Fermented Hemp Beverage

Abstract

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

Description

Technical Field

[0001] The present invention relates to a beverage composition containing fermented hemp or a fermented hemp extract. A further aspect of the present invention is a method for preparing the beverage composition.

Background Art

[0002] Hemp can be legally consumed in various forms in many jurisdictions. For example, the legal consumption of hemp by brewing an extract from the leaves and flowers of hemp is becoming increasingly common not only for pharmaceutical use but also as an enjoyable beverage that is recognized for its health benefits. However, hemp extract has an earthy and herbaceous taste that is not popular among consumers in general. Many people desire a new flavor in beverages but do not prefer to consume beverages with added flavors, especially synthetic flavors, or fruit juice beverages containing sugar. Therefore, there is commercial value in providing a hemp beverage that has a novel flavor and does not have a strong earthy / hemp note.

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

Summary of the Invention

[0004] The problem of the present invention is to improve the current state of the art and provide a novel product that at least to some extent overcomes the above-mentioned inconveniences, or at least an effective alternative. The object of the present invention is achieved by the subject matter of the independent claims. The dependent claims further develop the concept of the present invention.

[0005] Thus, in a first aspect, the present invention provides a beverage composition comprising fermented hemp or a fermented hemp extract, wherein the weight ratio of the total 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 apparatus, the container containing the beverage composition of the present invention.

[0007] In a third aspect, the present invention provides a method for manufacturing a beverage composition, the method comprising: a. immersing hemp in water to produce a hemp aqueous extract and immersed hemp; and b. fermenting the hemp aqueous extract, either in the presence or absence of the immersed hemp, wherein the fermentation is carried out by yeast including Pichia yeast.

[0008] A further aspect of the present invention provides the use of hemp fermented with Pichia kluyveri NYSC5485 (CNCM I-5525) for manufacturing a beverage.

[0009] Hemp leaves can be made into a beverage by soaking the dried leaves in either hot or cold water. These beverages have characteristic flavors such as hay, woody, earthy, anise seed, spicy and citrus notes. The inventors have surprisingly found that through fermentation with Pichia yeast, the ethanol in the hemp beverage remains low, the stewed herb, earthy and spicy notes are reduced, and a flavor with enhanced cool fruityness is generated. The pine-like, medicinal-like and herb-like flavors characteristic of hemp, which some consumers find unpalatable, are reduced by fermentation with Pichia yeast, particularly by fermentation with Pichia kluyveri NYSC5485.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Thus, the present invention relates in part to a beverage composition comprising (e.g., consisting of) fermented hemp or fermented hemp extract, wherein the total weight ratio of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000, for example greater than 4000, for example greater than 8000, for example greater than 10000, for example greater than 12000, for example greater than 14000, and further for example greater than 16000. The aldehyde 3-methylbutanal contributes to a malty, solvent-like note.

[0011] The ratio can be calculated as follows from the weight concentrations in ppm units.

Equation

[0012] During the fermentation of sugar by Pichia, higher esters such as 3-methylbutyl acetate are first produced, but as the fermentation progresses, higher alcohols such as 3-methylbutanol are produced. A high ratio of the total of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal corresponds to a pleasant aroma balance that reduces the malty, beer, and solvent notes in the fermented beverage and masks the hemp note.

[0013] In the context of the present invention, the term "hemp" refers to the flowers, seeds, and leaves of the plant Cannabis sativa (Cannabis sativa). Hemp may be, for example, chemotype II, III, IV, or V of Cannabis sativa. It should be understood that in the present invention, the term "hemp" generally does not refer to chemotype I of Cannabis sativa, which is commonly called marijuana. Hemp has low levels of the psychoactive component Δ

[0013] , 9 , , , , ,

[0012] , - It contains tetrahydrocannabinol (THC). For example, the hemp according to the present invention may contain less than 0.2% THC on a dry matter basis. Hemp contains cannabidiol (CBD) which is associated with therapeutic effects but has no psychoactive effects. Hemp is also a valuable source of polyphenols such as gallic acid, chlorogenic acid, caffeic acid, quercetin and ellagic acid.

[0014] In addition to those provided by fermented hemp or fermented hemp extract, the beverage composition of the present invention may contain cannabinoids. The cannabinoids may be isomers and / or derivatives of cannabinoids. The cannabinoid derivative may be a cannabinoid acid. In addition to those provided by fermented hemp or fermented hemp extract, the beverage composition of the present invention may contain cannabidiol.

[0015] In the context of the present invention, the term "fermentation" refers to a process in which the activity of microorganisms brings about a change (typically a desirable change) in a food or beverage. Fermentation can be by yeast and / or bacteria. Fermentation can be anaerobic or aerobic. Fermentation is one of the oldest techniques used for food preservation and enhancement.

[0016] The term "fermented hemp" refers to hemp that has been subjected to a process in which the activity of microorganisms such as yeast brings about a chemical change, typically a desirable change, in the organic components of the hemp. Fermented hemp can be fermented, for example, by yeast in an aqueous medium. Fermented hemp can be dried hemp that has been separated from the aqueous fermentation medium and dried after being added to and fermented in an aqueous fermentation medium. Fermented hemp may be fermented by "solid-state" fermentation, for example, by adding a starter culture of yeast to hemp leaves, for example, fresh hemp leaves.

[0017] The term "hemp extract" refers to a material extracted from hemp, e.g., an aqueous extract of hemp. The hemp extract may be, for example, an aqueous extract of hemp leaves and stalks after the oil has been removed from the leaves and stalks. The term "fermented hemp extract" refers to a hemp extract after the hemp extract has been fermented, e.g., an aqueous hemp extract after the aqueous extract has been fermented, such as with yeast. The fermented hemp extract may be a dried fermented hemp extract.

[0018] In one embodiment, the beverage composition (e.g., beverage) has a weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal greater than 2000, e.g., greater than 3500. The aldehyde 2-methylbutanal contributes a malty, solvent-like note. The ratio can be calculated from the weight concentrations in ppm as follows:

number

[0019] During the fermentation of sugars by Pichia, higher esters such as 2-methylbutyl acetate are initially produced, but as fermentation progresses, 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 aroma balance in the fermented beverage, reducing the malty, beer, and solvent notes and masking the hemp notes with these aroma markers.

[0020] 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 greater than 50, such as greater than 250, such as greater than 300, such as greater than 350, or even such as greater than 400. This ratio can be calculated from the weight concentrations in ppm as follows:

number

[0021] The compound 2,3-butanedione is also known as diacetyl. 2,3-butanedione provides buttery or fatty notes. A high ratio of the total 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 in fermentation.

[0022] In one embodiment, the beverage composition comprises less than 10 g / kg of acetic acid on a dry basis, such as less than 1 g / kg of acetic acid on a dry basis, and still more such as less than 0.5 g / kg of acetic acid on a dry basis. The "bite" due to acid in the flavor profile of beverages such as kombucha or kefir is not necessarily desired by consumers seeking a milder flavor profile.

[0023] Many consumers are seeking beverages with complex and refreshing tastes as alternatives 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 still more such as less than 0.05% by weight. The beverage composition may be selected from the group consisting of ready-to-drink beverages, beverage concentrates, soluble beverage powders, dried aromatic plant materials, and combinations thereof.

[0024] The Ready-to-Drink beverage can be fermented hemp extract or water flavored with fermented hemp. The Ready-to-Drink beverage may contain 2 to 30% by weight of hemp, for example 4 to 15% by weight of hemp. The Ready-to-Drink beverage may further contain other ingredients such as flavorings or stabilizers. The Ready-to-Drink beverage can be a carbonated beverage. The Ready-to-Drink beverage may further contain fruits or fruit juices such as strawberries, citrus fruits (e.g., oranges, grapefruit, lemons, limes, calamansi, bergamot, clementines, tangerines or yuzu), peaches, pomegranates, watermelons, blackberries, raspberries, persimmons or cranberries. The Ready-to-Drink beverage may further contain herbs such as mint, verbena or basil. The Ready-to-Drink beverage may further contain flower extracts such as hibiscus or roses. The Ready-to-Drink beverage may further contain vegetable juices or vegetable extracts such as tomatoes, cucumbers, kale and celery. The Ready-to-Drink beverage may further contain plant extracts. The Ready-to-Drink beverage may further contain dietary fibers such as inulin, wheat dextrin, resistant starch and β-glucan (e.g., derived from oats or barley). The Ready-to-Drink beverage may contain added vitamins and minerals. The Ready-to-Drink beverage may contain beneficial microorganisms that are replicating or non-replicating. The beneficial microorganisms may be, for example, probiotics. The Ready-to-Drink beverage may contain cannabidiol in addition to the cannabidiol provided by the fermented hemp or fermented hemp extract. The Ready-to-Drink beverage of the present invention may contain 10 to 100 mg of cannabidiol per liter in total, for example 20 to 60 mg of cannabidiol per liter.

[0025] One aspect of the present invention provides the use of the beverage composition of the present invention for preparing a beverage.

[0026] The beverage liquid concentrate may be a concentrated fermented hemp extract. The beverage liquid concentrate may further contain other ingredients such as flavors or stabilizers. The beverage liquid concentrate may contain 4 to 60% by weight of hemp, for example 8 to 30% by weight of hemp. The beverage liquid concentrate may further contain fruits or fruit juices such as strawberries, citrus fruits (e.g., oranges, grapefruit, lemons, limes, calamansi, bergamot, clementines, tangerines or yuzu), peaches, pomegranates, watermelons, blackberries, raspberries, persimmons or cranberries. The beverage liquid concentrate may further contain herbs such as mint, verbena or basil. The beverage liquid concentrate may further contain flower extracts such as hibiscus or rose. The beverage liquid concentrate may further contain vegetable juices or vegetable extracts such as tomatoes, cucumbers, kale and celery. The beverage liquid concentrate may further contain dietary fibers such as inulin, wheat dextrin, resistant starch and β-glucan (e.g., derived from oats or barley). The beverage liquid concentrate may further contain plant extracts. The beverage liquid concentrate may contain added vitamins and minerals. The beverage liquid concentrate may contain beneficial microorganisms, either replicative or non-replicative. The beneficial microorganisms may be, for example, probiotics.

[0027] A beverage preparation device (e.g., a beverage preparation machine) that houses extractable subdivided raw materials provides a convenient way to prepare beverages. Such subdivided raw materials are generally packed in containers configured as, for example, pods, pads, sachets, pouches, or capsules. One aspect of the present invention provides a container for use in a beverage preparation device that houses the beverage composition of the present invention. The container is for preparing a beverage when inserted into the beverage preparation device. The container may be, for example, a beverage capsule among various configurations. In one embodiment, the container houses the beverage composition of the present invention. For example, the container may contain dried fermented hemp or a dried fermented hemp extract. The container can contain dried fermented hemp and / or a dried fermented hemp extract, which may be combined with other dried raw materials such as tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee.

[0028] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising: a. immersing hemp in water to produce a hemp aqueous extract and immersed hemp; b. fermenting the hemp aqueous extract, in the presence or absence of the immersed hemp; The fermentation is carried out by yeast including Pichia yeast.

[0029] The yeast may mainly be Pichia yeast, for example, more than 50% of the microbial colony forming units present during fermentation may be Pichia. For example, more than 50% of the yeast colony forming units may be Pichia, for example, 60%, 70%, 80%, more than 90% of the yeast colony forming units may be Pichia. For example, essentially all yeast colony forming units present during fermentation may be Pichia, and as a further example, essentially all microbial colony forming units present during fermentation may be Pichia.

[0030] The beverage composition prepared by the method of the present invention can be a hemp beverage, for example, a beverage containing fermented hemp or a fermented hemp extract. The beverage composition prepared by the method of the present invention can have an ethanol content of less than 1.2 wt%, for example, less than 0.5 wt%, for example, less than 0.2 wt%, and further for example, less than 0.05 wt%. In one embodiment, the beverage composition prepared by the method of the present invention is the beverage composition of the present invention.

[0031] The term "immersion" refers to dipping a material into a liquid. Hemp may be immersed 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, and further for example at least 120 minutes. During the immersion process, the components of hemp are extracted into water. The immersion may 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. Hemp contains some fermentable carbohydrates, but additional fermentable carbohydrates, such as sucrose or glucose, may be added to the hemp aqueous extract. The fermentable carbohydrate can be fructose or glucose. The fermentable carbohydrate can be in the form of honey. For example, the fermentable carbohydrate may be added to the hemp aqueous extract at a level of 2 to 10% by weight, for example 3 to 7% by weight. Hemp may be dried and / or ground before immersion.

[0032] If fermentation is carried out in the absence of immersed hemp, the hemp can be immersed in water using, for example, a continuous countercurrent extraction system. A further option is to immerse the hemp in water using a batch process, and the immersed hemp is separated from the hemp aqueous extract using a filtration system. The dry-based hemp to water ratio during immersion can be 1:1 to 1:100 by weight, for example 1:2 to 1:95 by weight, for example 1:10 to 1:90 by weight, for example 1:30 to 1:70 by weight, and further for example 1:3 to 1:6.

[0033] After immersion, the temperature of the hemp aqueous extract is adjusted to a temperature suitable for yeast growth, such as 20°C to 37°C, for example 25°C to 35°C, for example 28°C to 32°C. The temperature can be adjusted, for example, using a laminar flow heat exchanger. Yeast is added to the hemp aqueous extract to initiate fermentation. The fermentation of the hemp aqueous extract may be carried out at a temperature of 25°C to 35°C, for example 28°C to 32°C. The fermentation of the hemp aqueous extract may 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.

[0034] The fermentation may be carried out at a pH of 3 to 8, for example 4 to 6.

[0035] The aqueous hemp extract may be fermented in the presence of other ingredients, such as flavorings or stabilizers. The aqueous hemp extract may be fermented in the presence of other ingredients, such as fruits or fruit juices, for example, strawberries, citrus fruits (e.g., oranges, grapefruits, lemons, limes, calamansi, bergamot, clementines, tangerines, or yuzu), peaches, pomegranates, watermelons, blackberries, raspberries, persimmons, or cranberries. The aqueous hemp extract may be fermented in the presence of other ingredients, such as herbs, for example, mint, verbena, or basil. The aqueous hemp extract may be fermented in the presence of other ingredients, such as flower extracts, for example, hibiscus or rose. The aqueous hemp extract may be fermented in the presence of other ingredients, such as vegetable juices or vegetable extracts, for example, tomato, cucumber, kale, or celery. The aqueous hemp extract may be fermented in the presence of other ingredients, such as plant infusions. The aqueous hemp extract may be fermented in the presence of other ingredients, such as added vitamins and minerals.

[0036] Fermentation may be carried out in the presence of a nitrogen source such as yeast extract or yeast peptone. Fermentation may also be carried out in the presence of amino acids such as valine, leucine, isoleucine, and / or phenylalanine, added at levels of, for example, 0.05% to 0.1% by weight of the aqueous hemp extract.

[0037] Fermentation may be carried out under anaerobic, microaerobic or aerobic conditions. For example, the oxygen partial pressure in the fermenter may be 0 to 30%, such as 1 to 20%, further such as 2 to 10%.

[0038] Preferably, the hemp has not been subjected to chemical treatments such as hydrolysis prior to fermentation.

[0039] Fermentation should be at least 10 3 CFU / g, e.g., at least 10 4 CFU / g, e.g., at least 10 5 CFU / g, e.g., at least 106 CFU / g, and more particularly for example at least 10 7 CFU / g of an initial mixed yeast level, and can be carried out by adding one or more yeasts.

[0040] Fermentation may be carried out in the absence of acetic acid bacteria in order to avoid the generation of an acid "puckery feeling" in the flavor profile, as seen in kombucha or kefir. In one embodiment, fermentation is carried out in the absence of lactic acid bacteria.

[0041] In one embodiment, fermentation is carried out in the absence of Saccharomyces subspecies. For example, fermentation can be carried out in the absence of Saccharomyces cerevisiae. S. cerevisiae is used in wine production and beer brewing. In addition to producing alcohol that may not be desirable, S. cerevisiae produces a "fermented", wine-like, yeast-like flavor note rather than a fresh, fruity note.

[0042] In one embodiment, fermentation is carried out in the absence of yeasts selected from the group consisting of Zygosaccharomyces bailii, Schizosaccharomyces pombe, Torulaspora delbreukii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis, and Candida stellate. These yeasts are commonly present in kombucha "tea-fungus" cultures.

[0043] The inventors have found that fermentation of hemp by Pichia kudriavzevii gives particularly good results. A beverage composition containing hemp or a hemp extract fermented with Pichia kudriavzevii has a pleasant fruity flavor with less smoky hemp notes than the unfermented beverage. Pichia kudriavzevii is naturally present worldwide, for example, in olives, grapes and coffee. In one embodiment, the Pichia yeast is mainly Pichia kudriavzevii, for example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kudriavzevii. For example, more than 50% of the yeast colony forming units can be Pichia kudriavzevii, for example, more than 60%, 70%, 80%, 90% of the yeast colony forming units can be Pichia kudriavzevii. For example, essentially all yeast colony forming units present during fermentation can be Pichia kudriavzevii, and as a further example, essentially all microbial colony forming units present during fermentation can be Pichia kudriavzevii.

[0044] Pichia kudriavzevii NCYC246 (or named CBS188) is the reference strain of Pichia kudriavzevii. The reference strain is publicly available, for example, from the National Collection of Yeast Cultures, Quadram Institute Bioscience (Norwich, UK).

[0045] In one embodiment, the Pichia yeast is Pichia kudriavzevii NCYC246. For example, more than 50% of the yeast colony forming units present during fermentation can be Pichia kudriavzevii NCYC246. For example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kudriavzevii NCYC246. For example, essentially all yeast colony forming units present during fermentation can be Pichia kudriavzevii NCYC246. As a further example, essentially all microbial colony forming units present during fermentation can be Pichia kudriavzevii NCYC246.

[0046] Pichia kudriavzevii NCYC2781 (alternatively named Maize Silage Isolate 86) is publicly available, for example, from the National Collection of Yeast Cultures, Quadram Institute Bioscience (Norwich, UK).

[0047] In one embodiment, the Pichia yeast is Pichia kudriavzevii NCYC2781. For example, more than 50% of the yeast colony forming units present during fermentation can be Pichia kudriavzevii NCYC2781. For example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kudriavzevii NCYC2781. For example, essentially all of the yeast colony forming units present during fermentation can be Pichia kudriavzevii NCYC2781. As a further example, essentially all of the microbial colony forming units present during fermentation can be Pichia kudriavzevii NCYC2781. Pichia kudriavzevii is widely distributed in nature and can be found, for example, in fruits such as olives, grapes and coffee. Pichia kudriavzevii NYSC5, isolated from a coffee fermentation plant in Nicaragua, was deposited on June 23, 2020 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France and received the deposit number CNCM I-5525.

[0048] In one embodiment, the yeast is Pichia kluyveri NYSC5485 (CNCM I-5525). For example, greater than 50% of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). For example, greater than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). For example, essentially all of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). As a further example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525).

[0049] The inventors have found that fermenting hemp with Pichia kluyveri NYSC 5485 produces particularly good results. Fermenting hemp with Pichia kluyveri NYSC 5485 results in a higher weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal, which corresponds to an improved aroma balance.

[0050] Fermentation should be at least 10 3 CFU / g, e.g., at least 10 4 CFU / g, e.g., at least 10 5 CFU / g, e.g., at least 10 6 CFU / g, further e.g. at least 10 7 This can be done by adding Pichia kluyveri, for example Pichia kluyveri 5485 (CNCM I-5525), at an initial yeast level of CFU / g.

[0051] The gene sequence of Pichia kudriavzevii NYSC5485 (CNCM I-5525) was analyzed using PacBio sequencing technology. DNA purification was performed using the Qiagen Gentra Puregene Yeast kit. Twelve DNA fragments were identified. These fragments may be individual chromosomes, namely 11 chromosomes and 1 mitochondrial DNA (SEQ ID NO: 4). The gene sequences are submitted in electronic form as SEQ ID NOs: 1 to SEQ ID NOs: 12.

[0052] In one embodiment of the method of the present invention, the Pichia yeast comprises a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 1, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 2, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 3, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 5, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 6, a gene sequence having at least 99% identity to SEQ ID NO: 7, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 8, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 9, a gene sequence having at least 99% identity to SEQ ID NO: 10, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 11, or a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 12.

[0053] In one embodiment of the method of the present invention, Pichia yeast includes a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 1, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 2, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 3, a gene sequence having at least 99.5% identity to SEQ ID NO: 4, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 5, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 6, a gene sequence having at least 99% identity to SEQ ID NO: 7, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 8, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 9, a gene sequence having at least 99% identity to SEQ ID NO: 10, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 11, and a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO: 12.

[0054] In one embodiment, Pichia yeast includes a gene sequence having at least 98% (e.g., at least 99%) overall nucleotide identity to the combination of SEQ ID NOS: 1 to 12. The term "combination of SEQ ID NOS: 1 to 12" means that sequence identity comparison is performed for all individual DNA fragments as if they were linked to a single sequence.

[0055] In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 1. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 2. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 3. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 5. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 6. In one embodiment, the Pichia yeast contains a gene sequence having at least 99% identity to SEQ ID NO: 7. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 8. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 9.In one embodiment, the Pichia yeast contains a gene sequence having at least 99% identity to SEQ ID NO: 10. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 11. In one embodiment, the Pichia yeast contains a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 9

[0056] <000021>The name referring to yeast can change over time. It should be understood that the yeast by the method of the present invention can include the gene sequence identity described in the preceding paragraph for the Pichia yeast of the present invention regardless of the name applied to the yeast.

[0057] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising: a) immersing hemp in water to produce a hemp aqueous extract and immersed hemp; and b) fermenting the hemp aqueous extract, either in the presence or absence of the immersed hemp, wherein the fermentation is carried out by yeast comprising a gene sequence having at least 98% (e.g., at least 99%) overall nucleotide identity to the combination of SEQ ID NOs: 1-12.

[0058] In one embodiment, the hemp aqueous extract is separated from the immersed hemp after fermentation. For example, the immersed hemp can be separated from the hemp aqueous extract using a filtration system. After fermentation, the hemp aqueous extract becomes a fermented hemp extract.

[0059] The fermented hemp extract may be packaged as a ready-to-drink beverage, for example, packaged in a bottle or can. The fermented hemp extract may have a concentration of total dissolved solids of 2-60% by weight, for example, a concentration of total dissolved solids of 2-25% by weight. After adding additional raw materials such as flavors and preservatives to the fermented hemp extract, it may be packaged as a ready-to-drink beverage. The fermented hemp extract may be heat-treated to inactivate or kill yeast and any spoilage organisms prior to packaging. The fermented hemp extract may be passed through a filter to remove yeast and spoilage organisms prior to packaging.

[0060] The fermented hemp extract may be combined with other raw materials such as fruits or fruit juices, herb extracts, flower extracts, vegetable juices or vegetable extracts, plant infusions, vitamins and minerals, and beneficial microorganisms. Cannabidiol may be added to the fermented hemp extract, for example, cannabidiol may be emulsified in an aqueous solution of the fermented hemp extract using an emulsifier.

[0061] In one embodiment, the hemp aqueous extract is concentrated after fermentation and packaged, for example, as a liquid concentrate suitable for the consumer to dilute before ingestion. The hemp aqueous extract may be concentrated, for example, as an intermediate product for the production of ready-to-drink products at different locations. The fermented hemp extract may have a concentration of total dissolved solids of 25 to 60% by weight, for example, a concentration of total dissolved solids of 50 to 60% by weight. The dissolved solids can also be measured, for example, by passing the extract through a microfilter to remove the 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 raw materials such as flavors and preservatives may be added to the liquid concentrate.

[0062] The hemp aqueous extract after fermentation (for example, fermented hemp extract) may be concentrated to produce a powder such as a soluble beverage powder. For example, the hemp aqueous extract after fermentation (fermented hemp extract) may be dried by spray drying or freeze drying to produce a soluble beverage powder. For example, the hemp aqueous extract after fermentation may be spray dried together with a carrier such as maltodextrin to produce a soluble beverage powder.

[0063] The hemp aqueous extract after fermentation may be spray dried together with other raw materials such as fruits or fruit juices, herb extracts, flower extracts, vegetable juices or vegetable extracts, plant infusions, vitamins and minerals, and beneficial microorganisms.

[0064] The soluble powder beverage may be packaged and sold as is, or may be used, for example, as an intermediate product for the production of ready-to-drink products at different locations. The soluble beverage powder may be filled into a container for use in a beverage preparation device. The soluble beverage powder may be combined with other raw materials, such as dried raw materials like tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee. The soluble powder beverage may be combined with other raw materials such as fruit powder, herbs, vegetable powder, vitamins, minerals, and beneficial microorganisms. For example, the soluble beverage powder may be combined with other raw materials and filled into a container for use in a beverage preparation device. The soluble beverage powder may be combined with milk powder or beverage creamer powder and optionally sugar to produce a beverage mix. After fermenting the hemp aqueous extract in the presence of immersed hemp, the immersed hemp becomes fermented hemp. In one embodiment, the immersed hemp present during the fermentation of the hemp aqueous extract is separated from the hemp aqueous extract after fermentation and then dried, for example, using a rotary dryer. The dried immersed hemp may be filled into a container for use in a beverage preparation device, or may be filled into a permeable bag such as a "tea bag". The dried immersed hemp may be combined with other dried raw materials such as tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee. In the context of the present invention, the term "tea" refers to the dried leaves, leaf buds, twigs or stems of the plant Camellia sinensis. The tea can be, for example, C. sinensis of the sinensis variety (var.sinensis), or C. sinensis of the assamica variety (var.assamica). The term "herbal tea" refers to an infusion made from the dried flowers, fruits, leaves, seeds or roots of plants other than Camellia sinensis.

[0065] In one embodiment, the immersed hemp present during the fermentation of the hemp aqueous extract is dried, together with the hemp aqueous extract after fermentation, for example by freeze-drying or spray-drying. The dried immersed hemp (e.g., dried fermented hemp) may be filled into a container for use in a beverage preparation device, together with the dried hemp aqueous extract after fermentation (e.g., dried fermented hemp extract), or may be filled into a liquid-permeable bag such as a "tea bag". The dried immersed hemp and the dried hemp aqueous extract after fermentation may be combined with other dried raw materials such as tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee.

[0066] One aspect of the present invention provides the use of hemp fermented with Pichia kudriavzevii NYSC5485 (CNCM I-5525) for manufacturing a beverage.

[0067] The fermented hemp according to the present invention can be obtained, for example, in a "solid" fermentation by adding a starter culture of Pichia yeast to hemp leaves, for example fresh hemp leaves that have not been dried. The term "solid" fermentation refers to fermentation carried out on solid materials. Although liquid water is present in solid fermentation, this term is used in contrast to liquid fermentation that can occur in an aqueous solution in a fermentation vessel.

[0068] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising a step of adding a starter culture of yeast to hemp leaves, wherein the starter culture supplies at least 10 4 CFU (e.g., at least 10 5 CFU, and further for example at least 10 6 CFU) of yeast per gram of hemp leaves, a step of fermenting the hemp leaves, a step of drying the fermented hemp leaves to produce fermented hemp, a step of extracting the fermented hemp with water to produce a ready-to-drink beverage, or The step of filling a container for preparing a beverage, such as a beverage preparation device, with fermented hemp when inserted into the container, or any of them is included.

[0069] The starter culture of yeast may be added to hemp leaves as a liquid or powder. The hemp leaves may have a moisture content of 45-55% by weight when the starter culture of yeast is added. The leaves may be treated with steam before fermentation to reduce the amount of environmental microorganisms before adding yeast. Fermentation may occur at a solids content of more than 40% by weight, such as more than 45% by weight, and further such as more than 50% by weight.

[0070] During a certain period of continuous fermentation, the drying of the fermented hemp may be started. For example, the hemp leaves may be placed in a container such as a tank, the starter culture may be added, and then after allowing the fermentation to proceed for 1-4 days, the fermented hemp may be taken out of the container and dried. As a further example, after adding the starter culture to the hemp leaves, the hemp leaves may be gradually dried, for example, in a rack or hanging tray. Once fermentation has started, the hemp leaves may be gradually dried while continuing the fermentation. For example, the fermented hemp leaves may be dried to a moisture content of less than 20% by weight (such as less than 15% by weight) over a period of 1-30 days, such as 4-2 days. The drying temperature may be less than about 40 °C, such as about 25 °C - 35 °C. A fan may be used to blow dry air onto the material until the hemp leaves are dry. The fermented hemp leaves may be dried in sunlight.

[0071] The yeast may be Pichia, such as Pichia kudriavzevii, and further such as Pichia kudriavzevii NYSC5485.

[0072] When inserted into a beverage preparation device, the container for beverage preparation may be in the form of a pod, pad, sachet, pouch, or capsule, etc.

[0073] The ready-to-drink beverage may be filled in a bottle or a can. The fermented hemp may be combined with other extractable materials such as tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee before being extracted with water to produce the ready-to-drink beverage.

[0074] The fermented hemp may be combined with tea, herbal tea, dried fruits, soluble coffee or roasted ground coffee in a container for beverage preparation when inserted into a beverage preparation device.

[0075] Those skilled in the art will understand that all features of the invention disclosed herein can be freely combined. In particular, the features described for the products of the invention may be combined with the methods of the invention, and vice versa. Furthermore, features described for different embodiments of the invention may be combined. If known equivalents exist for a particular feature, such equivalents are incorporated as if specifically recited herein.

[0076] Further advantages and features of the invention are apparent from the drawings and non-limiting examples.

Examples

[0077] Example 1: Fermentation method of hemp Using organic hemp leaves (Coop Naturaplan, Switzerland) as the starting material, a leachate was prepared. First, 20 g of the dry starting material was added to 1000 mL of water at 100 °C and immersed for 10 minutes to produce a high-temperature leachate. Then, the leachate was placed in an ice bath until the temperature dropped below 30 °C. Next, the leachate was filtered through a sieve to remove further small pieces and / or leaves. The filtrate (hemp aqueous extract) was dispensed in the same amount as a control (Ref) or for various fermentation samples.

[0078] Fermented hemp was produced by fermenting the leachate with glucose (2% m / m) and various starter cultures including Pichia kudriavzevii NYSC5485 (CNCM I-5525), P. kudriavzevii NCYC246 (the type strain of Pichia kudriavzevii), P. kudriavzevii NCYC2781, commercially available brewing yeast Saccharomyces cerevisiae (SafAle S-33, LeSaffre, France), and a symbiotic culture of bacteria and yeasts (SCOBY) pellicle derived from kombucha tea fermentation. All strains (except SCOBY) were grown from glycerol vials in edible yeast-peptone liquid medium and inoculated into the reference hemp brewing liquid at an optical density (OD600nm) of 0.5. All inoculated leachates were stored at 30 °C for 24 hours under microaerobic conditions. After incubation, each fermented leachate was filter sterilized (0.2 μm).

[0079] Example 2: Sensory analysis of hemp fermented with various yeasts The sensory profiles of various variations of hemp leachate were tasted. The control had a hemp flavor with smoky, hayish, woody, and earthy notes. The hemp leachate fermented with Saccharomyces cerevisiae had strong malty and sour characteristics along with off-notes. The hemp fermented with SCOBY had a very strong sour taste. The hemp leachates fermented with two different Pichia yeasts were fruity and had significantly reduced hemp / smoky notes compared to the control. Butter-like, sour, and fatty off-notes were not identified in the hemp fermented with Pichia yeast. The hemp fermented with Pichia kudriavzevii NYSC5485 could be considered to have a more balanced aroma profile than the hemp fermented with Pichia kudriavzevii NCYC246.

[0080] Example 3: Analysis of flavor profiles focusing on the aromatic compounds present in hemp fermented with various yeasts Quantitative profiling of the volatile substances in hemp leachate was carried out by headspace / solid-phase microextraction (HS / SPME-GC-MS) combined with gas chromatography and mass spectrometry. The apparatus used was a gas chromatograph (Agilent Technology 8890 GC System) connected to a mass spectrometer (Agilent Technology 7010B TQ).

[0081] First, 1.0 mL of the hemp leachate sample was incubated in a 10 mL screw-top headspace vial at 30 °C for 10 minutes, and then extracted using an SPME fiber (PDMS / DVB, Supelco) at 30 °C for 10 minutes. All vials were placed in a tray cooled to 6 °C before analysis. The volatile substances were thermally desorbed from the SPME fiber at 250 °C in splitless mode and separated on a capillary column (DB-WAX, Agilent). The gas chromatograph oven temperature was programmed to be initially 35 °C for 5 minutes, then increase at 4 °C / min to 230 °C, and then hold at 230 °C for 10 minutes. Helium was used as the carrier gas at a flow rate of 1 mL / min. The target compounds were identified by pure standards and quantified using MSD ChemStation software (Agilent). All samples were measured twice.

[0082] 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 after 24 hours are shown in the following table in ppm units. [Table 1]

[0083] The samples fermented with SCOBY had a high level of acetic acid corresponding to 190 g / kg on a dry basis.

[0084] The weight ratios A, B, and C of the aroma components are shown in the following table. [Number] [Table 2]

[0085] Samples fermented with Pichia yeast (NYSC5485, NCYC2781, and NCYC246) showed higher ratios of A, B, and C than the control and samples fermented with S. cerevisiae or SCOBY. Pichia kudriavzevii NYSC5485 resulted in higher values for ratios of A, B, and C than the reference strain Pichia kudriavzevii NCYC246 and higher values for ratios of A and B than Pichia kudriavzevii NCYC2781.

[0086] The levels of terpenes and terpenoids present in the samples were measured. The following target compounds were identified by the NIST library and retention indices. The peak areas of each compound were integrated over the whole sample, and the ratios of fermented samples to non-fermented samples are shown below. All of these compounds have been reported and detected in hemp extracts or hemp oil [V.R.L.J. Bloemendal et al., Org. Biomol. Chem., 18, 3203 - 3215 (2020). DOI 10.1039 / D0OB00464B][A.S. 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].

[0087] All samples were measured twice. [Table 3]

[0088] Terpenes and terpenoids are major contributing factors to the "hempiness" characteristic, which is perceived as a combination of pine-like, medicinal-like, and grassy flavor notes. After fermentation by Pichia kudriavzevii, the hempiness was significantly reduced. P. kudriavzevii NYSC5485 was found to reduce these compounds related to the hempiness characteristic to a greater extent than the reference strain P. kudriavzevii NYSC246.

[0089] Example 4: Influence on sensory by aroma ratio investigated by spike Solutions were prepared by adding 3-methylbutyl acetate, 3-methylbutanol, and 3-methylbutanal at different levels. In one set of solutions, the compounds were added to water, and in the other set, the compounds were added to an unfermented hemp extract control (Ref).

[0090] Samples were tasted by six professional taste panels, and comments were collected. The samples and comments are shown below. [Table 4]

[0091] It is important to have an appropriate balance between esters / alcohols and aldehydes. In hemp leachate, it was observed that when the ester level is high and the aldehyde level is low, for example, when ratio A exceeds 2000, the "hemp-like" notes can be masked.

[0092] Example 5: Comparison of the gene sequence of P. kudriavzevii NYSC5485 and the gene sequence of the reference strain P. kudriavzevii NCYC246 The gene sequences of Pichia kudriavzevii NYSC5485 (CNCM I-5525) and P. kudriavzevii NCYC246 were analyzed using PacBio sequencing technology. DNA purification was performed using the Qiagen Gentra Puregene Yeast kit. Twelve DNA fragments were identified for P. kudriavzevii NYSC5485, and fifteen fragments were identified for the reference strain P. kudriavzevii NCYC246. The gene sequence of P. kudriavzevii NYSC5485 was submitted in electronic form as SEQ ID NO: 1 to SEQ ID NO: 12, and for P. kudriavzevii NCYC246, it was submitted as SEQ ID NO: 13 to SEQ ID NO: 27. The mitochondrial DNA is SEQ ID NO: 4 for P. kudriavzevii NYSC5485 and SEQ ID NO: 20 for P. kudriavzevii NCYC246.

[0093] The gene sequence of Pichia kudriavzevii NYSC5485 (CNCM I-5525) was compared with P. kudriavzevii NCYC246 using the software OrthoANIu. The overall average nucleotide identity was found to be 97.91%.

[0094] The correspondence relationships between various sequences are shown in the following table. Multiple sequences of Pichia kudriavzevii NYSC5485 have the closest correspondence with combinations of the sequences of P. kudriavzevii NCYC246, and vice versa.

Table 5

[0095] The reference strain P. kudriavzevii NCYC246 contained gene sequences with the following maximum identities for each of SEQ ID NO: 1 to 12: 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 hemp or a fermented hemp extract, wherein the total weight ratio of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000.

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

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

4. The 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 ready-to-drink beverages, beverage concentrates, soluble beverage powders, dried aromatic plant materials, and combinations thereof.

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

7. A method for preparing a beverage composition, comprising: a. Immersing hemp in water to produce a hemp aqueous extract and immersed hemp; b. Fermenting the hemp aqueous extract in the presence or absence of the immersed hemp, wherein the fermentation is carried out by yeast including Pichia yeast.

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

9. The method according to claim 7 or 8, wherein the Pichia yeast comprises a gene sequence having at least 98% overall nucleotide identity to the combination of SEQ ID NOs: 1 to 12.

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

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

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

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

15. Use of hemp fermented with Pichia kudriavzevii NYSC5485 (CNCM I-5525) for producing a beverage.

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