Method for fermenting cultured plant cells

Abstract

Herein is provided a method in which sterile cultured plant cells suitable for human consumption are fermented with fermenting microbe(s) suitable for human consumption in a fermentation mixture to obtain fermented plant cells. Fermented cultured plant cells obtainable by the method are also provided.

Description

[0001] METHOD FOR FERMENTING CULTURED PLANT CELLS

[0002] TECHNICAL FIELD

[0003] The present disclosure generally relates to fermentation of cultured plant cells. The disclosure relates particularly, though not exclusively, to microbial fermentation of cultured plant cells.

[0004] BACKGROUND

[0005] This section illustrates useful background information without admission of any technique described herein representative of the state of the art.

[0006] Recently, cell cultures have been suggested as more sustainable alternatives for conventional agriculture products. For example, biomass from cell cultures may reduce the need for land, water and / or other resources compared to products from conventional agriculture.

[0007] However, biomass from cell culture does not necessarily have all desired properties for the intended end use. Typically, biomass from cell cultures differs from conventional agricultural counterparts for example in their structure, appearance and / or flavor.

[0008] There is hence a need to find ways to process biomass from cell culture to improve or modify its properties in view of intended end use for example in food and / or beverage applications.

[0009] SUMMARY

[0010] The appended claims define the scope of protection. Any examples and technical descriptions of apparatuses, products and / or methods in the description and / or drawings not covered by the claims are presented as examples useful for understanding the invention.

[0011] According to a first example aspect there is provided a method comprising providing sterile cultured plant cells suitable for human consumption; and fermenting the cultured plant cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising the cultured plant cells 5-50 wt-%, based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture, and inoculated with 102-1010cfu / g of the fermenting microbes to obtain fermented plant cells.

[0012] In certain embodiments, the fermentation mixture comprises 5-30 wt-%, preferably 6-10 wt- %, more preferably 6-8 wt-% of the cultured plant cells, based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture; and / or the fermentation mixture is inoculated with 103-109cfu / g, preferably 104-108, more preferably 105-107cfu / g of the fermenting microbes.

[0013] In certain embodiments, the fermenting microbe(s) is selected from one or more of bacteria, yeasts and / or fungi, preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, yeasts belonging to Family Saccharomycetaceae, and / or ascomycetous filamentous fungi, more preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, and / or ascomycetous filamentous fungi.

[0014] In certain embodiments, the method is a method for making coffee comprising: providing sterile cultured coffee cells suitable for human consumption; fermenting the cultured coffee cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising 5-50 wt-% of the cultured coffee cells, based on the dry weight of the cultured coffee cells and the total weight of the fermentation mixture, and inoculated with 102- 1010cfu / g of the fermenting microbes to obtain fermented coffee cells; drying the fermented coffee cells or a portion thereof to obtained dried coffee cells; and roasting the dried coffee cells preferably at a temperature within a range from 80 °C to 350 °C, more preferably within a range from 150 °C to 300 °C, even more preferably within a range from 200 °C to 250 °C to obtain roasted coffee cells.

[0015] In certain embodiments, the method for making coffee comprises extracting coffee beverage from the roasted coffee cells.

[0016] According to a second example aspect there is provided fermented cultured plant cells obtainable or obtained by a method according to the first example aspect.

[0017] According to a further example aspect there is provided a food product or a beverage, preferably food product, comprising fermented cultured plant cells according to the second example aspect. According to a yet further example aspect there is provided use of fermented cultured plant cells according to the second example aspect to prepare a beverage or a food product, preferably beverage.

[0018] The present inventors have found that by subjecting sterile, cultured plant cells suitable for human consumption to microbial fermentation by food grade fermenting microbe(s) the flavor and / or odor of the cultured plant cells may be modified or improved, and may overcome a flavor gap compared to naturally occurring counterparts at least to some extent, or at least provide the cultured cells with a more pleasant flavor and / or odor.

[0019] Different non-binding example aspects and embodiments have been illustrated in the foregoing. The embodiments in the foregoing are used merely to explain selected aspects or steps that may be utilized in different implementations. Some embodiments may be presented only with reference to certain example aspects. It should be appreciated that corresponding embodiments may apply to other example aspects as well.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] Some example embodiments will be described with reference to the accompanying figures, in which:

[0022] Fig. 1 shows a picture of cultured Coffea arabica cells in a glass jar prior to fermentation;

[0023] Fig. 2 shows a picture of freeze dried fermented cultured Coffea arabica cells according to an example embodiment after roasting at 225 °C;

[0024] Fig. 3 shows a spider web chart of generic descriptive analysis of flavor and odor of coffee beverages according to example embodiments and according to a reference example; and Fig. 4 shows a Principal component analysis biplot of generic descriptive analysis of flavor and odor of coffee beverages according to example embodiments and according to a reference example.

[0025] Fig. 5 shows result the strain grouping based on volatile compound analysis as a Principal component analysis Scores plot.

[0026] Fig. 6 shows results from the volatile compound analysis as a Principal component analysis Loadings plot. Together with Fig. 6 it displays the strain dependent volatile compound production.

[0027] Fig. 7 shows the result of Hierarchical clustering on principal components and displays the similar and different strain groups based on the volatile compounds. DETAILED DESCRIPTION

[0028] Cellular agriculture typically refers to cultured cells used in production of agricultural commodities, such as food products, beverages, or ingredients thereof. In the context of the present disclosure, cellular agriculture components, ingredients, or products refer generally to components, ingredients, or products from cell and / or microbial culture(s), such as components, ingredients, or products grown or propagated in bioreactor(s).

[0029] As used herein, plant cell derived biomass refers to cultured plant cells harvested from cell culture. Coffee cell biomass refers herein to cultured coffee cells harvested from cell culture.

[0030] Microbial fermentation refers herein to a process in which metabolic activity of microorganism(s) brings about a change to a substrate, here cultured plant cells. In certain embodiments, the change is in flavor, odor, and / or structure.

[0031] Contained refers herein to cell cultivation or fermentation, as the case may be, that takes place in a dedicated environment, typically inside a bioreactor, so that contamination may be avoided and growth of target organism(s) supported.

[0032] As used herein, spontaneous fermentation refers to fermentation caused by indigenous microbes (microbes that are naturally present).

[0033] In the present disclosure, cfu refers to colony forming units, and cfu / g is expressed based on the total weight of the fermentation mixture.

[0034] Sterile refers herein to something being substantially free from bacteria or other living microbes, especially something being free from indigenous microbes.

[0035] As used herein, suitable for human consumption refers to compounds, species, components, food products and / or ingredients, and / or beverages and / or ingredients thereof that a human, preferably at least a human adult, may safely ingest. Suitable for human consumption generally refers herein to food grade or edible ingredients or components. In the context of this disclosure, suitable for human consumption or food grade does not necessarily refer to regulatory approval. For example, microbes, ingredients, and / or components that have a positive (yes) QPS (qualified assumption of safety) status may be considered suitable for human consumption.

[0036] As used herein, total weight refers to the wet weight of the composition or sample in question. In the context of this disclosure, a food product refers to food products suitable for human consumption. As used herein, beverage refers to beverages suitable for human consumption.

[0037] As used herein, fermentable sugars may include one or more of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose, lactose, and / or maltotriose.

[0038] Microbial fermentation is a biological way to alter or improve raw material or ingredients of food and / or beverages. Conventionally, spontaneous fermentation is mostly used. However, spontaneous fermentation may be unpredictable, and even cause undesired alterations and / or food safety concerns as the microbes present, their amount, and / or growth is not controlled.

[0039] Cellular agriculture products or ingredients may be seen as a more sustainable alternative to products or ingredients from conventional agriculture e.g. from a human and / or environmental perspective. However, replacing or providing substitutes for existing agricultural products or ingredients by biotechnologically produced cells is by no means easy or straight forward. Biotechnologically produced cells, such as cultured plant cells, typically differ from their naturally occurring counterparts e.g. in their structure and appearance, as well as chemical compositions. For example, cultured plant cells may contain phenolics, lipids, cholesterol, flavor precursors etc. in different amounts than their naturally occurring counterparts. Certain compounds present in cultured plant cells may not be detectable in naturally occurring counterparts and vice versa.

[0040] Differences in flavor and / or odor, as well as flavor and / or odor precursors, in cultured plant cells compared to naturally occurring counterparts may cause problems when looking e.g. for biotechnologically produced alternatives or substitutes for conventional food products and / or beverages. This difference is sometimes referred to as flavor gap.

[0041] The present inventors have now found that by subjecting sterile, food grade cultured plant cells to preferably contained and controlled microbial fermentation by food grade fermenting microbe(s) the flavor and / or odor of the cultured plant cells may be modified or improved. For example, controlled and contained microbial fermentation may develop desired flavor compounds from flavor precursors present in the cultured plant cells. Furthermore, additional flavor may be added from the fermenting microbes in a natural way. Suitably selecting the fermenting microbes and fermentation conditions enables flavor design which may overcome the flavor gap at least to some extent, or at least provide the cultured cells with a more pleasant flavor and / or odor.

[0042] In controlled cultivation or controlled fermentation, as the case may be, the cell cultivation process or the fermentation process may be controlled by defining or predetermining one or more, typically more than one, preferably all, parameters such as temperature, agitation, aeration, pH, and, in case of fermentation, species and / or strain of the fermenting microbes, and adjusting the parameter(s) as necessary or desired at any time during the cultivation or the fermentation.

[0043] According to a first example aspect, herein is provided a method comprising: providing sterile cultured plant cells suitable for human consumption; fermenting the cultured plant cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising 5-50 wt-%, preferably 5-30 wt-%, more preferably 6-10 wt-%, even more preferably 6-8 wt-% of the cultured plant cells, based on the dry weight of the cultured plant cells and total weight of the fermentation mixture, and inoculated with 1O2-1 O1ocfu / g, preferably 103-109cfu / g, further preferably 104-108, more preferably 105- 107cfu / g of the fermenting microbes to obtain fermented cultured plant cells.

[0044] The present disclosure provides a method for microbial fermentation of cultured plant cells for food and beverage applications. The present inventors have found that biotechnologically produced plant cells, i.e. cultured plant cells, suitable for human consumption, such as cultured coffee cells and / or cultured cocoa cells, preferably from contained cultivation in bioreactors can be subjected to fermentation with microbial species or strains (individual species / strains or mixture of species / strains) to modify sensory and structural properties, especially flavor and / or odor of the biotechnologically produced biomass (cultured plant cells).

[0045] Accordingly, the present method typically comprises fermenting the cultured plant cells with the fermenting microbe(s) to modify sensory and / or structural properties, preferably at least flavor, more preferably at least flavor and odor, of the cultured plant cells. Preferably, the fermenting comprises modifying flavor compounds and / or precursors thereof.

[0046] The present method provides a biological way of modifying or improving cultured plant cells. Thus, the need for additives (chemicals) for example in an end product may be reduced, or chemical additives may even be omitted. Preferably, the method comprises: providing fermenting microbe(s) suitable for human consumption; inoculating the cultured plant cells with 102-1O10cfu / g, preferably, 103-109cfu / g, more preferably 104-108, even more preferably 105-107cfu / g of the fermenting microbes to obtain a fermentation mixture, fermenting the cultured plant cells in the fermentation mixture to obtain fermented cultured plant cells.

[0047] According to a second example aspect, herein is provided use of microbial fermentation to modify flavor and / or odor of cultured plant cells, such as cultured coffee cells, preferably in food and / or beverage applications.

[0048] The cultured plant cells (plant cell biomass from cell culture) provided in the present method may be produced by conventional means for example in bioreactors, such as wave bioreactors, followed by harvesting using conventional methods. The cultured plant cells may be from continued culture and / or from batch culture. Preferably, the cultured plant cells provided in the present method are from controlled and contained cultivation.

[0049] Typically, plant cells or plant cell biomass from cell cultures is sterile. In the present method, providing sterile plant cell or plant cell biomass enables controlled fermentation in the fermenting step. Presence of undesired microbes in the provided cells may cause spontaneous fermentation in the fermenting step, which is in the present method to be avoided as it may have an undesired effect on e.g. sensory properties such as taste and / or odor. Growth of undesired and / or uncontrolled microbes on the plant cells may also be a food safety concern and even render cells unsuitable for human consumption. In the present method, the fermentation may be controlled and contained, preferably fully controlled and contained. Preferably, the present fermentation is performed without spontaneous fermentation.

[0050] In certain preferred embodiments, providing sterile cultured plant cells suitable for human consumption comprises: culturing plant cells suitable for human consumption in a cell culture medium followed by harvesting of cultured plant cells, the harvesting comprising separating cultured plant cells from spent cell culture medium and optionally washing the separated plant cells.

[0051] In the present method, food grade fermenting microbes may be cultivated on the provided cultured plant cells causing fermentation of the cultured plant cells. The fermenting microbes of the present method may be of individual species or strains, or may be a mixture of more than one species and / or strains, for example a mixture of two or more species and / or strains. The fermenting microbes employed in the present method may be for example bacteria and / or yeasts. In the present Examples successful fermentation of cultured plant cells modifying flavor and odor was demonstrated with fermenting bacteria and fermenting yeast of individual species, respectively, as well as with mixture of fermenting bacteria, and mixtures of fermenting bacterium and fermenting yeast. Eventually, the fermentation or growth of the fermenting microbes on the provided cultured plant cells may be stopped, and the fermented cultured plant cells or fermented plant cell biomass may be recovered and optionally further processed. The duration of the fermentation may vary. For example, the duration of the fermentation may be 24h-48h, but examples of the duration of the fermentation are not limited hereto.

[0052] The fermenting microbes(s) may be selected based on the cultured plant cells to be fermented, the intended end use, and / or desired flavor and / or odor attributes. In the present method, the fermenting microbes are suitable for human consumption, i.e. they may be considered food grade or edible. In certain preferred embodiments, the fermenting microbe(s) is / are selected from one or more of bacteria, yeasts and / or fungi, preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, yeasts including Family Saccharomycetaceae, and / or ascomycetous filamentous fungi, more preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, and / or ascomycetous filamentous fungi. For example, the fermenting microbe(s) may be selected from one or more of gram-positive lactic acid bacteria, such as Pediococccus pentosaceus, lactobacillus, such as Lactiplantibacillus plantarum, and / or ascomycetous yeasts, such as Kluyveromyces lactis, but examples of the fermenting microbes are not limited hereto. Benefits of these fermenting microbes may include production of flavor compounds as metabolites during fermentation, safety upon use in food and / or beverage applications, and modification or improvement of sensory properties of the plant cells, and in embodiments wherein at least one fermenting microbe is a bacterium, efficient acid production. Furthermore, these fermenting microbes may improve technofunctional properties of the cultured plant cells. As discussed in the foregoing, cultured plant cells are typically structurally very different from their naturally occurring counterparts, and there may be a need to modify or improve techno-functional properties of the cultured biomass. A techno-functional property may be any property, excluding nutritional value, affecting utilisation in food and / or beverage applications, such as one or more of emulsification, solubility, water and / or oil binding, foam capacity, stability, gelation, and / or viscosity. In certain embodiments, at least one fermenting microbe may be a probiotic strain or a micronutrient producing strain. Such fermenting microbes contribute to health benefits of the present fermented cultured plant cells.

[0053] Preferably, the fermenting microbes produce metabolites substantially without ethanol content. That is, preferably, the fermenting microbes are non-ethanol producing fermenting microbes. In certain preferred embodiments, the fermenting microbes are without Saccharomyces cerevisiae or Saccharomyces species content. Species of Saccharomyces may produce non-negligible amounts of ethanol as metabolites during fermentation. An objective of the present disclosure is to alter flavor and / or odor attributes, especially flavor attributes, of cultured plant cells for food and / or beverage applications. Although ethanol may sometimes be an acceptable or even desired metabolite, in most food and / or beverage applications ethanol is undesired and may render the food and / or beverage unsuitable for its intended use. Herein, the main objective is to modify the flavor and / or odor attributes without intoxicating side products, such as ethanol. For example, in coffee grounds or coffee beverage, ethanol is undesired.

[0054] The inoculum level is preferably within a range from 103cfu / g to 109cfu / g, more preferably from 104cfu / g to 108cfu / g, even more preferably from 105to 107cfu / g. Unless otherwise mentioned, inoculum or inoculum level fermentation mixture refers to the total content of fermenting microbe(s) added to the fermentation mixture during the fermentation. The inoculation may be performed for example using a liquid (starter) culture or a freeze dried (starter) culture, or both,. The inoculation may be performed either in one step in the beginning of the fermentation, or in more than one steps during the fermentation with at least a first step in the beginning of the fermentation. Especially if more than one (starter) cultures or more than one species and / or strains of fermenting microbes is used, the inoculation may be performed in more than one step. In embodiments in which the fermenting microbes comprise two or more species and / or strains, the ratio (cfu / g:cfu / g) between the different species and / or strains may vary for example depending on desired flavor and / or odor attributes, the selected fermenting microbes, cultured plant cells, and / or intended end use. In embodiments in which the fermenting microbes comprise two or more species and / or strains, each species and / or strain is preferably present in about equal amounts. For example, in embodiments wherein the fermenting microbes are a co-culture or mixture of two species or strains, the inoculum ratio may be about 50:50 (cfu / g to cfu / g).

[0055] Controlled and contained microbial fermentation of cultured plant cells or plant cell biomass from cell culture has previously not been suggested. As discussed in the foregoing, cultured plant cells generally differ from their naturally occurring counterparts in many aspects. Hence, plant cell biomass from cell culture cannot be expected to behave or to be processable in the same or even similar way as naturally occurring counterparts. The present inventors have now found that cultured plant cells are very well suited for microbial fermentation, especially as defined in the first example aspect. For example, the present disclosure provides fermentation conditions and parameters that are particularly well suited for microbial fermentation of plant cell biomass from cell culture, especially fermentation to modify flavor and / or odor attributes of said biomass.

[0056] Plant cell biomass from cell culture (cultured plant cells) has many benefits in view of the fermentation. For example, there is typically not any need to grind or otherwise mechanically process the harvested cells, and they may be provided to the fermentation or fermentation mixture as is. Plant cell biomass from cell culture is typically easy to inoculate with the fermenting microbe(s) simply by mixing, and the mixing is typically easy to perform. Furthermore, the water content of plant cell biomass from cell culture may be adjusted already in the harvesting and / or washing step. This may avoid a need to adjust water content of the fermentation mixture later or in a separate step. Also, in certain embodiments (spent) cell culture medium may be recovered from the cell culture of the cultured plant cells, typically in connection with harvesting of the cultured plant cells, and at least partially recycled to the fermentation mixture enabling use of residual nutrients in the cell culture medium, such as fermentable sugars, in the fermenting.

[0057] In certain preferred embodiments, the water content of the fermentation mixture is within a range from 50 to 95 wt-%, preferably from 70 to 95 wt-%, even more preferably from 90 to 94 wt-%, based on the total weight of the fermentation mixture. Such water contents have been found particularly suitable for microbial fermentation of cultured plant cells.

[0058] The desired water content of the fermentation mixture may be obtained in a harvesting step of the cultured plant cells, e.g. by adjusting or controlling straining or filtering of the cultured plant cells from the cell culture medium. Alternatively or additionally, the water content may be adjusted by adding or removing water in a separate step or when preparing the fermentation mixture.

[0059] In certain embodiments, the cultured plant cells are dried and rehydrated before the fermenting. Flavor compounds and / or flavor precursors may however evaporate and / or degrade during drying of the cultured plant cell, which may alter their sensory properties. Accordingly, the cultured plant cells are in the present method preferably provided without having been subjected to drying.

[0060] The fermenting is typically affected by the composition of the fermentation mixture, including the cultured plant cells and the fermenting microbe(s), and technical conditions, such as duration of the fermenting, temperature and pH. Preferably, the present method comprises fermenting the provided cultured plant cells with fermenting microbe(s) suitable for human consumption under suitable conditions to provide a fermentation product, preferably with alterations in flavor compounds compared to the provided cultured plant cells prior to fermentation.

[0061] In certain embodiments, the present method comprises adjusting one or more of temperature, pH, water content and / or solid matter content, especially content of the cultured plant cells, and / or O2 concentration of the fermentation mixture. The adjusting may be based on analysis data obtained from the fermentation process, for example one or more of measured temperature, measured pH, analysed water and / or solid matter content, and / or analysed O2 concentration.

[0062] In certain preferred embodiments, forming the fermentation mixture comprises inoculating the cultured plant cells with 102-1010cfu / g, preferably 103-109cfu / g, more preferably 104- 108, even more preferably 105- 107cfu / g of the fermenting microbes, said inoculation preferably comprising mixing the microbes with the cultured plant cells, and optionally adjusting water content and / or sugar content of the fermentation mixture, preferably to obtain a water content of 50-95 wt-%, more preferably 70-95 wt-%, even more preferably 90 to 94 wt-%, based on the total weight of the fermentation mixture, and / or a sugar content of 10-40 mg sugar / 100 mg dry weight of the fermentation mixture, more preferably 15-35 mg sugar / 100 mg dry weight of the fermentation mixture. The optional adjustment of water content and / or sugar content may be performed before or after, or before and after, the inoculation. The inoculation is expressed based on total weight of the fermentation mixture.

[0063] In the present method, the dry weight of the cultured plant cells in the fermentation mixture is within a range from 5-50 wt-%, preferably from 5 to 30 wt-%, more preferably from 6 to 10 wt-%, even more preferably from 6 to 8 wt-%, based on the total weight of the fermentation mixture. Such dry weight contents of the cultured plant cells have been found to contribute to successful fermentation of the cultured plant cells, especially providing alterations in flavor and / or odor, especially flavor, attributes. Typically, the fermentation mixture contains at least the cultured plant cells and the fermenting microbes in the herein described amounts. Water (H2O) content and sugar content of the fermentation mixture may be provided by the cultured plant cells, or adjusted by typically adding water (H2O) and / or sugar(s). The fermentation mixture may contain (spent) cell culture medium typically from the cultivation of the cultured plant cells. If desired, the fermentation mixture may contain for example added vitamins and / or minerals, typically in minor or trace amounts. Addition of vitamins and / or minerals to the fermentation mixture may be desired for example in food applications.

[0064] In certain preferred embodiments, the fermentation mixture substantially consist of the cultured plant cells, the fermentating microbes, and optionally cell culture medium from cultivation of the cultured plant cells, and the water content of the fermentation mixture is within a range from 50 to 95 wt-%, preferably from 70 to 95 wt-%, even more preferably from 90 to 94 wt-%, based on the total weight of the fermentation mixture.

[0065] Preferably, the fermenting is carried out at a temperature within a range from 20 to 40 °C, preferably from 20 to 37 °C, more preferably 25 to 30 °C. Such temperatures provide good growth and metabolic activity of the fermenting microbes.

[0066] In certain preferred embodiments, the pH of the fermentation mixture is within a range from 6 to 3, preferably from 5 to 3. A pH on the acidic side improves product stability and influences sensory properties. The pH may be adjusted to a predetermined value during fermentation, or the pH may be let to developed, typically decrease, during fermentation. During fermentation, the fermenting microbes, especially fermenting bacteria, may produce organic acids decreasing the pH of the fermentation mixture. In certain embodiments, the fermentation is carried out without adjusting the pH.

[0067] Preferably, the fermentation mixture contains 10-40 mg sugar / 100 mg dry weight of the fermentation mixture, preferably 15-35 mg sugar / 100 mg dry weight of the fermentation mixture, said sugar preferably comprising one or more of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose, lactose, and / or maltotriose, more preferably at least one or more of glucose, fructose and / or sucrose. Such sugar contents are beneficial in that they contribute to efficient microbial metabolism, and for example fast production of microbial metabolites, such as organic acids, in fermentation of the cultured plant cells. Unless otherwise mentioned, sugar or sugar content refers herein to the total sugar content, i.e. sum amount of the contents of the sugars present. The present inventors have found that the cultured plant cells as such may contain enough fermentable sugars to provide the fermentation mixture with desired sugar content. Hence, in certain preferred embodiments, the sugar content of the fermentation mixture is provided by the cultured plant cells. In certain embodiments, at least a portion of the sugar content of the fermentation mixture is provided by residual sugar from the cell culture medium. Residual sugar from the cell culture medium may be provided to the fermentation mixture for example by maintaining (recovering) a portion of the cell culture medium in the harvesting of the cultured plant cells and / or by recycling spent cell culture medium to the fermentation mixture. For example in these embodiments, added sugar is not necessarily needed.

[0068] In certain preferred embodiments, providing sterile cultured plant cells suitable for human consumption comprises: culturing plant cells (suitable for human consumption) in a cell culture medium (suitable for human consumption) followed by harvesting of cultured plant cells, the harvesting comprising separating cultured plant cells from spent cell culture medium and optionally washing the separated plant cells, and optionally recovering the spent cell culture medium and adding of the spent cell culture medium to the fermentation mixture to adjust sugar and / or water content, preferably both, of the fermentation mixture. Alternatively or additionally, sugar and / or water may be added to the fermentation mixture.

[0069] The fermentation may be aerobic fermentation or anaerobic fermentation. In certain embodiments, the fermentation may be sequential fermentation, i.e. the fermentation may comprise a first fermentation process followed by one or more further fermentation processes. In embodiments wherein the fermentation is sequential fermentation, anaerobic fermentation may be followed by aerobic fermentation, or vice versa. In certain embodiments wherein the fermentation is sequential fermentation, anaerobic fermentation processes may follow each other, or aerobic fermentation processes may follow each other. The fermentation processes in a sequential fermentation may in certain embodiments be of the same type, that is either aerobic or anaerobic.

[0070] In certain embodiments, the fermentation is aerobic fermentation. In certain embodiments, the fermentation is anaerobic fermentation. In certain embodiments, the fermentation comprises anaerobic fermentation and anaerobic fermentation, typically carried out sequentially.

[0071] The present method may comprise stirring the fermentation mixture during the fermentation.

[0072] The stirring may be for example continuous, intermittent, or periodical. The fermentation may be carried out in pilot scale or industrial scale reactor(s), and state of the art reactors may be suitable for carrying out the present fermentation.

[0073] The present method may be applied on a variety of edible, cultured plant cells. The cultured plant cells provided in the present process may be of one species or strain, or they may be a mixture of cultured plant cells of two or more species and / or strains.

[0074] Preferably, the cultured plant cells are one or more of, preferably one of, coffee cells, such as cultured Coffea arabica cells, cocoa cells (Theobroma cacao), vanilla cells (Vanil a planifoiia), tea cells (Camellia sinensis), cabbage cells (Brassica oleracea) and other Brassicaceae, soy bean cells (Glycine max) and other legume cells, onion / garlic cells (Allium spp.), chili cells (Capsicum spp.), cucumber cells (Cucumis sativus), mango cells (Mangifera indica), and / or other vegetable cells. More preferably, the cultured plant cells are one or more of, preferably one of, coffee cells, such as cultured Coffea arabica cells, cocoa cells (Theobroma cacao), vanilla cells (Vanilla planifoiia), tea cells (Camellia sinensis), cabbage cells (Brassica oleracea), soy bean cells (Glycine max), onion / garlic cells (Allium spp.), chili cells (Capsicum spp.), cucumber cells (Cucumis sativus), and / or mango cells (Mangifera indica). Even more preferably, the cultured plant cells are coffee cells, such as cultured Coffea arabica cells.

[0075] The present method may find use in various food and / or beverage applications. For example, the present fermentation may be used to alter flavor and / or odor, especially flavor, attributes of cultured cells of species typically serving as spices, such as cultured vanilla cells. For example, the present fermentation process may be used to alter flavor and / or odor, especially flavor, attributes of cultured vegetable cells and / or fruit cells e.g. for providing substitutes for conventional pickled vegetables and / or fruits.

[0076] The present inventors have found that the present method is particularly well suited for modifying flavor and odor profile, especially flavor profile, of cultured coffee cells. By fermenting cultured coffee cells as herein described, the cells may, typically after roasting, serve as well tasting substitute for conventional coffee grounds in coffee making, as demonstrated in the present Examples.

[0077] According to a further example aspect herein is provided fermented cultured plant cells obtainable or obtained with the method according to the first example aspect. Such fermented cells may have improved or more pleasant sensory properties, particularly flavor and / or odor, preferably both, and / or modified or improved mouthfeel, and / or techno- functional properties, such as one or more of emulsification, solubility, water and / or oil binding, foam capacity, stability, gelation, and / or viscosity, compared to corresponding cultured plant cells prior to fermentation.

[0078] The present method may comprise subjecting the fermented cultured plant cells to further processing. For example, the fermented cells may be frozen and stored for example at temperatures within a range from -15 °C to -200 °C, such as about -20°C. Typically, the frozen cells are thawed before use e.g. in food and / or beverage application. Alternatively or additionally, the fermented cultured plant cells may be dried, preferably freeze dried or lyophilized.

[0079] The fermented cultured plant cells may be mixed with other food and / or beverage ingredients, and / or subjected to process steps of making food product(s) and / or beverage(s). For example, the fermented cultured plant cells may be used as ingredients in cooking, baking, desert(s), yoghurt(s), plant-based yoghurt substitute(s), confectionary, snack product(s), and / or plant-based milk substitute(s).

[0080] The present method may comprise incorporating the fermented plant cells optionally after further processing, such as drying, heating, and / or homogenisation, in a food product or a beverage, preferably food product. Heating may be for example cooking, baking, boiling, roasting, or a combination thereof.

[0081] According to a further example aspect herein is provided a food product or a beverage, preferably a food product, comprising fermented cultured plant cells obtainable or obtained according to the method of the first example aspect. The food product may for example be a baked goods, a desert, a yoghurt, a plant-based yoghurt substitute, a confectionary, a snack product, and / or a plant-based milk substitute. The food product or beverage may contain from 0.001 wt-% to 100 wt-% of the fermented plant cells based on the total weight of the food product or beverage. The share of the fermented plant cells in the food product or beverage may depend on the plant cells and their purpose in the food product or beverage. For example, if the fermented plant cells are used as a spice, which could be the case for example in embodiments wherein the fermented plant cells are fermented vanilla cells, the share of the fermented cells may be minor. On the other hand, for example if the fermented cells provide a substitute for pickled vegetables, their share in the food product may be high, even approaching 100 wt-%. According to a yet further example aspect, herein is provided use of fermented cultured plant cells obtainable or obtained according to the method of the first example aspect to prepare a beverage or a food product, preferably beverage. The present fermented cultured plant cells may be used for example in making a beverage, such as making or extracting coffee.

[0082] In certain embodiments, the method comprises drying the fermented cultured plant cells and heating, preferably roasting, the dried fermented cultured plant cells, optionally followed by extracting or mixing with liquid, preferably water, the liquid preferably having a temperature within a range from 70 °C to 100 °C, more preferably from 80 °C to 100 °C, even more preferably from 90 °C to 100 °C.

[0083] For example, in embodiments wherein the cultured plant cells are coffee cells and / or cocoa cells, the fermented cultured coffee and / or cocoa cells may be freeze dried, the freeze dried cells then subjected to roasting.

[0084] In certain embodiments, the fermented plant cells may be subjected to homogenisation. For example, in case there are aggregates formed during cultivation and / or fermentation, a homogenisation step may be beneficial. Such homogenisation may be carried out before or after optional drying, or even before and after optional drying if desired.

[0085] In embodiments wherein the fermented, cultured plant cells are subjected to roasting, typically after freeze drying, the roasting is preferably carried out at a temperature within a range from 80 °C to 350 °C, more preferably within a range from 150 °C to 300 °C, even more preferably within a range from 200 °C to 250 °C, such as about 225 °C. Preferably, the roasting is carried out at a relative humidity of 0-15 %. The roasting time may vary, for example depending on the roasting temperature used. For example, the roasting time may be within a range from 2 min to 30 min, or from 2 min to 20 min, or from 3 min to 15 min, or from 3 min to 6 min. The roasting may include stirring of the fermented cells being roasted. Stirring the fermented cells contributes to more even roasting. In certain embodiments, the roasting is performed at conditions corresponding to those of roasting conventional counterparts, such as coffee beans and / or cocoa.

[0086] Subjecting the fermented cells to roasting, typically after drying, preferably freeze drying, may further modify or improve their sensory properties, preferably at least flavor and / or odor, more preferably both. Roasting may also modify the color of the fermented, optionally dried, plant cells, for example contribute to dark brown color of fermented, lyophilized coffee cells.

[0087] For example, in embodiments wherein the roasted cells are dried, preferably freeze dried, fermented coffee cells, the method may comprise passing a liquid, typically water, through the roasted cells to obtain coffee beverage. Coffee beverage brewed or extracted from fermented cultured coffee cells may have superior flavor profile as compared to coffee beverage brewed or extracted from non-fermented cultured coffee cells, and flavor profile may even be improved compared to conventional coffee beverage. The obtained beverage may be mixed with further ingredients, such as milk, cream, sugar, and / or syrup. The coffee beverage may be prepared from the fermented cultured coffee cells, preferably having been subjected to drying, preferably freeze drying, and roasting, by any conventional coffee making means using any conventional coffee making equipment for coffee grounds.

[0088] Preferably, the present method is a method for making coffee comprising: providing sterile cultured coffee cells suitable for human consumption; fermenting the cultured coffee cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising 5-50 wt-%, preferably 5-30 wt-%, more preferably 6-10 wt-%, even more preferably 6-8 wt-% of the cultured coffee cells, based on the dry weight of the coffee cells and total weight of the fermentation mixture, and inoculated with 102-1010cfu / g, preferably 103-109cfu / g, more preferably 104-108, even more preferably 105-107cfu / g of the fermenting microbes to obtain fermented cultured coffee cells; drying, preferably freeze-drying, the fermented cultured coffee cells to obtain dried, preferably freeze-dried, coffee cells, the freeze-drying optionally being preceded and / or followed by homogenisation; and roasting the freeze-dried coffee cells preferably at a temperature within a range from 80 to 350 °C, more preferably within a range from 150 °C to 300 °C, even more preferably within a range from 200 °C to 250 °C, to obtain roasted coffee cells.

[0089] Preferably, the method for making coffee comprises extracting preferably with water coffee beverage from the roasted coffee cells.

[0090] Method for making coffee may refer herein to a method yielding coffee grounds or a substitute for coffee grounds, or a method yielding coffee beverage, or a combination thereof.

[0091] In another embodiment providing the sterile cultured plant cells comprises culturing plant cells in a cell culture medium followed by harvesting. Optionally, the harvesting comprises separating cultured plant cells from spent cell culture medium, and the cultured plant cells are provided without having been subjected to drying. In a preferred embodiment the cultured plant cells are provided as fresh cultured plant cells.

[0092] In an embodiment the fermentation does not involve co-cultivation of the cultured plant cells.

[0093] In an embodiment the cultured plant cells are, or comprise, cultured coffee cells.

[0094] In an embodiment is provided a method for making roasted coffee cells comprising: providing sterile cultured coffee cells suitable for human consumption; fermenting the cultured coffee cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising the coffee cells 5-50 wt-% based on the dry weight of the cultured coffee cells and the total weight of the fermentation mixture, and inoculated with 102-1O10cfu / g of the fermenting microbes to obtain fermented coffee cells; drying the fermented coffee cells to obtained dried coffee cells; and roasting the dried coffee cells preferably at a temperature within a range from 80°C to 350°C, more preferably within a range from 150°C to 300°C, even more preferably within a range from 200°C to 250°C, to obtain roasted coffee cells

[0095] Implementation and embodiments are further disclosed in the following numbered embodiments:

[0096] Embodiment 1 : A method comprising: providing sterile cultured plant cells suitable for human consumption; fermenting the cultured plant cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising the cultured plant cells 5-50 wt-% based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture, and inoculated with 102-1O10cfu / g of the fermenting microbes to obtain fermented plant cells.

[0097] Embodiment 2: The method according to embodiment 1 , wherein the fermentation mixture comprises 5-30 wt-%, preferably 6-10 wt-%, more preferably 6-8 wt-% of the cultured plant cells, based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture, and / or wherein the fermentation mixture is inoculated with 103-109cfu / g, preferably 104-108, more preferably 105-107cfu / g of the fermenting microbes.

[0098] Embodiment 3:The method according to embodiment 1 or 2, wherein the fermenting microbe(s) is selected from one or more of bacteria, yeasts and / or fungi, preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, yeasts belonging to Family Saccharomycetaceae, and / or ascomycetous filamentous fungi, more preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, and / or ascomycetous filamentous fungi. Embodiment 4: The method according to any one of the preceding embodiments, wherein the fermentation mixture contains 20-40 mg sugar / 100 mg dry weight of the fermentation mixture, preferably 25-35 mg sugar / 100 mg dry weight of the fermentation mixture, said sugar preferably comprising one or more of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose, lactose, and / or maltotriose, more preferably at least one or more of glucose, fructose and / or sucrose.

[0099] Embodiment 5: The method according to embodiment 4, wherein the sugar in the fermentation mixture is provided by the cultured plant cells.

[0100] Embodiment 6: The method according to any one of the preceding embodiments, comprising adjusting one or more of temperature, pH, water content and / or solid matter content, especially content of the cultured plant cells, and / or O2 concentration of the fermentation mixture.

[0101] Embodiment 7: The method according to any one of the preceding embodiments, wherein forming the fermentation mixture comprises inoculating the cultured plant cells with 102-1010cfu / g, preferably 103-109cfu / g, more preferably 104-108cfu / g, even more preferably 105-107cfu / g of the fermenting microbe(s), said inoculation preferably comprising mixing the microbe(s) with the cultured plant cells, and optionally adjusting water content and / or sugar content of the fermentation mixture.

[0102] Embodiment 8: The method according to any one of the preceding embodiments, wherein providing sterile cultured plant cells suitable for human consumption comprises culturing plant cells in a cell culture medium followed by harvesting of cultured plant cells, the harvesting comprising separating cultured plant cells from spent cell culture medium and optionally washing the separated plant cells, and optionally recovering spent cell culture medium and adding of the spent cell culture medium to the fermentation mixture to adjust sugar and / or water content, preferably both, of the fermentation mixture.

[0103] Embodiment 9: The method according to any one of the preceding embodiments, comprising subjecting the fermented plant cells to further processing, such as homogenisation, drying, heating, and / or roasting.

[0104] Embodiment 10: The method according to any one of the preceding embodiments, comprising drying the fermented plant cells and roasting the dried fermented plant cells, optionally followed by extracting or mixing with liquid, preferably water. Embodiment 11 : The method according to any one of the preceding embodiments, wherein the method comprises incorporating the fermented plant cells optionally after further processing, such as drying, heating, roasting, and / or homogenisation, in a food product or a beverage, preferably food product.

[0105] Embodiment 12: The method according to any one of the preceding embodiments, wherein the cultured plant cells are cultured coffee cells, such as cultured Coffea arabica cells, or cultured cocoa cells.

[0106] Embodiment 13: The method according to embodiment 12, wherein the method is a method for making coffee comprising: providing sterile cultured coffee cells suitable for human consumption; fermenting the cultured coffee cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising 5-50 wt-% of the cultured coffee cells, based on the dry weight of the cultured coffee cells and the total weight of the fermentation mixture, and inoculated with 102- 1 O10cfu / g of the fermenting microbes to obtain fermented coffee cells; drying the fermented coffee cells to obtained dried coffee cells; and roasting the dried coffee cells preferably at a temperature within a range from 80 °C to 350 °C, more preferably within a range from 150 °C to 300 °C, even more preferably within a range from 200 °C to 250 °C to obtain roasted coffee cells.

[0107] Embodiment 14: The method according to embodiment 13, comprising extracting coffee beverage from the roasted coffee cells.

[0108] Embodiment 15: Fermented cultured plant cells obtainable by a method according to any one of embodiments 1-14.

[0109] Embodiment 16: A food product or a beverage, preferably food product, comprising fermented cultured plant cells according to embodiment 15.

[0110] Embodiment 17: Use of fermented cultured plant cells according to embodiment 15 to prepare a beverage or a food product, preferably beverage.

[0111] EXAMPLES

[0112] Example 1. Coffee cell cultivation

[0113] Callus was initiated from leaf explants of commercially available Coffea arabica seedlings in callus induction medium (Table 1 ) and incubated at 24 °C in darkness. Formed callus was cut off after two to four weeks and subcultured on callus establishment medium (Table 1 ). The calli were maintained by subculturing monthly on callus maintenance medium (Table 1 ). Cell suspensions were established in a culture medium with otherwise same composition as the callus maintenance medium but without gelling agent (Gelrite) in 250 ml Erlenmeyer flasks containing 70 ml of culture medium on an orbital shaker at 110 rpm and 24 °C in darkness, and the cells suspensions of the coffee {Coffea arabica) cells were subcultured every ten days.

[0114] For biomass production sterile coffea arabica cells were cultivated in a wave bioreactor using 20 I and 50 I wave bags with final working volumes of 10 I and 25 I, respectively. Inoculum for wave bag cultivation was prepared in shake flasks. Cultivation parameters were as follows: temperature 24 °C, angle 10°, rocking level 26 for 20 I wave and 24 for 50 I wave, aeration 300 ml / min, darkness. The culture medium contained water (H2O), sugars, macro- and micronutrients, vitamins and phytohormones and corresponded to the callus maintenance medium but without gelling agent (Gelrite). Coffea arabica cells were harvested from the cell culture by separating culture medium and coffea arabica cells by filtering with Miracloth in a Buchner funnel. The Coffea arabica cells recovered in the filtration were washed with sterile water. The thus obtained cultured Coffea arabica cells (coffee cell biomass) were stored at -20°C until fermentation.

[0115] Table 1. Cell culture media

[0116] Example 2. Sugar analysis

[0117] Cultured Coffea arabica cells (coffee cell biomass) was obtained as described in Example

[0118] 1 , with the exception that instead of storing the cells at -20°C the cultured Coffea arabica cells were freeze dried (lyophilized).

[0119] Freeze dried, cultured Coffea arabica cells from two replicates, A and B, were analysed for fermentable sugars. 50.2 mg freeze dried cells from each of replicates A and B were respectively mixed with 10 ml MilliQ water to prepare respective samples A and B (both samples having a concentration of 50.2 mg freeze dried cells per 10 ml MilliQ water).

[0120] Contents of fermentable sugars, in this example glucose, fructose, sucrose, maltose, and maltotriose, were measured from samples A and B, respectively, with HPAEC-PAD as described in Gibson et al, Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus, Yeast, 2013, 30, p. 255-266, using a Dionex ICS-6000 equipped with Carbopac PA1 column after water extraction and using corresponding standards. The results are shown in Table

[0121] 2.

[0122] Table 2. Analysed contents of fermentable sugars in freeze dried coffee cell biomass.

[0123] DW refers to dry weight of the sample.

[0124] As can be seen from Table 2, the contents of the fermentable sugars are quite similar between the two replicates A and B. Based on Table 2, it can be concluded that added sugar is not necessarily needed for fermentation of cultured Coffea arabica cells (coffee cell biomass), and that cultured coffee cell biomass obtained as described in Example 1 can be fermented without added sugar.

[0125] Example 3. Fermentation

[0126] Three microbial species having bioflavoring potential were chosen and employed as examples of fermenting microbes according to the present disclosure.

[0127] The selected fermenting microbes were 2 bacteria, namely gram-positive lactic acid bacteria Pediococccus pentosaceus and lactobacillus Lactiplantibacillus plantarum, and 1 yeast, namely ascomycetous yeast Kluyveromyces lactis.

[0128] The fermenting microbes selected for the present Examples had positive QPS (qualified assumption of safety) status and are hence considered suitable for human consumption. Suggested growth temperatures for these microbes were about 30 °C for Pediococccus pentosaceus and Lactiplantibacillus plantarum, and about 25 °C for Kluyveromyces lactis.

[0129] 105 g (wet weight) fermentations of cultured Coffea arabica cells (coffee cell biomass) obtained from wave bioreactor culture as described in Example 1 were performed in glass jars. Fig. 1 shows a picture of cultured Coffea arabica cells obtained from wave bioreactor culture as described in Example 1 in a glass jar prior to fermentation.

[0130] The dry weight of cultured coffea arabica cells (coffee cell biomass) in each jar was 7.3 wt- % based on the total weight (wet weight) of the respective fermentation mixture, the remainder of the fermentation mixture containing mostly H2O with minor amounts of cell culture medium as described in Example 1. The pH of the fermentation mixtures was about pH 5.3.

[0131] Fermentation mixture inocula was 1 %, i.e. 106cfu / g fermenting microbe(s) per g fermentation mixture (wet weight). After inoculation, the jars were incubated at 30 °C for 48 h. The pH was measured and sensory (odor) evaluation performed for each sample at 24 h and at 48 h incubation. Microbiological quality was assessed at 48 h incubation. The results are reported in Tables 4, 5, and 6, respectively.

[0132] In total, 6 samples and one reference sample were prepared and incubated: 3 samples with one species of fermenting microbes and 3 samples with co-cultures of two species of fermenting microbes, and one reference without fermenting microbes. The samples and reference are described in Table 3. In the co-cultures, the species were present in a ratio of 50:50 (cfu / g : cfu / g).

[0133] Table 3. Fermentation samples and reference sample (negative control). Table 4. pH at 24 h and 48 h incubation.

[0134] As seen from Table 4, the pH of all fermentation samples decreased compared to the initial pH 5.3 of the fermentation mixture. The pH of the fermentation samples, with the exception of 1 C, decreased further between the 24 h and 48 h measuring points. The pH of the reference sample 1 G without fermenting microbes remained at the initial value at 24 h and had decreased just slightly at 48 h. The pH of the reference sample 1 G was higher at 24h and 48h compared to the fermentation samples 1 A-1 F.

[0135] During fermentation the fermenting bacteria produced organic acids, and in samples 1A, 1 B, 1 D-1 F containing at least one bacterium species as fermenting microbe the decrease of pH indicated good fermentation. A pH below < 4.5 in the fermenting samples was also desired to ensure good microbiological quality. Samples 1A, 1 B, and 1 D-1 F had a pH well below 4.5 already at 24 h and the pH had decreased further at 48 h. Sample 1 C showed a less pronounced decrease in pH as it was fermented with a yeast in the absence of fermenting bacteria. However, as shown in Table 6 good microbial quality was nevertheless observed also for sample 1 C.

[0136] Table 5. Sensory odor evaluation at 24 h and 48 h incubation performed by two persons.

[0137] The odor of each fermenting sample as well as the reference sample was evaluated by sensory evaluation by two persons at 24 h and at 48 h incubation. As seen in Table 5, the odor was altered by the fermentation and at least to some extent its duration, and the odor was influenced by the fermenting microbe(s) in question.

[0138] The odors reported in T able 5 show that the odor of the reference sample without fermenting microbes and substantially without fermentation taking place, had an odor generally perceived as worse or less appealing compared to samples 1A-1 F with fermenting microbe(s) and in which fermentation occurred. Table 5 thus shows that sensory properties of the cultured Coffea arabica cells were successfully modified by controlled and contained microbial fermentation, especially, the sensory properties of the cultured coffee cell biomass were be improved, i.e. modified to have properties generally perceived as more pleasant (compared to a substantially unfermented negative control sample). Table 6. Microbiological quality at 48 h incubation.

[0139] In Table 6, PCA refers to plate count agar, TSA refers to tryptic soy agar, YM refers to yeast malt agar, MRS= refers to De Man, Rogosa and Sharpe agar, Chromocult refers to Chromocult® Coliform Agar, and Mossel refers to Bacillus cereus Agar Base. Table 6 shows or indicates growth of the inoculated microbes during the incubation. For example, comparing YM yeast + mold results reported in Table 6, reported cfu / g was much higher for the samples inoculated with a fermenting yeast, namely K. lactis, (samples 1 C, 1 E, and 1 F) compared to the negative control 1 G and samples 1 A, 1 B, and 1 D inoculated with fermenting bacteria. Comparing MRS lactic acid bacteria results, the samples 1A-1 F inoculated with fermenting microbe(s) showed much higher cfu / g counts compared to the reference sample 1 G. This further indicates growth of the inoculated fermenting microbes in the samples.

[0140] As also shown or indicated in Table 6, the microbial quality of all samples 1A-1 F was good. Numbers of aerobic bacteria, spore forming bacteria, contaminants, and Chromo cult E.coli, presumptive bacillus cereus group were low in the samples.

[0141] After the fermentation and sampling for the above studies, samples 1A-1 G were freeze dried. Freeze-dried samples were stored in airtight containers in a freezer at -20 °C until roasting.

[0142] Example 4. Coffee beverage Example 4.1 Preparation of coffee beverages

[0143] Each of the freeze-dried samples 1A-1 F as well as an additional reference sample (nonincubated negative control) 1 H was roasted. Reference sample 1 H was obtained by cultivating in wave bioreactor and harvesting therefrom Coffea arabica cells as described in Example 1 followed by freeze drying. The freeze-dried samples and reference sample were roasted, separately from each other, on a wire rack at 225 °C in a fan-assisted oven for 3- 6 min with occasional stirring. The roasted samples and their weight before roasting are reported in Table 7. Fig. 2 shows a picture of roasted, freeze-dried fermented coffee cells obtained according to Examples 1 and 3, and roasted as described above. Table 7. Samples for roasting

[0144] For sensory evaluation of coffee beverage, 5 samples and 3 reference samples of hot brewed coffee drinks were prepared. The samples are listed and described in Table 8. The reference examples included drinks brewed from two conventional, commercially available coffee grounds (samples S and R), as well as a drink brewed from nonfermented, freeze dried and roasted cultured Coffea arabica cells (sample 680).

[0145] Tabel 8. Samples for sensory evaluation of brewed coffee beverage

[0146] Coffee beverage was brewed by placing the grounds or powder in a filter bag and passing hot water through. Details of the brewing processes are reported in Table 9.

[0147] Table 9. Brewing details

[0148] The left over grounds in the filter after brewing as well as obtained coffee drink were evaluated for their color using L*a*b values. The L*a*b values are reported in Table 10.

[0149] Table 10. L*a*b colour values

[0150] Example 5.2 Sensory evaluation of coffee beverages

[0151] The coffee beverage samples obtained as described in Example 4.1 and Table 9 were evaluated using generic descriptive analysis. The evaluation was performed in a sensory laboratory by 4 trained panelists, and the results were processed with Eyequestion software.

[0152] A training session was conducted for the panelist earlier on the same day as the generic descriptive analysis. The training samples were commercial hot brewed coffee beverages (Juhla Mokka 20 % diluted (S), and Pelican Rouge 50% diluted (R)), one hot brewed coffee beverage from freeze dried, roasted, nonfermented cultured coffee cells (680 / 1 H), and hot brewed coffee beverage from freeze dried, roasted L. plantarum fermented coffee cells prepared and obtained as described in Examples 1 , and 3-4 (roasted, freeze-dried fermented samples of cultured coffea arabica cells).

[0153] References for some attributes and their intensities on 0-10 line scale are reported in Table 11.

[0154] Table 11. Reference attributes and their intensities on 0-10 line scale. aq=aqueous solution All coffee beverage samples 1A / 348, 1 C / 805, 1 D / 452, 1 E / 134, 1 F / 199, and reference sample 1 H / 680 were evaluated once for odor and flavor by each panelist. The results are plotted in Figs. 3 and 4.

[0155] As seen in Fig. 3, the fermentation altered both odor and flavor, and the selected fermenting microbe(s) contributed to flavor and odor profiles of the respective coffee beverages.

[0156] Example 6

[0157] The measurement of volatile compounds was adapted from J. Agric. Food Chem. 2023, 71 , 18478-18488 with small modifications.

[0158] After the fermentation of the coffee cells according to Example 3, the fermentates were frozen until analysis of volatile compounds. Additional reference sample non-incubated Coffea arabica cells was included. The frozen fermentates were thawed and mixed. One gram of each sample was weighed accurately in 20 ml headspace vials in duplicate. 50 ng of 3-octanol was added as internal standard in an aqueous solution and the headspace vials were closed.

[0159] The sample was first incubated at 60 °C for one minute. The volatiles were then extracted for 30 minutes at 60 °C with 250 rpm agitation (cycled with 5 s agitation, 30 s pause) to a 2 cm 50 / 30 pm DVB / Car / PDMS SPME fibre (Supelco, Bellafonte, PA). The volatiles compounds in the fibre were desorbed for 6 min in splitless mode (split opened after 1.5 min) in the split / splitless injection port at 250 °C. The instrument was an Agilent (Agilent Technologies, CA, US) GC-MS system with a 7890B GC instrument and a 5977B mass selective detector.

[0160] The volatiles were separated in a Restek Stabilwax-DA GC column (30 m x 250 pm x 0.25 pm). Helium was the carrier gas with 1 .5 mL constant flow. The temperature program was the following: initially held at 40 °C for 2 min, then raised from 40 °C to 240 °C at 10 °C min- 1 , and held at 240 °C for 8 min.

[0161] The quadrupole mass selective detector, with electronic impact ionization (ionization energy = 70 eV) was operated in scan mode with a mass range of 25-600 m / z.

[0162] The contents were calculated as 3-octanol equivalents, assuming a response factor of 1 for all compounds to allow comparison between samples. The resulting volatile compound dataset was then analysed with Principal component analysis (PCA). Mean-centering and unit variance scaling was used in the model. The results of this model are shown in Figures 5 and 6. The scores of the seven principal components were then used for hierarchical clustering with Ward’s clustering method and Squared Euclidean distance measurement. The result is shown in Figure 7.

[0163] These figures together show that all strains produce a characteristic volatile compound profile, but in particular K. lactis or combinations containing K. lactis produce a unique profile compared to other strains. This is demonstrated by the close distance of the aforementioned samples in Figure 5, and large relative distance of the joint cluster compared to the other clusters in Figure 7. Figure 6 shows that different esters including methyl acetate, ethyl acetate and ethyl propanoate were characteristic as well as different volatile acids including 2- and 3-methylbutanoic acid. Conversely, P. pentosaceus and L. plantarum produced similar volatile compound profiles, and caused a relatively small difference to the incubated negative control as indicated by the same cluster membership in Figure 7.

[0164] Various embodiments have been presented. It should be appreciated that in this document, words comprise, include, and contain are each used as open-ended expressions with no intended exclusivity.

[0165] The foregoing description has provided by way of non-limiting examples of particular implementations and embodiments a full and informative description of the best mode presently contemplated by the inventors for carrying out the invention. It is however clear to a person skilled in the art that the invention is not restricted to details of the embodiments presented in the foregoing, but that it can be implemented in other embodiments using equivalent means or in different combinations of embodiments without deviating from the characteristics of the invention, which are defined in the appended claims.

[0166] Furthermore, some of the features of the afore-disclosed example embodiments may be used to advantage without the corresponding use of other features. As such, the foregoing description shall be considered as merely illustrative of the principles of the present invention, which is defined in the appended claims, and not in limitation thereof. Hence, the scope of the invention is only restricted by the appended patent claims.

Claims

CLAIMS1 . A method comprising: providing sterile cultured plant cells suitable for human consumption; and fermenting the cultured plant cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising the cultured plant cells 5-50 wt-% based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture and inoculated with 102-1010cfu / g of the fermenting microbes to obtain fermented plant cells.

2. The method according to claim 1 , wherein the fermentation mixture comprises 5-30 wt-%, preferably 6-10 wt-%, more preferably 6-8 wt-% of the cultured plant cells, based on the dry weight of the cultured plant cells and the total weight of the fermentation mixture, and / or wherein the fermentation mixture is inoculated with 103- 109cfu / g, preferably 104-108, more preferably 105-107cfu / g, of the fermenting microbes.

3. The method according to claim 1 or 2, wherein the fermenting microbe(s) is selected from one or more of bacteria, yeasts and / or fungi, preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, yeasts belonging to Family Saccharomycetaceae, and / or ascomycetous filamentous fungi, more preferably from one or more of bacteria belonging to Order Lactobacillales, Family Acetobacteraceae, and / or ascomycetous filamentous fungi.

4. The method according to any one of the preceding claims, wherein the fermentation mixture contains 10-40 mg sugar / 100 mg dry weight of the fermentation mixture, preferably 15-35 mg sugar / 100 mg dry weight of the fermentation mixture, said sugar preferably comprising one or more of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose, lactose, and / or maltotriose, more preferably at least one or more of glucose, fructose, and / or sucrose.

5. The method according to claim 4, wherein the sugar in the fermentation mixture is provided by the cultured plant cells.

6. The method according to any one of the preceding claims, comprising adjusting one or more of temperature, pH, water content and / or solid matter content, especially content of the cultured plant cells, and / or O2 concentration of the fermentation mixture.

7. The method according to any one of the preceding claims, wherein forming the fermentation mixture comprises: inoculating the cultured plant cells with 102-1010cfu / g, preferably 103-109cfu / g, more preferably 104-108cfu / g, even more preferably 105-107cfu / g of the fermenting microbe(s), said inoculation preferably comprising mixing the microbe(s) with the cultured plant cells, and optionally adjusting water content and / or sugar content of the fermentation mixture.

8. The method according to any one of the preceding claims, wherein providing sterile cultured plant cells suitable for human consumption comprises: culturing plant cells in a cell culture medium followed by harvesting of cultured plant cells, the harvesting comprising separating cultured plant cells from spent cell culture medium and optionally washing the separated plant cells, and optionally recovering spent cell culture medium and adding of the spent cell culture medium to the fermentation mixture to adjust sugar and / or water content, preferably both, of the fermentation mixture.

9. The method according to any one of the preceding claims, comprising subjecting the fermented plant cells to further processing, such as homogenisation, drying, heating, and / or roasting.

10. The method according to any one of the preceding claims, comprising drying the fermented plant cells and roasting the dried fermented plant cells, optionally followed by extracting or mixing with liquid, preferably water.

11. The method according to any one of the preceding claims, wherein the method comprises incorporating the fermented plant cells optionally after further processing, such as drying, heating, roasting, and / or homogenisation, in a food product or a beverage, preferably food product.

12. The method according to any one of the preceding claims, wherein the cultured plant cells are cultured coffee cells, such as cultured Coffea arabica cells, or cultured cocoa cells.

13. The method according to claim 12, wherein the method is a method for making coffee comprising: providing sterile cultured coffee cells suitable for human consumption; fermenting the cultured coffee cells with fermenting microbe(s) suitable for human consumption in a fermentation mixture comprising 5-50 wt-% of the cultured coffeecells, based on the dry weight of the cultured coffee cells and the total weight of the fermentation mixture, and inoculated with 102- 1 O10cfu / g of the fermenting microbes to obtain fermented coffee cells; drying the fermented coffee cells to obtained dried coffee cells; and roasting the dried coffee cells preferably at a temperature within a range from 80 °C to 350 °C, more preferably within a range from 150 °C to 300 °C, even more preferably within a range from 200 °C to 250 °C, to obtain roasted coffee cells.

14. The method according to claim 13, comprising extracting coffee beverage from the roasted coffee cells.

15. Fermented cultured plant cells obtainable by a method according to any one of claims1-14.

16. A food product or a beverage, preferably food product, comprising fermented cultured plant cells according to claim 15.

17. Use of fermented cultured plant cells according to claim 15 to prepare a beverage or a food product, preferably beverage.

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