Fermented fructus cannabis beverage

Abstract

The present invention relates to a beverage composition comprising fermented cannabis or a fermented cannabis extract. A further aspect of the invention is a method of preparing a beverage composition.

Description

Technical Field

[0001] The present invention relates to a beverage composition comprising fermented hemp seed or fermented hemp seed extract. Another aspect of the present invention is a method of preparing the beverage composition. Background Art

[0002] In many jurisdictions, hemp can be legally consumed in various forms. The consumption of legal hemp (for example, by brewing an infusion from the leaves and flowers of hemp) is becoming increasingly popular not only for medicinal purposes but also as a pleasant beverage with perceived health benefits. However, hemp infusions have a strong earthy and herbal flavor that not all consumers like. Many people wish that beverages have new flavors, but are reluctant to drink beverages that add flavorings, especially synthetic flavorings or sugary juices. Therefore, providing a hemp beverage with a new flavor and a less intense earthy / hempy flavor would be commercially valuable.

[0003] Any reference in this specification to prior art documents should not be taken as an admission that such prior art is well-known or forms part of the common general knowledge in the art. As used in this specification, the words "include", "comprising" and similar words should not be construed as having an exclusive or exhaustive meaning. In other words, they are intended to mean "including, but not limited to". Summary of the Invention

[0004] One object of the present invention is to improve the prior art and to provide a new product that overcomes at least some of the above-mentioned inconveniences, or at least to provide a useful alternative. This object of the present invention is achieved by the subject matter of the independent claims. The dependent claims further develop the concept of the invention.

[0005] Therefore, in a first aspect, the present invention provides a beverage composition comprising fermented hemp seed or fermented hemp seed extract, wherein the weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000.

[0006] A second aspect of the present invention provides a container for use in a beverage preparation device, the container containing a beverage composition of the present invention.

[0007] In a third aspect, the present invention provides a method of preparing a beverage composition, the method comprising:

[0008] a. soaking hemp seed in water to form an aqueous hemp seed extract and soaked hemp;

[0009] as well as

[0010] b. fermenting the aqueous hemp seed extract in the presence or absence of the soaked hemp seed;

[0011] The fermentation is carried out by yeast including Pichia yeast.

[0012] Another aspect of the present invention provides use of hemp seeds fermented with Pichia kluyveri NYSC 5485 (CNCM I-5525) for producing a beverage.

[0013] By soaking the dried leaves in hot or cold water, hemp leaves can be made into beverages. These beverages have unique local flavors, including hay, wood, earth, anise, spicy and citrus notes. The inventors have surprisingly found that by fermenting with Pichia yeast, hemp beverages maintain a low ethanol content, but develop enhanced fresh fruity flavors, reducing the stewed herbs, earth and spicy notes. By fermenting with Pichia yeast, particularly by fermenting with Pichia kluyveri NYSC 5485, the pine woody, medicinal and grassy flavor characteristics of hemp that some consumers find unpalatable are reduced. DETAILED DESCRIPTION

[0014] Thus, the present invention relates in part to beverage compositions comprising (e.g., consisting of) fermented hemp seed or a fermented hemp seed extract, wherein the weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000, e.g., greater than 4000, e.g., greater than 8000, e.g., greater than 10000, e.g., greater than 12000, e.g., greater than 14000, further e.g., greater than 16000. The aldehyde 3-methylbutanal contributes a maltose, solvent-like aroma.

[0015] This ratio can be calculated based on the weight concentration in parts per million as follows:

[0016]

[0017] Higher esters, such as 3-methylbutyl acetate, are initially produced during the fermentation of sugars with Pichia pastoris, but as fermentation proceeds, higher alcohols, such as 3-methylbutanol, are produced. A high ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal corresponds to a pleasant flavor balance with reduced maltose, beer, and solvent notes in the fermented beverage, thereby masking the hemp note.

[0018] In the context of the present invention, the term hemp refers to the flowers, seeds and leaves of the plant Cannabis sativa. Hemp can be, for example, Cannabis sativa chemotype II, III, IV or V. It should be understood that the term hemp in the present invention does not refer to Cannabis sativa chemotype I, which is commonly known as marijuana. Hemp has low levels of the psychoactive component Δ 9 -Tetrahydrocannabinol (Δ9 -tetrahydrocannabinol (THC). For example, hemp according to the present invention may contain less than 0.2% THC on a dry matter basis. Hemp includes cannabidiol (CBD), which is associated with therapeutic effects but does not have psychoactive effects. Hemp is also a valuable source of polyphenols such as gallic acid, chlorogenic acid, coumaric acid, quercetin, and ellagic acid.

[0019] In addition to those cannabinoids provided by fermented hemp or fermented hemp extract, the beverage composition of the present invention may also include cannabinoids. Cannabinoids can be isomers and / or derivatives of cannabinoids. Cannabinoid derivatives can be cannabinoid acids. In addition to cannabidiols provided by fermented hemp or fermented hemp extract, the beverage composition of the present invention may also include cannabidiols.

[0020] In the context of the present invention, the term "fermentation" refers to a process in which the activity of microorganisms causes a change (usually a desired change) in a food or beverage. Fermentation can be carried out with yeast and / or bacteria. Fermentation can be anaerobic or aerobic. Fermentation is one of the oldest ways to preserve and fortify food.

[0021] The term "fermented hemp" refers to hemp that has undergone a process in which the activity of a microorganism, such as a yeast, causes chemical changes (usually desired changes) in the organic components of the hemp. Fermented hemp can be fermented in an aqueous medium, for example, with yeast. Fermented hemp can be dry hemp that is added to an aqueous fermentation medium and fermented, then separated from the aqueous fermentation medium and dried. Fermented hemp can be fermented in a "solid state," for example, by adding a starter culture of yeast to hemp leaves (e.g., fresh hemp leaves).

[0022] The term "hemp extract" refers to a substance extracted from hemp, such as an aqueous extract of hemp. The hemp extract can, for example, be an aqueous extract from hemp leaves and stems after oil has been removed from the leaves and stems. The term "fermented hemp extract" refers to a hemp extract in which the hemp extract has been fermented, such as an aqueous hemp extract in which the aqueous extract has been fermented (such as by yeast). The fermented hemp extract can be a dried fermented hemp extract.

[0023] In one embodiment, the weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal of a beverage composition (e.g., a beverage) is greater than 2000, such as greater than 3500. The aldehyde 2-methylbutanal contributes maltose, solvent-like notes. This ratio can be calculated in parts per million based on weight concentration as follows:

[0024]

[0025] Higher esters, such as 2-methylbutyl acetate, are initially produced during the fermentation of sugars with Pichia pastoris, but as fermentation proceeds, higher alcohols, such as 2-methylbutanol, are produced. A high ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal corresponds to a pleasant flavor balance with reduced maltose, beer, and solvent notes in the fermented beverage, thereby masking the hemp note with these flavor markers.

[0026] In one embodiment, the beverage composition (e.g., beverage) has a weight ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate, and 2-phenylethanol to 2,3-butanedione of greater than 50, such as greater than 250, such as greater than 300, such as greater than 350, and further such as greater than 400. This ratio can be calculated based on the weight concentration in parts per million as follows:

[0027]

[0028] The compound 2,3-butanedione is also known as diacetyl. It contributes to buttery or fatty notes. A high ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate, and 2-phenylethanol to 2,3-butanedione corresponds to enhanced fruity and floral notes without the buttery and fatty notes that can be perceived as off-flavors during fermentation.

[0029] In one embodiment, the beverage composition comprises less than 10 g / kg acetic acid on a dry basis, for example less than 1 g / kg acetic acid on a dry basis, and further for example less than 0.5 g / kg acetic acid on a dry basis. The sour "note" in the flavor profile of beverages such as kombucha or kefir is not always desired by consumers seeking a milder flavor profile.

[0030] Many consumers seek beverages with complex, fresh flavors as an alternative to alcoholic beverages. In one embodiment, the beverage composition has an ethanol content of less than 1.2% by weight, such as less than 0.5% by weight, such as less than 0.2% by weight, and further such as less than 0.05% by weight. The beverage composition can be selected from the group consisting of a ready-to-drink beverage, a beverage liquid concentrate, a soluble beverage powder, a dried aromatic plant material, and combinations thereof.

[0031] Ready-to-drink beverage can be fermented hemp seed infusion or water flavored with fermented hemp seed. Ready-to-drink beverage can comprise hemp seed between 2 % by weight and 30 % by weight, for example, hemp seed between 4 % by weight and 15 % by weight. Ready-to-drink beverage can comprise other components in addition, such as flavoring agent or stabilizer. Ready-to-drink beverage can be carbonated. Ready-to-drink beverage can comprise fruit or juice in addition, such as strawberry, citrus fruit (for example, orange, grapefruit, lemon, sour orange, four seasons orange, bergamot, clementine, tangerine or Japanese pomelo), peach, pomegranate, watermelon, blackberry, raspberry, persimmon or cranberry. Ready-to-drink beverage can comprise herbal medicine in addition, such as mint, verbena or basil. Ready-to-drink beverage can comprise flower extract in addition, such as hibiscus or rose. Ready-to-drink beverage can comprise vegetable juice or vegetable extract in addition, such as tomato, cucumber, kale and celery. Ready-to-drink beverage can comprise plant infusion in addition. Ready-to-drink beverages may additionally contain dietary fiber, such as inulin, wheat dextrin, resistant starch and beta-glucan, for example beta-glucan from oats or barley. Ready-to-drink beverages may contain added vitamins and minerals. Ready-to-drink beverages may contain replicating or non-replicating beneficial microorganisms. Beneficial microorganisms may be, for example, probiotics. Ready-to-drink beverages may also contain cannabidiol in addition to the cannabidiol provided by fermented hemp seeds or fermented hemp seeds extract. Ready-to-drink beverages of the present invention may contain a total of between 10 mg and 100 mg of cannabidiol / liter, for example between 20 mg and 60 mg of cannabidiol / liter.

[0032] One aspect of the present invention provides use of the beverage composition of the present invention for preparing a beverage.

[0033] The beverage liquid concentrate can be a concentrated fermented hemp seed infusion. The beverage liquid concentrate can also include other components, such as flavorings or stabilizers. The beverage liquid concentrate can include between 4% by weight and 60% by weight of the hemp seed, for example, between 8% by weight and 30% by weight of the hemp seed. The beverage liquid concentrate can also include fruit or juice, such as strawberry, citrus fruit (for example, orange, grapefruit, lemon, sour orange, citrus fruit, bergamot, clementine, tangerine or Japanese pomelo), peach, pomegranate, watermelon, blackberry, raspberry, persimmon or cranberry. The beverage liquid concentrate can also include herbs, such as mint, verbena or basil. The beverage liquid concentrate can also include flower extracts, such as hibiscus or rose. The beverage liquid concentrate can also include vegetable juice or vegetable extracts, such as tomato, cucumber, kale and celery. The beverage liquid concentrate may further comprise dietary fiber, such as inulin, wheat dextrin, resistant starch, and beta-glucan, for example, from oats or barley. The beverage liquid concentrate may further comprise a plant infusion. The beverage liquid concentrate may comprise added vitamins and minerals. The beverage liquid concentrate may comprise replicating or non-replicating beneficial microorganisms. The beneficial microorganisms may be, for example, probiotics.

[0034] The beverage preparation device (for example beverage preparation machine) that holds extractable portioning ingredients provides a convenient method for preparing beverage.This type of portioning ingredients is usually packaged in a container, and this container is constructed as for example pod, pad, bag, pouch, capsule etc. An aspect of the present invention provides the container for the beverage preparation device, and this container contains beverage composition of the present invention.Container is used for preparing beverage when inserting in the beverage preparation device.Container can for example be beverage capsule, and other configurations. In one embodiment, container contains beverage composition of the present invention.For example, container can contain dry fermented fructus hempen or dry fermented fructus hempen extract.This container can contain dry fermented fructus hempen and / or dry fermented fructus hempen extract, and these can be combined with other dry ingredients (such as tea, herbal tea, dried fruit, soluble coffee or roasted and ground coffee).

[0035] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising:

[0036] a. soaking hemp seed in water to form an aqueous hemp seed extract and soaked hemp seed; and

[0037] b. fermenting the aqueous hemp seed extract in the presence or absence of the soaked hemp seed;

[0038] The fermentation is carried out by yeast including Pichia pastoris.

[0039] The yeast may be primarily Pichia pastoris, for example, more than 50% of the microbial colony forming units present during fermentation may be Pichia pastoris. For example, more than 50% of the yeast colony forming units may be Pichia pastoris, for example, more than 60%, 70%, 80%, or 90% of the yeast colony forming units may be Pichia pastoris. For example, substantially all of the yeast colony forming units present during fermentation may be Pichia pastoris, and as a further example, substantially all of the microbial colony forming units present during fermentation may be Pichia pastoris.

[0040] The beverage composition prepared by the method of the present invention can be a hemp seed beverage, for example, a beverage comprising fermented hemp seed or fermented hemp seed extract. The beverage composition prepared by the method of the present invention can have an ethanol content of less than 1.2% by weight, for example, less than 0.5% by weight, for example, less than 0.2% by weight, further for example, less than 0.05% by weight. In one embodiment, the beverage composition prepared by the method of the present invention is a beverage composition of the present invention.

[0041] The term soaking refers to submerging or immersing a substance in a liquid. The hemp seed can be soaked in water for at least 2 minutes, for example, at least 3 minutes, for example, at least 4 minutes, for example, at least 5 minutes, for example, at least 10 minutes, for example, at least 15 minutes, for example, at least 20 minutes, for example, at least 30 minutes, for example, at least 60 minutes, or further, for example, at least 120 minutes. During the soaking process, the components of the hemp seed are extracted into the water. The soaking can be carried out at a temperature between 4°C and 98°C, for example, between 20°C and 95°C, for example, between 60°C and 95°C. Although the hemp seed contains some fermentable sugars, additional fermentable sugars, such as sucrose or glucose, can be added to the aqueous hemp seed extract. The fermentable sugar can be fructose or glucose. The fermentable sugar can be in the form of honey. For example, the fermentable sugar can be added to the aqueous hemp seed extract at a level between 2% and 10% by weight, for example, between 3% and 7% by weight. The hemp seed can be dried and / or ground before soaking.

[0042] When fermentation will be carried out under the situation that does not exist through soaking fructus hemp, for example, can use continuous countercurrent extraction system that fructus hemp is soaked in water.Another kind of selection is to use batch process to soak fructus hemp in water, uses filtering system to separate the fructus hemp through soaking from aqueous fructus hemp extract.During soaking period, the fructus hemp of butt and the ratio of water can be between by weight 1:1 and 1:100, for example, between by weight 1:2 and 1:95, for example, between by weight 1:10 and 1:90, for example, between by weight 1:30 and 1:70, further for example between 1.3 and 1:6.

[0043] After soaking, the temperature of the aqueous hemp seed extract is adjusted to a temperature suitable for yeast growth, for example, between 20°C and 37°C, for example, between 25°C and 35°C, for example, between 28°C and 32°C. The temperature can be adjusted, for example, using a laminar flow heat exchanger. Yeast is added to the aqueous hemp seed extract to start fermentation. The fermentation of the aqueous hemp seed extract can be carried out at a temperature between 25°C and 35°C, for example, between 28°C and 32°C. The fermentation of the aqueous hemp seed extract can be carried out for at least 2 hours, for example, at least 6 hours, for example, at least 12 hours, for example, at least 24 hours, and further for example, at least 48 hours.

[0044] Fermentation may be carried out at a pH between 3 and 8, such as between 4 and 6.

[0045] Aqueous hemp seed extract can be fermented in the presence of other components (such as flavorings or stabilizers). Aqueous hemp seed extract can be fermented in the presence of other components (such as fruits or juices, for example strawberries, citrus fruits (for example, oranges, grapefruits, lemons, sour oranges, four-season oranges, bergamots, clementines, tangerines or Japanese pomelo), peaches, pomegranates, watermelons, blackberries, raspberries, persimmons or cranberries). Aqueous hemp seed extract can be fermented in the presence of other components (such as herbs, for example mint, verbena or basil). Aqueous hemp seed extract can be fermented in the presence of other components (such as flower extracts, for example hibiscus or rose). Aqueous hemp seed extract can be fermented in the presence of other components (such as vegetable juices or vegetable extracts, for example tomato, cucumber, kale or celery). Aqueous hemp seed extract can be fermented in the presence of other components (such as plant infusions). Aqueous hemp seed extract can be fermented in the presence of other components (such as added vitamins and minerals).

[0046] Fermentation can be carried out in the presence of a nitrogen source such as yeast extract or yeast peptone. Fermentation can be carried out in the presence of added amino acids such as valine, leucine, isoleucine and / or phenylalanine, for example, at a level of between 0.05% and 0.1% by weight of the aqueous hemp seed extract.

[0047] Fermentation can be carried out under anaerobic, microaerophilic or aerobic conditions. For example, the oxygen partial pressure in the fermenter can be 0% to 30%, such as 1% to 20%, and further such as 2% to 10%.

[0048] Preferably, the hemp seeds are not subjected to chemical processing such as hydrolysis prior to fermentation.

[0049] Fermentation can be carried out by 3 CFU / g, for example at least 10 4 CFU / g, for example at least 10 5 CFU / g, for example at least 10 6 CFU / g, further such as at least 10 7 The initial combined yeast level of CFU / g was determined by adding one or more yeasts.

[0050] The fermentation may be carried out in the absence of acetic acid bacteria to avoid producing a sour "note" in the flavor profile, such as that found in kombucha or kefir. In one embodiment, the fermentation is carried out in the absence of lactic acid bacteria.

[0051] In one embodiment, the fermentation is carried out in the absence of a Saccharomyces subspecies. For example, the fermentation can be carried out in the absence of Saccharomyces cerevisiae. Saccharomyces cerevisiae is used in winemaking and beer brewing. In addition to producing alcohol, which may be undesirable, Saccharomyces cerevisiae produces "fermented," wine-like, yeasty flavor notes rather than fresh, fruity notes.

[0052] In one embodiment, the fermentation is performed in the absence of yeast selected from the group consisting of Zygosaccharomyces bailii, Schizosaccharomyces pombe, Torulaspora delbreuckii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis, and Candida stellate. These yeasts are typically present in kombucha "tea" cultures.

[0053] The inventors have found that fermenting hemp seeds with Pichia kluyveri produces particularly good results. Beverage compositions containing hemp seeds or hemp seed extracts fermented with Pichia kluyveri have a pleasant fruity flavor with less smoky hemp notes compared to unfermented beverages. Pichia kluyveri is naturally found throughout the world, for example on olives, grapes, and coffee. In one embodiment, the Pichia yeast is primarily Pichia kluyveri, for example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri. For example, more than 50% of the yeast colony forming units can be Pichia kluyveri, for example, more than 60%, 70%, 80%, 90% of the yeast colony forming units can be Pichia kluyveri. For example, substantially all of the yeast colony forming units present during fermentation can be Pichia kluyveri. For further example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri.

[0054] Pichia kluyveri NCYC 246 (alternatively named CBS188) is a type strain of Pichia kluyveri and is publicly available from, for example, the National Collection of Yeast Cultures, Quadram Institute Bioscience, Norwich, UK.

[0055] In one embodiment, the Pichia yeast is Pichia kluyveri NCYC 246. For example, greater than 50% of the yeast colony forming units present during fermentation can be Pichia kluyveri NCYC 246. For example, greater than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri NCYC 246. For example, substantially all of the yeast colony forming units present during fermentation can be Pichia kluyveri NCYC 246. For further example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri NCYC 246.

[0056] Pichia kluyveri NCYC 2781 (alternatively named Maize Silage Isolate 86) is publicly available, for example from the National Collection of Yeast Cultures, Quadram Institute Bioscience, Norwich, UK.

[0057] In one embodiment, the Pichia yeast is Pichia kluyveri NCYC 2781. For example, greater than 50% of the yeast colony forming units present during fermentation can be Pichia kluyveri NCYC 2781. For example, greater than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri NCYC 2781. For example, substantially all of the yeast colony forming units present during fermentation can be Pichia kluyveri NCYC 2781. For further example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri NCYC 2781. Pichia kluyveri is widely distributed in nature and is found, for example, on fruits such as olives, grapes, and coffee. Pichia kluyveri NYSC 5485, collected and isolated from coffee fermentation in Nicaragua, was deposited on June 23, 2020, at the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, France, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, with the deposit number CNCM I-5525.

[0058] In one embodiment, the yeast is Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example, more than 50% of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example, substantially all of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM 1-5525), and further for example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM 1-5525).

[0059] The present inventors have found that particularly good results are obtained by fermenting hemp seeds with Pichia kluyveri NYSC 5485. Fermenting hemp seeds with Pichia kluyveri NYSC 5485 results in a higher weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal, corresponding to an enhanced flavor balance.

[0060] Fermentation can be carried out by 3 CFU / g, for example at least 10 4 CFU / g, for example at least 10 5 CFU / g, for example at least 10 6 CFU / g, further such as at least 10 7 The initial combined yeast level of CFU / g is carried out by adding Pichia kluyveri, for example Pichia kluyveri NYSC 5485 (CNCM 1-5525).

[0061] The genetic sequence of Pichia kluyveri NYSC 5485 (CNCM I-5525) was analyzed using PacBio sequencing technology. DNA was purified using the Qiagen Gentra Puregene Yeast Kit. Twelve DNA fragments were identified. These DNA fragments may represent individual chromosomes; 11 chromosomes and one mitochondrial DNA (SEQ ID NO:4). The genetic sequences are electronically submitted as SEQ ID NO:1-SEQ ID NO:12.

[0062] In one embodiment of the methods of the invention, Pichia pastoris comprises a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 1, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 2, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 3, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 5, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 6, a genetic sequence that is at least 99% identical to SEQ ID NO: 7, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 8, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 9, a genetic sequence that is at least 99% identical (e.g., at least 99% identical) to SEQ ID NO: 10, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 11, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 12, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 13, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 14, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 15 NO:11 has a genetic sequence that is at least 98% identical (eg, at least 99% identical) and a genetic sequence that is at least 98% identical (eg, at least 99% identical) to SEQ ID NO:12.

[0063] In one embodiment of the methods of the invention, the Pichia pastoris comprises a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 1, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 2, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 3, a genetic sequence that is at least 99.5% identical to SEQ ID NO: 4, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 5, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 6, a genetic sequence that is at least 99% identical to SEQ ID NO: 7, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 8, a genetic sequence that is at least 98% identical (e.g., at least 99% identical) to SEQ ID NO: 9, a genetic sequence that is at least 99% identical (e.g., at least 99% identical) to SEQ ID NO: 10, a genetic sequence that is at least 99% identical (e.g., at least 99% identical) to SEQ ID NO: 11. NO:11 has a genetic sequence that is at least 98% identical (eg, at least 99% identical) and a genetic sequence that is at least 98% identical (eg, at least 99% identical) to SEQ ID NO:12.

[0064] In one embodiment, the Pichia pastoris comprises a genetic sequence having at least 98% (e.g., at least 99%) overall nucleotide identity to the combination of SEQ ID NOs: 1-12. The term "combination of SEQ ID NOs: 1-12" means that all individual DNA fragments are compared for sequence identity as if joined into a single sequence.

[0065] In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 1. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 2. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 3. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 5. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 6. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 99% identical to SEQ ID NO: 7. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 8. In one embodiment, Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 9. In one embodiment, the Pichia pastoris comprises a genetic sequence that is at least 99% identical to SEQ ID NO: 10. In one embodiment, the Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 11. In one embodiment, the Pichia pastoris comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 12.

[0066] The name of the yeast may change over time. It will be appreciated that the yeast according to the methods of the invention may comprise the genetic sequence identity described in the preceding paragraphs for the Pichia pastoris of the invention, regardless of the name applied to the yeast.

[0067] One aspect of the present invention provides a method for preparing a beverage composition, comprising: a) soaking hemp seeds in water to form an aqueous hemp seed extract and soaked hemp seeds; and b) fermenting the aqueous hemp seed extract in the presence or absence of the soaked hemp seeds; wherein the fermentation is performed by a yeast comprising a genetic sequence having at least 98% (e.g., at least 99%) overall nucleotide identity to a combination of SEQ ID NOs: 1-12.

[0068] In one embodiment, after fermentation, the aqueous hemp seed extract is separated from the soaked hemp seed. For example, a filtration system can be used to separate the soaked hemp seed from the aqueous hemp seed extract. After fermentation, the aqueous hemp seed extract becomes a fermented hemp seed extract.

[0069] Fermented fructus hemp extract can be packaged as instant drink, for example, is packaged in a bottle or a jar. Fermented fructus hemp extract can have the concentration of the total dissolved solids between 2 % by weight and 60 % by weight, for example the concentration of 2 % by weight to 25 % by weight total dissolved solids. Before the fermented fructus hemp extract is packaged as instant drink, can add extra batching, such as flavoring and preservative. Fermented fructus hemp extract can be heat-treated before packaging to deactivate or kill yeast and any corrupt organisms. Fermented fructus hemp extract can before packaging, pass through a filter to remove yeast and any corrupt organisms.

[0070] The fermented hemp seed extract can be combined with other ingredients such as fruit or fruit juice, herbal extracts, flower extracts, vegetable juice or extracts, plant extracts, vitamins and minerals, and beneficial microorganisms. Cannabidiol can be added to the fermented hemp seed extract, for example, by emulsifying the cannabidiol into an aqueous solution of the fermented hemp seed extract with the aid of an emulsifier.

[0071] In one embodiment, the aqueous hemp seed extract is concentrated after fermentation, for example, packaged as a liquid concentrate suitable for being diluted by the consumer before consumption. The aqueous hemp seed extract can be concentrated into an intermediate product for, for example, manufacturing ready-to-drink products at different locations. The fermented hemp seed extract can have a concentration of 25 % by weight to 60 % by weight total dissolved solids, further for example a concentration of 50 % by weight to 60 % by weight total dissolved solids. The dissolved solids can, for example, be measured by: passing the extract through a fine filter to remove undissolved solids, weighing the filtrate to determine the total weight, evaporating the filtrate, and then weighing the residue to obtain the weight of the dissolved solids. Additional ingredients (such as flavorings and preservatives) can be added to the liquid concentrate.

[0072] The aqueous hemp seed extract (for example fermented hemp seed extract) after fermentation can be concentrated to form a powder, such as a soluble beverage powder. For example, the aqueous hemp seed extract (fermented hemp seed extract) after fermentation can be dried by spray drying or freeze drying to form a soluble beverage powder. For example, the aqueous hemp seed extract after fermentation can be spray dried together with a carrier (such as maltodextrin) to form a soluble beverage powder.

[0073] The fermented aqueous hemp seed extract can be spray-dried with other ingredients such as fruit or juices, herbal extracts, flower extracts, vegetable juices or extracts, plant infusions, vitamins and minerals, and beneficial microorganisms.

[0074] The soluble beverage powder can be packaged and sold as is, or it can be used as the intermediate product for, for example, manufacturing instant drinkable products in different locations. The soluble beverage powder can be filled in the container for the beverage preparation device. The soluble beverage powder can be combined with other ingredients (for example dry ingredients, such as tea, herbal tea, dried fruit, soluble coffee or roasted and ground coffee). The soluble beverage powder can be combined with other ingredients (such as fruit powder, herbal medicine, vegetable powder, vitamins, minerals and beneficial microorganisms). For example, the soluble beverage powder can be combined with other ingredients and filled in the container for the beverage preparation device. The soluble beverage powder can be combined with milk powder or beverage creamer powder and optional sugar to form a beverage mix. After fermenting the aqueous hemp seed extract in the presence of soaked hemp seed, the soaked hemp seed becomes the fermented hemp seed. In one embodiment, after fermentation, from the aqueous hemp seed extract, separate the soaked hemp seed that exists during the fermentation of the aqueous hemp seed extract, for example use a rotary dryer to dry it then. The dried hemp seed through soaking can be filled into the container for beverage preparation device, or filled into permeable bag (such as " tea bag "). The dried hemp seed through soaking can be combined with other dry ingredients (such as tea, herbal tea, dried fruit, soluble coffee or roasted and ground coffee). In the context of the present invention, the term tealeaves refers to the dry leaf, leaf bud, twig or stem of plant tea tree (Camellia sinensis). Tea can be, for example, Chinese tea (C.sinensis var.sinensis) or Assam tea (C.sinensis var.assamica). The term "herbal tea" refers to the infusion made from the dry flower, fruit, leaf, seed or root of the plant except tea tree.

[0075] In one embodiment, for example, by freeze drying or spray drying, the soaked fructus hemp existing during the aqueous fructus hemp extract fermentation is dried together with the aqueous fructus hemp extract after the fermentation. The dry fructus hemp through soaking (for example dry fermented fructus hemp) can be filled into a container for a beverage preparation device together with the aqueous fructus hemp extract (for example dry fermented fructus hemp extract) after fermentation, or filled into a permeable bag (such as a "tea bag"). The dry fructus hemp through soaking and the aqueous fructus hemp extract after fermentation can be combined with other dry ingredients (such as tea, herbal tea, dried fruit, soluble coffee or roasted and ground coffee).

[0076] One aspect of the present invention provides use of hemp seeds fermented with Pichia kluyveri NYSC 5485 (CNCM I-5525) for producing a beverage.

[0077] Fermented hemp according to the present invention can be obtained in a "solid-state" fermentation, for example by adding a starter culture of Pichia pastoris to hemp leaves (e.g., fresh hemp leaves that have not yet dried). The term "solid-state" fermentation refers to fermentation performed on solid material. Liquid water is present in solid-state fermentation, but this term is used in contrast to liquid fermentation, which can occur in an aqueous solution in a fermentation vessel.

[0078] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising:

[0079] A starter culture of yeast is added to the hemp leaves, wherein the starter culture provides at least 10 4 CFU (e.g. at least 10 5 CFU, further such as at least 10 6 CFU) of yeast / gram of hemp leaf;

[0080] fermented hemp leaves;

[0081] Drying fermented hemp leaves to form fermented hemp; and any of the following;

[0082] Extracting the fermented hemp with water to form a ready-to-drink beverage, or

[0083] The fermented hemp is filled into a container, such as a container that is used to prepare a beverage when inserted into a beverage preparation device.

[0084] The yeast starter culture can be added to the hemp leaves as a liquid or powder. When the yeast starter culture is added, the hemp leaves can have a moisture content of between 45% and 55% by weight. The leaves can be treated with steam before fermentation to reduce the amount of environmental microorganisms before the yeast is added. Fermentation can be performed at a solids content greater than 40% by weight, such as greater than 45% by weight, and further, such as greater than 50% by weight.

[0085] When some fermentation continues, the fermented hemp can be dried. For example, hemp leaves can be placed in a container (such as a jar), a starter culture is added, and then the fermentation is carried out for 1 to 4 days, after which the fermented hemp is taken out of the container and dried. Further for example, before the hemp leaves are slowly dried, for example on a shelf or on a hanging tray, a starter culture can be added to the hemp leaves. Once fermentation begins, the hemp leaves can be slowly dried while continuing to ferment. For example, the fermented hemp leaves can be dried to a moisture content of less than 20 weight % (for example, less than 15 weight %) within a period between 1 day and 30 days, for example, between 4 days and 20 days. The drying temperature can be lower than about 40 ° C, for example, between about 25 ° C and 35 ° C. A fan can be used to blow dry air onto the material until the hemp leaves are dry. The fermented hemp leaves can be dried in the sun.

[0086] The yeast may be Pichia pastoris, such as Pichia kluyveri, further such as Pichia kluyveri NYSC5485.

[0087] The container for preparing a beverage, when inserted into a beverage preparation device, may be in the form of a capsule, a pad, a sachet, a pouch, a capsule or the like.

[0088] The ready-to-drink beverage can be filled into bottles or cans. The fermented hemp can be combined with other extractable materials (such as tea, herbal tea, dried fruit, soluble coffee or roast and ground coffee) and then extracted with water to form the ready-to-drink beverage.

[0089] When inserted into a beverage preparation device, the fermented hemp can be combined with tea, herbal tea, dried fruit, soluble coffee, or roast and ground coffee in a container for preparing a beverage.

[0090] Those skilled in the art will appreciate that they are free to combine all features of the present invention disclosed herein. Specifically, features described for the product of the present invention may be combined with methods of the present invention, and vice versa. In addition, features described for different embodiments of the present invention may be combined. If known equivalents exist for specific features, such equivalents are incorporated herein, as if these equivalents were explicitly mentioned in this specification.

[0091] Further advantages and features of the invention will become apparent upon reference to the accompanying drawings and non-limiting examples.

[0092] Example

[0093] Example 1: Fermented Hemp Method

[0094] Organic hemp leaves (Coop Naturaplan, Switzerland) were used as the starting material for the preparation of the infusion. A hot infusion was first performed by adding 20 g of the dry starting material to 1000 ml of water at 100° C. and soaking for 10 minutes. The infusion was then placed in an ice bath until the temperature dropped below 30° C. The infusion was then filtered through a sieve to remove excess debris and / or leaves. The filtrate (aqueous hemp extract) was divided equally between the control (Ref) and the different fermentation variants.

[0095] Fermented hemp was prepared by fermenting infusions with glucose (2% m / m) and different starter cultures, including: Pichia kluyveri NYSC 5485 (CNCM I-5525), Pichia kluyveri NCYC 246 (a type strain of Pichia kluyveri), Pichia kluyveri 2781, the commercial brewing yeast Saccharomyces cerevisiae (SafAle S-33, LeSaffre, France), and a symbiotic culture of bacteria and yeast (SCOBY) pellicles from kombucha fermentation. All strains (except the SCOBY) were grown in food-grade yeast-peptone broth in glycerol vials and inoculated into a reference hemp brewery with an optical density (OD at 600 nm) of 0.5. All inoculated infusions were stored at 30°C under microaerophilic conditions for 24 hours. After incubation, each fermented infusion was filter-sterilized (0.2 μm).

[0096] Example 2: Sensory Analysis of Hemp Fermented with Different Yeasts

[0097] The sensory profiles of the different variations of hemp infusions were tasted. The control tasted hemp with smoky, herbal, woody, and earthy notes. The hemp infusion fermented with S. cerevisiae had a strong maltose and acidic profile with off-flavors. The hemp fermented with the SCOBY had a very strong acidity. The hemp infusions fermented with the two different Pichia yeasts were fruity, with greatly reduced hemp / smoky notes compared to the control. No buttery, sour, or fatty off-flavors were identified in the hemp fermented with Pichia yeast. The hemp fermented with Pichia kluyveri NYSC 5485 can be considered to have a more balanced flavor profile than the hemp fermented with Pichia kluyveri NCYC 246.

[0098] Example 3: Analysis of flavor profiles focused on the presence of aromatic compounds in hemp fermented with different yeasts .

[0099] Quantitative volatile profiling of hemp infusions was performed by headspace / solid phase microextraction coupled to chromatography and mass spectrometry (HS / SPME-GC-MS) using a gas chromatograph (Agilent Technologies 8890 GC system) coupled to a mass spectrometer (Agilent Technology 7010BTQ).

[0100] Initially, 1.0 ml of hemp infusion sample was incubated in a 10-mL screw-cap headspace vial at 30°C for 10 minutes, followed by extraction using an SPME fiber (PDMS / DVB, Chromatography (Supelco)) at 30°C for 10 minutes. Prior to analysis, all vials were placed in a tray cooled at 6°C. Volatiles were thermally desorbed from the SPME fiber at 250°C in splitless mode and separated using a capillary column (DB-WAX, Agilent). The gas chromatograph oven temperature was initially set to 35°C for 5 minutes, then increased to 230°C at 4°C / min, and then held at 230°C for 10 minutes. Helium was used as the carrier gas with a flow rate of 1 mL / min. Target compounds were identified by pure standards and quantified in MSD ChemStation software (Agilent Technologies). All samples were measured in duplicate.

[0101] The following table lists the levels of 3-methylbutyl acetate, 3-methylbutanol, 3-methylbutanal, 2-methylbutyl acetate, 2-methylbutanol, 2-methylbutanal, isobutyl acetate, 2-phenylethyl acetate, 2-phenylethanol, 2,3-butanedione, ethanol, and acetic acid in parts per million after 24 hours.

[0102]

[0103] The samples fermented with the SCOBY had high levels of acetic acid, corresponding to 190 g / kg on a dry basis.

[0104] The weight ratios of the aroma components A, B and C are given in the table below.

[0105]

[0106]

[0107]

[0108] Samples fermented with Pichia pastoris (NYSC5485, NCYC 2781, and NCYC246) showed higher ratios A, B, and C than the control and samples fermented with S. cerevisiae or SCOBY. Pichia kluyveri NYSC 5485 provided higher values ​​for the ratios A, B, and C than the model strain Pichia kluyveri NCYC 246 and higher values ​​for the ratios A and B than Pichia kluyveri NCYC 2781.

[0109] The levels of terpenes and terpenoids present in the samples were measured. The following target compounds were identified using the NIST library and retention indices. The peak area for each compound was integrated for the samples, and the ratio of fermented to unfermented samples is shown below. All of these compounds have been reported and detected in hemp extracts or hemp oil [VRLJ Bloemendal et al., Organic and Biomolecular Chemistry (Org. Biomol. Chem.), 18, 3203-3215 (2020). DOI 10.1039 / D0OB00464B] [AS Wanas et al., "Chemical Composition of Volatile Oils of Fresh and Air-Dried Buds of Cannabis chemovars, Their Insecticidal and Repellent Activities", Natural Product Communications, May 19, 2020. DOI 10.1177 / 1934578X20926729].

[0110] All samples were measured in duplicate.

[0111]

[0112] Terpenes and terpenoids are the main contributors to the "hempiness" characteristic, which is perceived as a combination of pine, medicinal, and grassy notes. After fermentation with Pichia kluyveri, the hempiness was significantly reduced. Pichia kluyveri NYSC 5485 was found to reduce these compounds associated with the hempiness characteristic more than the model strain Pichia kluyveri NCYC 246.

[0113] Example 4: Sensory Impact of Fragrance Ratios by Spiking Studies

[0114] Solutions were prepared by adding different levels of 3-methylbutyl acetate, 3-methylbutanol, and 3-methylbutanal. In one set of solutions, the compounds were added to water, while in another set, the compounds were added to an unfermented hemp extract control (Ref).

[0115] A six-person technical tasting panel tasted the samples and compiled their notes. The samples and notes are below.

[0116]

[0117] It is important to have the right balance between esters / alcohols and aldehydes. It has been observed that in hemp infusions, high levels of esters and low levels of aldehydes (e.g. a ratio A greater than 2000) can mask the "hemp-like" notes.

[0118] Example 5: Genetic sequence of Pichia kluyveri NYSC 5485 and model strain Pichia kluyveri Comparison of NCYC246 .

[0119] The genetic sequences of Pichia kluyveri NYSC 5485 (CNCM I-5525) and Pichia kluyveri NCYC 246 were analyzed using PacBio sequencing technology. DNA purification was performed using the Qiagen Gentra Puregene Yeast Kit. Twelve DNA fragments were identified for Pichia kluyveri NYSC 5485, and 15 DNA fragments were identified for the model strain Pichia kluyveri NCYC 246. The genetic sequence of Pichia kluyveri NYSC 5485 is electronically provided as SEQ ID NO:1–SEQ ID NO:12, and the genetic sequence of Pichia kluyveri NCYC 246 is provided as SEQ ID NO:13–SEQ ID NO:27. The mitochondrial DNA of Pichia kluyveri NYSC 5485 is provided as SEQ ID NO:4, and the mitochondrial DNA of Pichia kluyveri NCYC 246 is provided as SEQ ID NO:20.

[0120] The software OrthoANIu was used to compare the genetic sequence of Pichia kluyveri NYSC 5485 (CNCM 1-5525) with the genetic sequence of Pichia kluyveri NCYC 246. An overall average nucleotide identity of 97.91% was found.

[0121] The correspondence between the different sequences is given in the table below: Some sequences of Pichia kluyveri NYSC 5485 correspond most closely to combinations of sequences of Pichia kluyveri NCYC 246 and vice versa.

[0122]

[0123]

[0124] The model strain Pichia kluyveri NCYC 246 contains genetic sequences with the greatest identity to each of SEQ ID NOs: 1-12, as follows: SEQ ID NO: 1, 97.85%; SEQ ID NO: 2, 97.40%; SEQ ID NO: 3, 97.86%; SEQ ID NO: 4, 99.02%; SEQ ID NO: 5, 98.05%; SEQ ID NO: 6, 97.93%; SEQ ID NO: 7, 98.12%; SEQ ID NO: 8, 97.07%; SEQ ID NO: 9, 97.20%; SEQ ID NO: 10, 98.33%; SEQ ID NO: 11, 97.526%; SEQ ID NO: 12, 97.77%.

Claims

1. A beverage composition comprising fermented fructus hempen or fermented fructus hempen extract, wherein the weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000, wherein the beverage composition does not contain Δ 9 -Tetrahydrocannabinol (Δ 9 -tetrahydrocannabinol, THC).

2. The beverage composition according to claim 1, wherein the weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal is greater than 2000.

3. The beverage composition according to claim 1 or claim 2, wherein the weight ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate and 2-phenylethanol to 2,3-butanedione is greater than 50.

4. A beverage composition according to any one of claims 1 to 3, having an ethanol content of less than 1.2% by weight.

5. The beverage composition according to any one of claims 1 to 4, wherein the beverage composition is selected from the group consisting of: a ready-to-drink beverage, a beverage liquid concentrate, a soluble beverage powder, a dried aromatic plant material, and combinations of these.

6. A container for use in a beverage preparation device, the container containing a beverage composition according to any one of claims 1 to 5.

7. A method of preparing a beverage composition, the method comprising: a. soaking hemp seed in water to form an aqueous hemp seed extract and soaked hemp seed; b. fermenting the aqueous hemp seed extract in the presence or absence of the soaked hemp seed; The fermentation is carried out by yeast including Pichia.

8. The method according to claim 7, wherein the Pichia pastoris is Pichia kluyveri.

9. The method of any one of claims 7 to 8, wherein the Pichia pastoris comprises a genetic sequence having at least 98% overall nucleotide identity to the combination of SEQ ID NOs: 1-12.

10. The process according to any one of claims 7 to 9, wherein the fermentation is carried out in the absence of Saccharomyces cerevisiae.

11. The process according to any one of claims 7 to 10, wherein the fermentation is carried out in the absence of acetic acid bacteria.

12. The method of any one of claims 7 to 11, comprising separating the aqueous hemp seed extract from the soaked hemp seed after fermentation.

13. The method according to any one of claims 7 to 11, wherein the soaked hemp seeds present during the fermentation of the aqueous hemp seed extract are dried together with the aqueous hemp seed extract after fermentation.

14. The method according to any one of claims 7 to 13, wherein the beverage composition is according to any one of claims 1 to 5.

15. Use of hemp seeds fermented with Pichia kluyveri NYSC 5485 (CNCM I-5525) for producing beverages.

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