of the cell extract. The content of CGAs may be measured according to the method described in the article of Wale, et al., “Level of caffeine, trigonelline and chlorogenic acids in green coffee (Coffea arabica L.) beans from Amhara region, Ethiopia”, Journal of Agriculture and Food Research, Volume 16, 2024, 101082, on biomass samples which are preferably dried until their moisture content reaches about 10 to about 11 wt.%. In other terms, “CGA-rich” means having a chlorogenic acids content of at least 5.0 mg / g, based on the total weight of the dry biomass, preferably at least 6.0 mg / g, at least 7.0 mg / g or at least 9.0 mg / g.

[0033] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0034] According to one aspect, the present invention is directed to a method of producing an in vitro coffee plant cell biomass for preparing a coffee drink, the method comprising:a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, optionally cocultivating with at least one microorganism;b) drying the biomass from step a) at least partially; andc) optionally, roasting the biomass from step b).

[0035] According to another aspect, the present invention is directed to a method of producing a coffee drink based on in vitro coffee plant cell biomass, the method comprising:a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, optionally cocultivating with at least one microorganism;b) drying the biomass from step a) at least partially;c) roasting the biomass from step b); andd) brewing the biomass from step c) with water to create the coffee drink.

[0036] According to one aspect, the present invention is directed to a method of producing an in vitro coffee plant cell biomass for preparing a coffee drink, the method comprising:a) co-cultivating an in vitro coffee plant cell biomass with at least one microorganism;b) drying the biomass from step a) at least partially; andc) optionally, roasting the biomass from step b).

[0037] According to another aspect, the present invention is directed to a method of producing a coffee drink based on in vitro coffee plant cell biomass, the method comprising:a) co-cultivating an in vitro coffee plant cell biomass with at least one microorganism;b) drying the biomass from step a) at least partially;c) roasting the biomass from step b); andd) brewing the biomass from step c) with water to create the coffee drink.

[0038] By conducting the methods of the invention utilizing coffee plant cell biomass comprising SE and follow-up steps, it has been discovered that it is possible to produce coffee drinks that are comparable to traditional coffee bean drinks. The biomass may also be co-cultivated with at least one microorganism to lead to these results.

[0039] In these methods, step a) preferably consists in co-cultivating an in vitro coffee plant cell biomass with at least one microorganism, wherein the biomass comprises somatic embryos.

[0040] While the prior art generally describes bioreactor-grown coffee cells that are dried and roasted to create coffee -like aroma and flavor, with chemical and sensory evaluations, the present invention is based on utilizing somatic embryos cell materials that are produced in vitro for the production of coffee drinks. The biomass utilized in the methods of the present invention are cultivated to contain caffeine, lipids, chlorogenic acids and / or trigonelline, preferably a combination of all, and lead to coffee drinks made from cell-cultured material aligning with consumer and market expectations. The sensory experience and flavor profile of the coffee drinks of the present invention, based on cell-cultured coffee plant material, are comparable to traditional coffee.

[0041] The biomass may be obtained by a method using direct somatic embryogenesis (DSE), indirect somatic embryogenesis (ISE) or a combination of both. DSE involves the direct conversion of somatic cells into embryos without an intermediate callus stage. ISE requires the formation of callus tissue from somatic cells, which then differentiates into embryos.

[0042] The biomass cell material used herein preferably has a caffeine content of at least 2.0 mg / g, based on the total weight of the cell material. In some embodiments, the cell material of the present invention has a caffeine content of at least 2.3 mg / g, 2.6 mg / g or at least 3.0 mg / g, based on the total weight of the cell material.

[0043] The biomass cell material used herein preferably has a sucrose content of at least 2 wt.%, based on the total weight of the dry biomass of the cell extract. In some embodiments, the cell material of the present invention has a sucrose content of at least 3 wt.%, at least 4 wt.% or at least 5 wt.%, based on the total weight of the cell material.

[0044] The biomass cell material used herein preferably has an amino acid (AAs) content of at least 4 wt.%, based on the total weight of the dry biomass of the cell extract. In some embodiments, the cell material of the present invention has an amino acid content of at least 5 wt.%, at least 7 wt.% or at least 9 wt.%, based on the total weight of the cell material.

[0045] The biomass cell material used herein preferably has a lipid content of at least about 1 wt.%, based on the total weight of the dry cell material. As described above, the lipid content of a cell material may be determined on dry materials, i.e., by weighing the extracted lipids after the sample has been dried in case of biomass, for example using AOAC 922.06 standard protocol. The lipid content may also be measured on wet samples, for example in the case of wet biomass, in case the moisture content of the sample is known (e.g., moisture content of 50 wt.% or 80 wt.%). In some embodiments, the cell material of the present invention has a lipid content of at least about 1.2 wt.%, at least about 1.3 wt.%, or at least about 1.5 wt.%, based on the total weight of the dry cell material.

[0046] The lipid content of a material is measured on dry materials (dry intact biomass), i.e., by weighing the extracted lipids after the sample has been dried using AOAC 922.06 standard protocol. The lipid content may also, alternatively, be measured according to the method described in Oliva-Cruz M, et al., “Total Fat Content and Fatty Acid Profile of Fine-Aroma Cocoa From Northeastern Peru”. Front Nutr. 2021 Jul 5;8:677000. The lipid content may also be measured on wet samples, in case the moisture content of the sample is known (e.g., moisture content of 50 wt.% or 80 wt.%).

[0047] The biomass cell material used herein preferably produces at least one secondary metabolite among trigonelline and chlorogenic acids; preferably both secondary metabolites. In particular, the biomass cell material used herein preferably has a chlorogenic acid (CGAs) content of at least 0.5 wt.%, based on the total weight of the dry biomass of the cell extract. In some embodiments, the cell material of the present invention has a chlorogenic acid content of at least 1 wt.%, at least 2 wt.% or at least 5 wt.%, based on the total weight of the cell material. The content of these secondary metabolites may be measured according to the method described in the article of Wale, et al., “Level of caffeine, trigonelline and chlorogenic acids in green coffee (Coffea arabica L.) beans from Amhara region, Ethiopia”, Journal of Agriculture and Food Research, Volume 16, 2024, 101082, on biomass samples which are preferably dried until their moisture content reaches about 10 to about 11 wt.%.

[0048] Co-cultivation step a) (optional)

[0049] The methods of the present invention may comprise a step a) which consists of co-cultivating an in vitro coffee plant cell biomass with at least one microorganism. Step a) may be repeated several times as needed. For example, the method of the invention may advantageously comprise at least two successive steps of co-cultivation with one microorganism or two distinct microorganisms.

[0050] In some embodiments, the microorganism is selected from the group consisting of fungi and bacteria, in particular mold, yeast, lactic acid bacteria, acetic acid bacteria and mixtures thereof.

[0051] In some embodiments, the co-cultivation step a) consists in incubating the biomass in a liquid medium comprising the microorganism.

[0052] In some embodiments, the microorganism contains enzymes effective for the degradation of taste and aromatic precursor molecules.

[0053] In some embodiments, the method of the invention involves fermentation. The fermentation step of the present invention may be carried out using appropriate methods.

[0054] In some embodiments, the fermentation is selected from the group consisting of solid-state fermentation (SSF), wherein microorganisms grow on a moist solid substrate without free-flowing water; submerged fermentation (SmF), wherein cells are cultured in a liquid nutrient medium under controlled agitation and aeration; semi-solid or semi-submerged fermentation, i.e., intermediate between SSF and SmF, often used for molds requiring surface contact; surface fermentation, wherein microbial growth occurs on the surface of a liquid medium, with oxygen supplied at the interface; static fermentation, which is performed without agitation, allowing natural gradients of nutrients and oxygen to form; agitated or stirred fermentation, which is conducted under continuous mixing to maintain homogeneity and oxygen transfer; batch fermentation, wherein microorganisms are cultivated in a fixed volume of medium without additional nutrient feed after inoculation; fed-batch fermentation, wherein nutrients are added intermittently or continuously during cultivation to prolong growth and product formation; continuous fermentation, wherein fresh medium is supplied and culture broth is removed continuously to maintain cells in steady-state growth; co-culture or mixed-culture fermentation; which involves two or more microbial species acting synergistically to transform the substrate; sequential fermentation, with successive inoculation of different microorganisms in separate phases of the process; anaerobic fermentation, which is conducted in the absence of oxygen, often producing organic acids, alcohols, or flavor compounds; aerobic fermentation, which requires oxygen for growth and metabolism, typical of fungi and acetic acid bacteria; fermentation assisted by immobilized cells, wherein cells are entrapped or attached to carriers to improve stability and reusability; and enzyme-assisted fermentation, wherein enzymatic pretreatment or co-application to enhance substrate accessibility or flavor precursor release.

[0055] More precisely, the microorganism may comprise yeast, such as Saccharomyces spp., Candida spp., and / or Pichia spp., for example, Saccharomyces cerevisiae, Candida milleri, Candida krusei, Pichia kudriavzevii, and Pichia pastoris', molds, such as Aspergillus spp. and Rhizopus spp., for example, Aspergillus niger, Aspergillus oryzae (Koji mold) and Rhizopus stolonifer, bacteria, such as Bacillus spp.., for example, Bacillus subtilis and Bacillus pumilus; lactic acid bacteria, such as Leuconostoc spp., Weissella spp., Lactobacillus spp. and Bifidobacterium spp., for example, Leuconostoc mesenteroides, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus rhamnosus, and Bifidobacterium bifidum, acetic acid bacteria, such as Acetobacter spp. and Gluconacetobacter spp., for example, Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans and Gluconacetobacter xylinus', and other fungi used for enzyme production in food, such asTrichoderma spp., Penicillium spp., and Neurospora spp., for example, Trichoderma reesei, Penicillium roqueforti and Neurospora crassa.

[0056] The enzyme which may be used in the present invention may comprise at least one of a protease, a glycosidase, and / or a lipase. The enzyme used in the present invention may be specifically selected from the group consisting of proteases, glycosidases, lipases and combinations thereof.

[0057] Solid-state fermentation (SSF) may be used in the method of the invention. SSF refers to the cultivation of plant cells or cell-derived biomass on a moist solid substrate in the absence of free-flowing liquid. The solid material serves both as a support matrix and as a source of nutrients or structural components facilitating cell growth and metabolic activity. In particular, SSF may be applied to coffee cell biomass to promote the development of aroma and flavor precursors under controlled moisture and temperature conditions. This process can mimic certain aspects of post-harvest fermentation occurring in natural coffee processing, while offering improved control over environmental parameters and reproducibility. Suitable substrates include agricultural by-products (such as coffee pulp, husk, or bran) or inert supports (such as perlite or cellulose-based materials) that can maintain adequate humidity for cell metabolism. SSF offers several advantages, including low water and energy consumption, high product concentration, and the potential to generate complex flavor precursors such as amino acids, reducing sugars, and chlorogenic acid derivatives, contributing to a roasted coffee-like aroma profile upon subsequent thermal treatment.

[0058] Alternatively, submerged fermentation (SmF) may be used in the method of the invention. SmF refers to a fermentation process in which the microbial or enzymatic conversion takes place in a liquid medium containing the biomass or its extract in suspension. The microorganisms grow and metabolize in the aqueous phase, allowing efficient control of environmental parameters such as temperature, pH, aeration, and nutrient availability. SmF is particularly suitable for plant cell or coffee biomass fermentation, as it facilitates homogeneous contact between cells, enzymes, and soluble substrates, thereby promoting the formation of desirable flavor, aroma, and bioactive compounds.

[0059] Washing step (optional)

[0060] In some embodiments, the biomass obtained from step a) is rinsed and / or washed several times in water, for example sterile water. This step may take place before the drying step b), if any.

[0061] Drying step b) (at least partially)

[0062] The methods of the present invention comprise a step b) which consists of drying the biomass at least partially.

[0063] In some embodiments, step b) consists of drying the biomass up to a residual moisture content of less than about 50 wt.%, less than about 40 wt.%, less than about 30 wt.%, less than about 20 wt.%,less than about 10 wt.%, less than about 5 wt.%, less than about 1 wt.% or less than about 0.1 wt.%, based on the total weight of the biomass.

[0064] According to preferred embodiments, the biomass may be dried until the moisture content is less than about 10 wt.%, for example less than about 9 or about 8 wt.%, based on the total weight of the biomass.

[0065] In some embodiments, the step of drying b) is carried by using hot-air or heat drying at a temperature ranging from about 30 to about 100°C, preferably from about 50 and about 90°C, more preferably at about 75-85°C.

[0066] Roasting step c)

[0067] The methods of the present invention may comprise a step c) which consists of roasting the biomass from step b).

[0068] Roasting is typically conducted at high temperatures. Several types of reactions generally occur during roasting (i.e., Maillard reaction, Amadori rearrangement, and Strecker-type reactions), contributing to the complex mixture of compounds (including pyrazines, furans, pyrroles, aldehydes, ketones, thiophenes, phenols), which creates a unique flavor and aroma profile.

[0069] In some embodiments, the step of roasting c) is carried out using drum roasting, air roasting, tangential roasting, packed bed roasting, continuous roasting, batch roasting, infrared roasting, continuous belt roasting, centrifugal roasting or a com bi nation of these methods.

[0070] In some embodiments, the step of roasting c) is carried out at temperatures ranging from about 100°C to about 260 °C, preferably from about 120°C to about 200 °C.

[0071] The brewing method c) may comprise or be selected among drip, pour over, immersion, steeping, cold method, percolation method, espresso extraction, pressure methods, gas infused, vacuum extraction, spray drying, freeze drying, agglomeration, micro-grinding, concentrated drying, vacuum drying, drum drying or a combination of these methods.

[0072] Brewing step d)

[0073] In some embodiments, the.step of brewing d) is carried out using drip, pour over, immersion, steeping, cold method, percolation method, espresso extraction, pressure methods, gas infused, vacuum extraction, spray drying, freeze drying, agglomeration, micro-grinding, concentrated drying, vacuum drying, drum drying or a combination of these methods.

[0074] Production of the biomass comprising somatic embryos

[0075] The methods of the present invention may comprise a preliminary step of producing somatic embryos from coffee explant material by direct or indirect somatic embryogenesis (DSE or ISE). The methods of the invention may comprise several steps of somatic embryos production. For example, these steps of somatic embryos production may be as follows:i) producing somatic primary embryos from coffee explant material by direct or indirect somatic embryogenesis;ii) producing somatic secondary embryos from the primary somatic embryos of step i) by direct or indirect somatic embryogenesis; andiii) optionally repeating step ii) to produce somatic nth embryos.

[0076] In these embodiments, the variable “n” means an integer greater than 2 and the term “nth” refers to the corresponding generation. For example, when n = 4, “nth” denotes the fourth generation.

[0077] Step iii) may be implemented up to 20 times, up to 15 times, up to 10 times or up to 5 times. For example, step iii) is implemented 1, 2, 3, 4 or 5 times.

[0078] In some embodiments, step i) consists in inducing callus from coffee explant material on a first medium and developing primary somatic embryos on a second medium by indirect somatic embryogenesis (ISE),wherein the first medium comprises at least one plant growth regulator (PGR) or hormone and wherein the second medium comprises no PGRs and no hormones, or an amount of PGRs or hormones which is less than 1 wt.%, based on the total weight of the medium.

[0079] In some other embodiments, step i) consists in generating embryos from coffee explant material on a first medium and developing primary somatic embryos on a second medium by direct somatic embryogenesis (DSE),wherein the first medium comprises at least one plant growth regulator (PGR) or hormone and wherein the second medium comprises no PGRs and no hormones, or an amount of PGRs or hormones which is less than 1 wt.%, based on the total weight of the medium.

[0080] The appropriate media environment allows to maximize the production of caffeine (and lipids, trigonelline and / or chlorogenic acids in certain embodiments) by the biomass and obtain a cell product having a caffeine content comparable, or even higher, to coffee beans, with all the advantages associated.

[0081] In the methods of the present invention, the plant tissue culture media may comprise basal inorganic salts encompassing all essential elements necessary for cellular growth. Typical examples used for somatic embryogenesis include but are not limited to Murashige & Skoog (MS) salts, Driver and Kuniyuki Walnut (DKW) salts and / or Gamborg B-5 salts. Salts can be employed at their full-strength concentration or diluted to varying concentrations to achieve the desired results. In addition, organic molecules such as glycine, myo-inositol, nicotinic acid, pyridoxine, and thiamine, often referred to as “vitamins”, are often added. A fixed carbon source, usually sucrose, is added at concentrations typically between 1.5 % and 4 % to drive growth. Plant Growth Regulators (PGR) such as auxins, cytokinins, gibberellin, abscisic acid (ABA), jasmonic acid (JA), ethylene, and similar substances, can be added toachieve desired results. The media can be solidified using agar or other polymerizing compounds, or they can be utilized in a liquid state.

[0082] The culture media used in the method of the invention may include various media, for example induction media (or inducing media), development media (or developing media) and / or maturation media (maturing media). Some of these media may for example be used sequentially.

[0083] The compositions of the media are not limited herein. They can include salts, vitamins, growth hormones, and an energy source such as mannitol, fructose, glucose, saccharose or sucrose.

[0084] In the context of the present invention concerned with coffee somatic embryogenesis, “induction media” are designed to reprogram somatic cells, inducing their transition into a single-cell state or multicellular pro embryonic masses (PEMs) that are capable of following a developmental pattern that mimics the growth of zygotic embryo within a seed. Typically, induction media contain plant growth regulators (PGR) at a sufficient concentration to initiate cellular reprogramming and division of somatic cells within the explant tissue. In some embodiments, induction is accomplished with a single medium composition. In some other embodiments, tissues are incubated sequentially on two or more distinct media compositions to accomplish induction. It should be noted that these subsequent media types are sometimes referred to as “expression media”. In some embodiments, induction media do not support the growth and development of somatic embryos through all the normal morphological stages observed within zygotic embryos (including globular, heart, torpedo, cotyledon).

[0085] Specifically, induction media can include one or more plant growth regulators (PGR) or hormones, including, but not limited to, ethephon, kinetin, putrescine, spermidine, hydrogen peroxide, 6-(γ,γ-dimethylallylamino)purine (2iP), and gibberellic acid / gibberellin. The PGR or hormones may be present in concentrations varying form at least about 0.01 mg / L to less than about 10 mg / L, from example from about 0.05 mg / L to about 9 mg / L, from about 0.10 mg / L to about 8 mg / L, from about 0.20 mg / L to about 7 mg / L, or from 0.50 mg / L to about 3.0 mg / L.

[0086] In the context of the present invention, “development media” may be designed to promote and allow the growth and development of induced cells or PEMs through the normal morphological stages observed within a zygotic embryo. A combination of cell division, cell expansion, and cellular reprogramming leads to the formation of a cotyledon stage somatic embryo that is morphologically and functionally equivalent to a zygotic embryo with an apical meristem, cotyledons, body, and root meristem. Development media differ from induction media in that they contain no PGRs or no hormones, or low PGRs or hormones concentrations. Typically, development media contain no PGRs and no hormones, or an amount of PGRs or hormones which is less than about 1 wt.%, less than about 0.5 wt.%, less than about 0.1 wt.% or even less than about 0.01 wt.%, based on the total weight of the medium.

[0087] In the context of the present invention, “maturation media” may be designed to promote the accumulation of caffeine and / or lipids (e.g., triglycerides) and seed storage proteins within the somatic embryo cotyledon tissues. These compounds serve as nutrient and energy storage reserves to sustain early growth of the developing plant immediately after germination. In the present invention, maturation media may contain concentrations of osmoticum, e.g., sugar (such as sucrose, sorbitol, mannitol) or polymeric compounds such as polyethylene glycol (PEG), that increase the osmotic pressure outside the plant cells. This leads to cell dehydration as water leaves the somatic embryo cells through osmosis, cessation of cell division, and biosynthesis of seed storage proteins and lipids. Alternatively, or additionally, the hormone abscisic acid (ABA) which is naturally induced under high osmotic conditions, can be added to the maturation medium (for example in the absence of osmoticum or in combination with high osmotic pressure) to achieve the same result.

[0088] In some embodiments of the present invention, at least one of the media employed in the method comprises casein, glutathione, ascorbic acid, gibberellins, jasmonic acid, salicylic acid, or a derivative thereof or a derivative thereof. The term “derivative” refers to a chemically distinct compound that maintains the properties of the original substance, while differing in certain structural or functional groups.

[0089] In some embodiments, step i) consists in inducing callus from coffee explant material on a first medium (which may also be called callus induction medium) and developing primary somatic embryos on a second medium (which may also be called development medium) by indirect somatic embryogenesis (ISE), wherein the first medium comprises at least one plant growth regulator (PGR) or hormone and wherein the second medium comprises no PGRs and no hormones, or an amount of PGRs or hormones which is less than about 1 wt.%, less than about 0.5 wt.%, less than about 0.1 wt.% or even less than about 0.01 wt.%, based on the total weight of the medium. According to these embodiments, step a) may comprise the following sub steps:il) inducing primary embryos from the coffee explant material on the first medium for a period of time sufficient to obtain induced primary embryos;iil) transferring the induced primary embryos into the second medium and culturing the induced primary embryos with exposure to light for a period sufficient to produce primary somatic embryos biomass by indirect somatic embryogenesis.

[0090] In some other embodiments, step i) consists in generating embryos from coffee explant material on a first medium (which may also be called callus induction medium) and developing primary somatic embryos on a second medium (which may also be called development medium) by direct somatic embryogenesis (DSE), wherein the first medium comprises at least one plant growth regulator (PGR) or hormone and wherein the second medium comprises no PGRs and no hormones, or an amount of PGRsor hormones which is less than about 1 wt.%, less than about 0.5 wt.%, less than about 0.1 wt.% or even less than about 0.01 wt.%, based on the total weight of the medium.

[0091] The culture media used in steps i)-iii) may be the same or different.

[0092] According to the methods of the invention, step(s) i)-iii) is(are) independently performed with exposure to light, in the dark or a combination of both. Preferably, step(s) i)-iii) is(are) performed in the dark.

[0093] According to the invention, the somatic embryos utilized for preparing coffee drinks have preferably been cultured under conditions effective to produce caffeine, and lipids, trigonelline and / or chlorogenic acids in certain embodiments. Conditions effective to produce a plant cell biomass producing caffeine, and lipids, trigonelline and / or chlorogenic acids in certain embodiments, may preferably involve an osmoticum.

[0094] As used herein, the term “osmoticum” means a substance, combination of substances or solution that influences the movement of water across the plant cell membrane through osmosis, specifically in the context of creating an osmotic gradient. The osmoticum utilized in the present invention may encompass any substance or combination of substances recognized in the field for their capacity to elevate osmotic pressure in the plant cells and / or inhibit the uptake of liquids from the medium, leading to the dehydration / desiccation of the somatic embryos.

[0095] Suspension cell culture may be used in the method of the present invention, for example for growing the callus in any of step i) or for culturing the somatic embryos. Suspension cell culture refers to a method of culturing or growing cells in a liquid medium where the cells are freely suspended and not attached to a substrate (such as the bottom of a culture dish or flask). In contrast to adherent cell culture, where cells attach to a surface, suspension cell culture involves maintaining cells in a state where they float or are suspended in the culture medium. While the term “suspension culture” may be associated with the idea single undifferentiated cell or small aggregates of undifferentiated cells in suspension, this does not apply to the present invention, where the biomass is expected to multiply and grow, for example in the form of aggregates, as an organized tissue and / or as differentiated tissue.

[0096] The biomass may be cultured in a bioreactor. An overview of bioreactors applied to coffee somatic embryogenesis for plant regeneration is provided in Ducos, el al., “Bioreactors for coffee mass propagation by somatic embryogenesis.”, 2007, International Journal of Plant Developmental Biology 1(1), 1-12. The bioreactor used in the method of the present invention may for example be chosen among stainless steel stirred bioreactors, columns, a stirred tank reactor (STR), temporary immersion system (TIS), an air-lift bioreactor, an air-culture bioreactor, a wave bag bioreactor, a photo-bioreactor, a bubble column bioreactor, or a mist or fog bioreactor, i.e., in bioreactors of 1 to 1000 L, or even more, such as up to 106 L. In some embodiments, step e) is implemented in bioreactors with a volume capacity of at least1, 10, 25, 50, 75, 100, 500 or 1000 L. Step b) is preferably carried out in a TIS or in a mist or fog bioreactor.

[0097] The concentration of the caffeine, and lipids, trigonelline and / or chlorogenic acids in certain embodiments in the material may be monitored in order to ensure that the cell material meet the expected specifications, for example that the biomass has a caffeine content of at least 5 mg / g based on the total weight of the extract. For example, at an industrial scale, such monitoring may be performed automatically by a control system connected to a computer, in order for example to adjust the composition of the medium according to trajectory setpoints.

[0098] Although usable on a smaller scale, the methods of the invention are preferably implemented on an industrial scale for commercial production.

[0099] In vitro coffee plant cell biomass

[0100] The present invention is also directed to in vitro coffee plant cell biomass, preferably obtained from the method described herein, comprising SE. As demonstrated in the examples, the flavor and aroma profde of the coffee plant cell biomass of the present invention, comprising somatic embryos, is unique and differentiates from coffee plant cell biomass based on callus, which are nondifferentiated, totipotent somatic, non-embryogenic cells.

[0101] The cell biomass of the invention may notably be characterized by its volatile and nonvolatile compound profiles.

[0102] In some embodiments, the cell biomass of the invention may notably be characterized by its content in pyrazines. Pyrazines are critical for the roasty, nutty, and coffee-like aromas that define a high-quality coffee flavor.

[0103] The cell biomass of the invention may notably be characterized by its content in furfural, 5-methylfurfural, 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, ethylpyrazine and / or methylpyrazine, which are significantly higher than those of coffee plant cell biomass based on callus.

[0104] The cell biomass of the invention may also be characterized by its low content in ethylbenzene, nonanal, and / or heptanal. These compounds are considered undesirable, and their low levels contribute to avoiding off-flavors in the resulting product. In some embodiments, the cell biomass is characterized by having substantially no ethylbenzene, no nonanal, and / or no heptanal, or no detectable levels of these compounds (below detection limits in GC-MS).

[0105] The cell biomass of the invention may notably be characterized by its caffeine content, its amino acid content, its sucrose content and its chlorogenic acid content. In particular, the caffeine content of the biomass may be at least about 2.0 mg / g, based on the total weight of the dry biomass, for example at least about 3.0 mg / g or at least about 7.0 mg / g.

[0106] In some embodiments, the in vitro coffee plant cell biomass of the present invention comprises:- a caffeine content of at least about 2.0 mg / g, based on the total weight of the dry biomass, preferably at least about 2.3 mg / g or at least about 3.0 mg / g;- an amino acids content of at least about 4 wt.%, based on the total weight of the dry biomass, preferably at least about 5 wt.% or at least about 6 wt.%;- a sucrose content of at least about 2 wt.%, based on the total weight of the dry biomass, preferably at least about 3 wt.%, at least about 4 wt.% or at least about 5 wt.%; and / or- a chlorogenic acids content of at least about 5.0 mg / g, based on the total weight of the dry biomass, preferably at least about 6.0 mg / g or at least about 7.0 mg / g.

[0107] The soil heavy metal accumulation leads to farm products presenting undesirable heavy metal contents. The biomass of the present invention may be advantageously characterized by its low content in heavy metals, including Cadmium (Cd) and Lead (Pb). For example, the content in Cd and / or Pb in the biomass is less than about 10 ppb, less than about 5 ppb, less than about 1 ppb or even less than about 0.5 ppb, based on the total weight of the dry biomass. Cd and Pb contents may be measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). In the ICP-MS method, the prepared sample is aerosolized and introduced into a high-temperature plasma, where the atoms are ionized. The mass spectrometer then separates and quantifies the ions based on their mass-to-charge ratio. In the ICP-OES method, inductively coupled plasma is used to atomize the sample. The emitted light at characteristic wavelengths is then measured to determine the concentration of cadmium. Reference may be made to the article of Martin Rose, et al., “A Review of Analytical Methods for Lead, Cadmium, Mercury, Arsenic and Tin Determination Used in Proficiency Testing”, J. Anal. At. Spectrom., 2001,16, 1101-1106.

[0108] Preferably, the in vitro coffee plant cell biomass of the invention has a high caffeine content, a high sucrose content, a high amino acid content and a high chlorogenic acids content.

[0109] In some embodiments, the in vitro coffee plant cell biomass of the invention is characterized in that it comprises somatic embryos (SE).

[0110] The in vitro coffee plant cell biomass of the invention may for example be obtained by the methods described herein.

[0111] Coffee drinks and products

[0112] The present invention is also directed to a coffee drink or product based on an in vitro coffee plant cell biomass described herein. Such in vitro coffee plant cell biomass may be obtained from the method of the present invention.

[0113] In some embodiments, the coffee drink is selected among espresso, americano, cappuccino, latte, flat white, macchiato, cortado, mocha, affogato, cafe au lait, cold brew, iced coffee, nitro cold brew, Turkish coffee, Vietnamese coffee, cafe Cubano, frappe, and instant coffee. Instant coffee productionmethods include spray-drying, freeze-drying, agglomeration, centrifugal extraction, vacuum evaporation, high-pressure extraction, vacuum freeze concentration, fluidized bed drying, supercritical CO2extraction, microwave vacuum drying, foam-mat drying, or any hybrid method of any of these.

[0114] In some embodiments, the coffee drink or product further comprises proteins, lipids, sweeteners natural or artificial flavors, fibers, antioxidants, vitamins, minerals, stabilizers, thickeners, preservatives, colorants, plant-based milks (e.g., almond milk, oat milk, soy milk), plant extracts, probiotics, prebiotics, spices (e.g., cinnamon, nutmeg, cardamom), salt, acids (e.g., citric acid), caffeine, flavonoids, hydrocolloids, or mixtures thereof.

[0115] The coffee product may also comprise additional plant-derived products, such as coffee husks, grains, or pomace, or any fruit and vegetable by-products, which represent valuable agro-industrial byproducts rich in carbohydrates and amino acids. These additional plant-derived products may be used as substrate for fermentation, and / or they may be mixed with the dried biomass of the invention. Such byproducts may be rich in polyphenols, for example flavanol. They may be cleaned, dried, and milled to obtain a uniform particle size suitable for fermentation or mixing. Moisture content can be adjusted to optimize microbial growth, for example by rehydration or partial drying. In certain embodiments, they may also be sterilized or pasteurized to reduce the presence of undesired microorganisms prior to inoculation.10116 ] Use of an in vitro coffee plant cell biomass

[0117] The present invention is also directed to the use of plant cell biomass produced in vitro and comprising somatic embryos to produce a coffee drink.

[0118] In some embodiments, the biomass is obtained by a method comprising the production of somatic embryos from coffee explant material by direct or indirect somatic embryogenesis, the cultivation of the somatic embryos under conditions effective to produce a plant cell biomass producing caffeine, and optionally the separation of the plant cell biomass from the cell culture medium.

[0119] In some embodiments, the biomass is characterized in that the content in Cd and / or Pb in the biomass is less than about 10 ppb, less than about 5 ppb, less than about 1 ppb or even less than about 0.5 ppb, based on the total weight of the dry biomass.

[0120] EXAMPLES

[0121] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which arc encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0122] The flavor profiles of three coffee samples Peet's Major Dickason's dark ground coffee (Peet), callus coffee (callus), and SE coffee were evaluated through gas chromatography-mass spectrometry (GC-MS) for volatile compounds (Example 1) and compositional analysis for nonvolatile components (Example 2). The volatile data, expressed as peak areas and relative percentages against the maximum peak area for each compound, reveal differences in aroma profiles, while nonvolatile data highlight variations in amino acids, sugars, lipids, and other compounds critical to flavor development. This analysis explains why callus CC coffee produces a less flavorful cup compared to SE coffee, which closely resembles the superior flavor of Peet’s and traditional Arabica coffee.

[0123] Example 1 - Volatile composition

[0124] Materials and methods

[0125] Callus cell culture

[0126] To produce coffee cell biomass at laboratory scale, suspension cultures of Coffea arabica lines were selected for upscaling. Inoculum material was prepared from five to six shake flasks that had been grown under the same conditions described previously. From each culture, 10 mL of suspension were transferred into pre-weighed centrifuge tubes and spun for approximately ten minutes at 2800 x g. After decanting the supernatant, the packed cell volume (PCV) was recorded to determine inoculation density. Based on these values, cell material equivalent to roughly 40 ± 3 g per liter was used to seed a wave-type bioreactor system (Biostat RM, Sartorius, Germany) equipped with 10-liter disposable CultiBag RM vessels. Each cultivation was started with a working volume of about five liters, beginning at the minimum fill of one liter or more to allow for subsequent expansion.

[0127] Additional medium was introduced after approximately nine to twelve days when sugar depletion became evident. The rocking platform was set to oscillate at 20 rpm at the stall and gradually increased to about 22 rpm after three to four days of cultivation. The rocking angle was maintained at eight degrees for one-liter cultures and increased to ten degrees once the volume was expanded. The laboratory temperature fluctuated naturally between 24 and 25 °C, so active temperature control was unnecessary; aeration was supplied continuously at about 300 mL per minute.

[0128] Each line was grown through four consecutive batches to yield the targeted dry biomass of around 50 grams per culture. Under these conditions, total batch times ranged from roughly eight to twenty-five days depending on growth behavior and medium supplementation. While the disposable wave system successfully produced the desired amount of biomass, the relatively low shear environment favored aggregate formation within the cultures. Because this setup offers limited control and monitoring compared with stirred-tank systems, some variation in growth was observed between batches particularly when the inoculum originated directly from shake-flask cultures rather than being pre-adapted to the rocking bioreactor conditions.

[0129] Somatic embryos culture

[0130] To maintain suspension cultures, cells were periodically subcultured to isolate and enrich aggregates ranging from roughly 250 to 1000 micrometers in diameter. Before each subculture, total wet mass and soluble solids (°Brix) could be recorded for reference. The culture was first passed through a sterile 1000 μm mesh that had been attached to a 50 mL centrifuge tube containing CP medium. The liquid passing through the mesh was collected as the working fraction, while the larger material was either discarded or, in some cases, retained as inoculum for new flasks. Aggregates exceeding one millimeter often contained dense, white-to-yellow rounded structures typical of embryogenic material, serving as a useful visual cue for competence.

[0131] The collected filtrate was then screened through a sterile 250 μm mesh, and the retained material in that size range was used for subsequent culture. In an alternate approach, rather than using mesh filters, the suspension was drawn into a 10 mL closed-tip pipette (approximately 1 mm tip opening). Gentle agitation and a brief settling period allowed heavier cell masses to separate at the bottom of the vessel, from which denser aggregates were collected.

[0132] The harvested fraction was adjusted to a density of around 5 grams per liter, equivalent to approximately 50 milligrams of wet cell mass per 10 milliliters of medium. Smaller flasks of 125 mL were used for volumes under 60 mL, while 250 mL flasks were chosen for larger volumes. For more rapid growth, inoculation densities between 8 and 10 g / L were used. Cultures were maintained at about 26 °C on a rotary shaker set to 100 rpm in complete darkness. Media were refreshed and cell density was rebalanced roughly every two weeks.

[0133] For regeneration, cell fractions within the 250-1000 μm range were transferred to R medium. Depending on the cell line, aggregates exceeding 1000 μm sometimes also produced embryos and could be cultured under similar conditions. In liquid form, regeneration cultures were started at a density close to 1 g / L (about 50 mg cell mass in 50-100 mL of medium) using 250 mL flasks; when larger vessels were needed, 500 mL flasks were used with up to 200 mL of medium. After about 30 days, the medium was replaced with fresh R medium. After 60 days, total biomass and °Brix were measured and recorded.

[0134] For regeneration on solid medium, approximately 1 mL of the liquid suspension was distributed evenly onto 60 x 15 mm petri dishes containing solidified R medium. Any excess liquid was removed to ensure even contact. Each plate was labeled with the date and flask identification. Plates were then incubated under the same temperature and lighting conditions, and cultures were evaluated for embryo development after roughly three to four weeks.

[0135] Unroasted coffee somatic embryo biomass (SE) was harvested from Coffea arabica cell culture and dried in an Excalibur 6-tray food dehydrator (Excalibur Dehydrator Corporation, Sacramento, CA,USA) at 40.6 °C (105 °F) for 4 hours to achieve dry weight basis for compositional analysis. The dried biomass was then submitted for analysis.

[0136] Results

[0137] The GC-MS analysis identified a range of volatile compounds, including furans, pyrazines, aldehydes, and other aroma-active molecules, each contributing to the sensory profile of the coffee samples. The dataset includes compounds with sensory descriptors such as caramel / toasty (e.g., furfural, 5-methylfurfural, 2-furanmethanol), roasty / nutty / coffee (e.g., 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, ethylpyrazine), almond (e.g., hexanal, benzaldehyde), fresh-roast (e.g., furfuryl methyl sulfide, dimethyl trisulfide), buttery (e.g., 2,3-pentanedione), and smoky / woody (e.g., phenol). Relative peak areas (%) were calculated for each compound by dividing the component area in each sample by the maximum component area observed across the three samples, providing a normalized comparison of compound abundance.Table 1Table 2

[0138] Callus coffee: a reduced flavor profile

[0139] Callus coffee exhibits a distinct volatile profile that suggests a less flavorful cup compared to Peet's and SE coffee. This is primarily due to its high levels of certain aldehydes and furans, coupled with a lower abundance of pyrazine compounds, which are critical for the characteristic roasty, nutty coffee aroma. Specifically, callus coffee shows elevated levels of:Ethylbenzene (100% relative peak area, compared to 0% in Peet and SE): Ethylbenzene contributes a harsh, solvent-like aroma, which can impart an undesirable chemical note to the coffee;Hexanal (100% relative peak area, compared to 0% in Peet and 36.31% in SE): Hexanal is associated with a green, grassy odor, which may detract from the rich, roasted coffee character;Nonanal (100% relative peak area, compared to 0% in Peet and SE): Nonanal imparts a waxy, soapy note, further reducing the appeal of the aroma;Heptanal (100% relative peak area, compared to 0% in Peet and SE): Heptanal has a fatty, rancid odor, which can negatively impact the sensory experience; and2-Pentylfuran (100% relative peak area, compared to 0% in Peet and 62.13% in SE): While 2-Pentylfuran contributes a buttery or green note, its dominance in callus coffee may overshadow more desirable roasted aromas.

[0140] These compounds, often associated with lipid oxidation or off-flavors, dominate the volatile profile of callus coffee, suggesting a less balanced and less appealing sensory experience. Additionally, callus coffee shows significantly lower levels of pyrazines, such as 2,5-dimethylpyrazine (20.35%> vs. 100% in Peet and 46.86% in SE), ethylpyrazine (3.17% vs. 100% in Peet and 20.92% in SE), and methylpyrazine (7.56% vs. 100% in Peet and 23.95% in SE). Pyrazines are critical for the roasty, nutty, and coffee -like aromas that define a high-quality coffee flavor. Their reduced presence in callus coffee likely results from lower amino acid content in the raw beans or processing conditions that limit Maillard reaction products, which are key precursors to pyrazine formation during roasting. The Maillard reaction, a chemical interaction between amino acids and reducing sugars, is responsible for generating these desirable aroma compounds, and a deficiency in amino acids would suppress pyrazine production, leading to a less robust flavor profile.

[0141] SE coffee: superior flavor and similarity to Peet’s coffee

[0142] SE coffee demonstrates a volatile profile more akin to Peet’s Major Dickason's dark ground coffee, suggesting a superior flavor quality compared to callus coffee. SE coffee contains higher levels of key aromatic compounds that align closely with Peet’s, particularly those associated with roasty, nutty, and caramel / toasty notes. Notable similarities include:Furfural (100% in SE vs. 40.33% in Peet, 2.67% in callus): This caramel / toasty compound is significantly more abundant in SE, contributing to a sweet, toasted aroma;5-Methylfurfural (100% in SE vs. 20.39% in Peet, 2.50% in callus): Another caramel / toasty compound, its high abundance enhances the rich, sweet notes in SE;2,5-Dimethylpyrazine (46.86% in SE vs. 100% in Peet, 20.35% in callus): This roasty / nutty compound is present at substantial levels, supporting a robust coffee flavor;2-Ethyl-3-methylpyrazine (100% in SE vs. 0% in Peet's and callus): Exclusive to SE, this pyrazine adds a potent roasty / nutty character, enhancing flavor complexity;Ethylpyrazine (20.92% in SE vs. 100% in Peet, 3.17% in callus): A key roasty compound, its presence in CC SE supports a strong coffee aroma; andMethylpyrazine (23.95% in SE vs. 100% in Peet, 7.56% in callus): Another pyrazine contributing to the nutty, roasted profile, present at higher levels than in callus.

[0143] The elevated levels of pyrazines in SE coffee suggest a higher amino acid content in the raw beans or optimized roasting conditions that promote Maillard reaction pathways, leading to greater pyrazine formation. This results in a flavor profile that closely mirrors the rich, complex aroma of Peet's, characterized by roasty, nutty, and caramel notes. Additionally, SE coffee has lower levels of undesirable compounds like ethylbenzene (0%), nonanal (0%), and heptanal (0%), avoiding the off-flavors that dominate callus coffee. While SE has a moderate level of hexanal (36.31% vs. 100% in callus), its impact is likely mitigated by the abundance of positive aroma compounds.

[0144] Example 2 - Nonvolatile composition

[0145] Nonvolatile components, including amino acids, sugars, lipids, organic acids, and alkaloids, were quantified as percentages of dry weight (% DW) and compared to traditional Arabica coffee (Table 3). These compounds influence flavor through their roles as precursors in the Maillard reaction, caramelization, and other roasting processes (Flament, I. (2002) Coffee Flavor Chemistry. John Wiley & Sons Ltd, Chichester). Key differences in amino acid and sugar content between CC SE and CC Callus Coffee provide insights into their flavor disparities.

[0146] Materials and methods10147 ] Same as in Example 1.

[0148] Results

[0149] The nonvolatile composition of the somatic embryo biomass (SE) of the present invention was compared with the benchmark unroasted callus derived cell cultured biomass reported in Khushvakov, et al., ACS Food Science & Technology 20244 (8), 1890-1903. The results are reported in Table 1.Table 3 - Nonvolatile composition (% DW): SE vs callus vs traditional arabica

[0150] Amino acids, sugars, lipids, organic acids, and alkaloids influence flavor through their roles as precursors in the Maillard reaction, caramelization, and other roasting processes (Flament, 2002). Key differences in amino acid and sugar content between SE and callus coffee provide insights into their flavor disparities.

[0151] The SE biomass of the present invention shows several shifts in key precursor free amino acids and sucrose that contribute to increassing formation of desirable roast aroma compounds (Strecker aldehydes and alkylpyrazines). The callus shows very low amino acids and atypically highmonosaccharides. Overall, the SE line shows a precursor profile that should translate into a fuller, more coffeelike aroma after roasting compared with callus derived material.

[0152] The callus material reports a very small pool of free amino acids (about 0.07% DW) with multiple amino acids (AAs) at or below detection. The SE line of the invention in contrast presents elevated contents in total protein and several free amino acids, in particular, high levels of branched chain amino acids (valine, leucine, isoleucine) and phenylalanine, which contribute to increased formation of impact Strecker aldehydes 2-methylpropanal, 3 -methylbutanal, and 2-methylbutanal (malty / cocoa) and phenylacetaldehyde (honey / floral). This contributes to the roasted, nutty, and coffee notes that are often muted in callus coffee.

[0153] Regarding sulfur containing precursors, the increases in methionine and cysteine materially affect sulfur impact odorants (methional, 2-furfurylthiol) that contribute to the recognizable fresh coffee or herbal character. The callus material shows largely nondetectable sulfur amino acids. Any measurable SE levels here are likely advantageous.

[0154] Regarding sugars, callus shows an atypically high fraction of free glucose / fructose and almost no sucrose. That profile biases roasting toward furfurals and caramel aromas. In coffee roasting, the balance between sucrose and free sugars is critical. Glucose and fructose are the actual drivers of the Maillard reaction, producing the pyrazines and Strecker aldehydes that give coffee its malty, nutty, and cocoa notes. Sucrose itself does not react directly, but during roasting it steadily breaks down into glucose and fructose, acting like a reservoir that feeds these reactions over time. This staged release helps build complexity and keeps the roast from tipping too far into simple caramel or burnt flavors. By contrast, when glucose and fructose are present in very high amounts from the start, as in callus, the chemistry is front-loaded, pushing more furfural formation and leaving less room for pyrazine development. The SE line, with its higher sucrose and more balanced monosaccharides, is characterized in that it roasts closer to traditional beans, supporting the complex flavor backbone that callus coffee tends to miss.

[0155] Caffeine levels also differentiate the SE material from callus in a meaningful way. Beyond its role as a stimulant, caffeine contributes to the taste profile by reinforcing bitterness and balancing sweetness and acidity. The callus samples analyzed in the article carried only a fraction of the caffeine normally found in Arabica, which reduces both the functional impact and the sensory balance of the brew. In contrast, the SE line of the invention contains caffeine at levels consistent with conventional coffee, restoring the characteristic bite and physiological effect that consumers expect. This is a key factor in producing a product that not only resembles coffee in composition but also delivers a recognizably authentic drinking experience.

[0156] Chlorogenic acids (CGAs) are one of the dominant phenolic groups in green coffee and play a central role in shaping the quality of the final cup. They act as important precursors during roasting, firstforming chlorogenic acid lactones that contribute a pleasant, mild bitterness in light and medium roasts, and then breaking down further into phenylindanes that bring a stronger, more intense bitterness in darker roasts (Liu, etal., 2016; Ludwig, et al., 2014). This stepwise transformation helps explain why coffees with sufficient CGAs tend to taste more structured and complex, while those with very low CGA content can taste thin or flat. Beyond serving as flavor precursors, chlorogenic acids also influence the balance of acidity and astringency in the brew, giving coffee its characteristic brightness and bite (Angeloni, et al., 2018). In addition, the ratio of CGAs to caffeine has been used in quality studies as a marker for both bean type and roast degree, reflecting how these compounds degrade differently under heat (Caprioli, et al., 2015). Taken together, elevated CGA levels in our somatic embryo line mean that the material has the potential to yield a cup with greater aromatic depth, balanced bitterness, and a sensory structure much closer to that of high-quality Arabica beans, whereas callus coffee, with its very low CGAs, lacks this critical foundation.

[0157] Khushvakov, J.; Opitz, S. E. W.; Plüss, N.; Sun, J.; Manthey, L. J.; Rischer, H.; Yeretzian, C. Analytical Platform to Determine Similarities and Dissimilarities between Cell-Cultured Coffee and Farm-Grown Coffee. ACS Food Sci. Technol. 2024, 4 (10), 1890-1903.

[0158] Frank, O.; Blumberg, S.; Kunert, C.; Hofmann, T. Structure Determination of 4- Vinylcatechol Oligomers Formed by Oxidative Coupling of 4- Vinylcatechol and Identification of New Bitter Compounds Formed in Roasted Coffee. J. Agric. Food Chem. 2007, 55 (5), 1945-1954.

[0159] Liu, C.; Wang, Y.; Wang, Z.; Chen, L.; Wang, S. Effect of Chlorogenic Acids on Bitterness and Astringency in Coffee. Food Chem. 2016, 207, 339-345.https: / / pubmed.ncbi.nlm.nih.gov / 27719962 / .

[0160] Ludwig, I. A.; Clifford, M. N.; Lean, M. E. J.; Ashihara, H.; Crozier, A. Coffee: Biochemistry and Potential Impact on Health. Food Funct. 2014, 5 (8), 1695-1717.https: / / www.science.org / content / article / coffees-bitter-mystery

[0161] Caprioli, G.; Cortese, M.; Maggi, F.; Minnetti, C.; Odello, L.; Sagratini, G.; Vittori, S.Quantification of Caffeine, Chlorogenic Acids, and Trigonelline in Coffee: A Comparison of Different Methods of Analysis. Molecules 2015, 20 (10), 18001-18012. https: / / doi.org / 10.3390 / molecules201018001 (PMC6526205).

[0162] Angeloni, G.; Guerrini, L.; Masella, P.; Innocenti, M.; Bellumori, M.; Parenti, A. What Kind of Coffee Do You Drink? An Investigation on Effects of Eight Different Extraction Methods. Food Res. Int.2018, 116, 1327-1335. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC10867520 / .

[0163] Van Boxtel, Jos, and Marc Berthouly. "High frequency somatic embryogenesis from coffee leaves: factors influencing embryogenesis, and subsequent proliferation and regeneration in liquid medium." Plant Cell, Tissue and Organ Culture 44 (1996): 7-17.

Claims

1. WHAT IS CLAIMED IS:

1. A method of producing an in vitro coffee plant cell biomass for preparing a coffee drink, the method comprising:3.a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, wherein the cultivation is optionally performed in the presence of at least one microorganism; b) drying the biomass from step a) at least partially; and4.c) optionally, roasting the biomass from step b).

2. A method of producing a coffee drink based on in vitro coffee plant cell biomass, the method comprising:6.a) cultivating an in vitro coffee plant cell biomass comprising somatic embryos, wherein the cultivation is optionally performed in the presence of at least one microorganism;7.b) drying the biomass from step a) at least partially;8.c) roasting the biomass from step b); and9.d) brewing the biomass from step c) with water to create the coffee drink.

3. The method of claims 1 or 2, wherein the cultivation step a) consists in co-cultivating the in vitro coffee plant cell biomass in the presence of at least one microorganism, wherein the microorganism is selected from the group consisting of mold, yeast, lactic acid bacteria, acetic acid bacteria and mixtures thereof.

4. I’he method of any one of claims 1-3, wherein the cultivation step a) consists in incubating the biomass in a liquid medium comprising the microorganism.

5. The method of any one of claims 1-4, wherein the microorganism contains enzymes effective for the degradation of taste and aromatic precursor molecules.

6. The method of any one of claims 1-5, wherein the drying step b) is carried out using hot-air or heat drying, vacuum drying, drum drying, rotary drying, freeze-drying, microwave drying or a combination thereof.

7. The method of any one of claims 1-6, wherein the drying step b) is carried by using hot-air or heat drying at a temperature ranging from about 30 to about 100°C, preferably from about 50 and about 90°C, more preferably at about 75-85°C.

8. The method of any one of claims 1-7, wherein the roasting step c) is carried out using drum roasting, air roasting, tangential roasting, packed bed roasting, continuous roasting, batch roasting, infrared roasting, continuous belt roasting, centrifugal roasting or a combination of these methods.

9. The method of any one of claims 2-8, wherein the brewing step d) is carried out using drip, pour over, immersion, steeping, cold method, percolation method, espresso extraction, pressure methods, gas infused, vacuum extraction, spray drying, freeze drying, agglomeration, microgrinding, concentrated drying, vacuum drying, drum drying or a combination of these methods.

10. The method of any one of claims 1-9, wherein the biomass has a caffeine content of at least about 2.0 mg / g, based on the total weight of the dry biomass, preferably at least about 3.0 mg / g or at least about 7.0 mg / g.

11. The method of any one of claims 1-10, wherein the biomass has an amino acid content of at least 4 wt.%, based on the total weight of the dry biomass, preferably at least about 5 wt.% or at least about 6 wt.%.

12. The method of any one of claims 1-11, wherein the biomass produces at least one secondary metabolite among trigonelline and chlorogenic acids; preferably both of these secondary metabolites.

13. The method of any one of claims 1-12, wherein the biomass has a sucrose content of at least 0.03 wt.%, based on the total weight of the dry biomass, preferably at least 0.5 wt.%, at least 1 wt.% or at least 5 wt.%.

14. The method of any one of claims 1-13, wherein the biomass has a chlorogenic acids content of at least about 7.0 mg / g, based on the total weight of the dry biomass, preferably at least about 8.0 mg / g or at least about 9.0 mg / g.

15. The method of any one of claims 1-14, wherein the biomass is obtained by a method comprising the production of somatic embryos from coffee explant material by direct or indirect somatic embryogenesis, the cultivation of the somatic embryos under conditions effective to produce a plant cell biomass producing caffeine, and optionally the separation of the plant cell biomass from the cell culture medium.

16. The method of any one of claims 1-15, wherein the biomass is characterized in that the content in Cd and / or Pb in the biomass is less than about 10 ppb, less than about 5 ppb, less than about 1 ppb or even less than about 0.5 ppb, based on the total weight of the dry biomass.

17. An in vitro coffee plant cell biomass comprising somatic embryos obtained from the method of any one of claims 1, 3-16.

18. The biomass of claim 17, comprising:25.- a caffeine content of at least about 2.0 mg / g, based on the total weight of the dry biomass, preferably at least about 3.0 mg / g or at least about 7.0 mg / g;26.- an amino acid content of at least about 4 wt.%, based on the total weight of the dry biomass, preferably at least about 5 wt.% or at least about 6 wt.%; - a sucrose content of at least 2 wt.%, based on the total weight of the dry biomass, preferably at least 3 wt.%, at least 4 wt.% or at least 5 wt.%;27.- a chlorogenic acid content of at least about 7.0 mg / g, based on the total weight of the dry biomass, preferably at least about 8.0 mg / g or at least about 9.0 mg / g;28.- a trigonelline content of at least 0.3 mg / g, based on the total weight of the dry biomass, preferably at least 0.32 mg / g, at least 0.35 mg / g or at least 0.5 mg / g; and / or29.- a content in Cd and / or Pb of less than 10 ppb, less than 5 ppb, less than 1 ppb or even less than 0.5 ppb, based on the total weight of the dry biomass.

19. The biomass of any one of claims 17-18, wherein the biomass is obtained by a method comprising the production of somatic embryos from coffee explant material by direct or indirect somatic embryogenesis, the cultivation of the somatic embryos under conditions effective to produce a plant cell biomass producing caffeine, and optionally the separation of the plant cell biomass from the cell culture medium.

20. The biomass of any one of claims 17-19, wherein the biomass is characterized in that the content in Cd and / or Pb in the biomass is less than about 10 ppb, less than about 5 ppb, less than about 1 ppb or even less than about 0.5 ppb, based on the total weight of the dry biomass.

21. A coffee drink or coffee product based on in vitro coffee plant cell biomass obtained from the method of any one of claims 2-16.

22. A coffee drink or coffee product based on in vitro coffee plant cell biomass of any one of claims 17-20.

23. The drink of any one of claims 21-22, being selected among espresso, americano, cappuccino, latte, flat white, macchiato, cortado, mocha, affogato, cafe au lait, cold brew, iced coffee, nitro cold brew, Turkish coffee, Vietnamese coffee, cafe Cubano, frappe, and instant coffee.

24. The drink or product of any one of claims 21-23, further comprising proteins, lipids, sweeteners natural or artificial flavors, fibers, antioxidants, vitamins, minerals, stabilizers, thickeners, preservatives, colorants, plant-based milks (e.g., almond milk, oat milk, soy milk), plant extracts, probiotics, prebiotics, spices (e.g., cinnamon, nutmeg, cardamom), salt, acids (e.g., citric acid), caffeine, flavonoids, hydrocolloids, or mixtures thereof.

25. The product of claim 22 or 24, comprising a plant-derived product, preferably a plant-derived product selected among coffee husks, grains, pomace, or any fruit and vegetable by-products.

26. Use of an in vitro coffee plant cell biomass comprising somatic embryos to produce a coffee drink.

27. The use of claim 26, wherein the biomass is characterized in that it comprises:- a caffeine content of at least about 2.0 mg / g, based on the total weight of the dry biomass, preferably at least about 2.3 mg / g, at least about 2.6 mg / g or at least about 3.0 mg / g;38.- an amino acid content of at least about 4 wt.%, based on the total weight of the dry biomass, preferably at least about 5 wt.% or at least about 6 wt.%;39.- a sucrose content of at least 2 wt.%, based on the total weight of the dry biomass, preferably at least 3 wt.%, at least 4 wt.% or at least 5 wt.%;40.- a chlorogenic acid content of at least about 7.0 mg / g, based on the total weight of the dry biomass, preferably at least about 8.0 mg / g or at least about 9.0 mg / g; and / or41.- a trigonelline content of at least 0.3 mg / g, based on the total weight of the dry biomass, preferably at least 0.32 mg / g, at least 0.35 mg / g or at least 0.5 mg / g.

28. The use of any one of claims 26-27, wherein the biomass is obtained by a method comprising the production of somatic embryos from coffee explant material by direct or indirect somatic embryogenesis, the cultivation of the somatic embryos under conditions effective to produce a plant cell biomass producing caffeine, and optionally the separation of the plant cell biomass from the cell culture medium.

29. The use of any one of claims 26-28, wherein the biomass is characterized in that the content in Cd and / or Pb in the biomass is less than about 10 ppb, less than about 5 ppb, less than about 1 ppb or even less than about 0.5 ppb, based on the total weight of the dry biomass.

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