Fermented acerola cherry beverage

Abstract

The present invention relates to a beverage composition comprising fermented acerola cherry or a fermented acerola cherry 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 acerola or a fermented acerola extract. Another aspect of the present invention is a method of preparing a beverage composition. Background Art

[0002] Acerola is a rich natural source of ascorbic acid and contains a number of phytonutrients such as carotenoids, phenols, anthocyanins and flavonoids. The flavor profile is typically sour and astringent, and the taste may vary with seasonal fluctuations. Acerola often needs to be mixed with sweetened juices and purees (such as pineapple and mango), or sweetened with sugar to increase its appeal.

[0003] Many people desire new flavors and sweetness in beverages, but are reluctant to drink beverages with added flavorings, especially synthetic flavorings or sugary fruit juices. Therefore, it would be commercially valuable to provide acerola cherry beverages with increased flavor complexity and less astringency and sourness.

[0004] Any reference in this specification to prior art documents should not be taken as an admission that such prior art is well-known technology or forms part of the common general knowledge in the field. As used in this specification, the words "include", "comprises" and similar words should not be understood to have an exclusive or exhaustive meaning. In other words, they are intended to mean "including, but not limited to". Summary of the invention

[0005] It is an object of the present invention 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. 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.

[0006] Therefore, the present invention provides in a first aspect a beverage composition comprising fermented acerola cherry or fermented acerola cherry extract, 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 150.

[0007] 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.

[0008] In a third aspect, the present invention provides a method of preparing a beverage composition comprising fermenting an aqueous acerola extract, wherein the fermentation is performed by a yeast comprising Pichia pastoris.

[0009] Another aspect of the present invention provides use of acerola cherry fermented with Pichia kluyveri NYSC 5485 (CNCM I-5525) for producing a beverage.

[0010] Acerola cherries can be made into beverages by extracting juice from the berries or mashing the berries to form a "thickness". These beverages are sour and astringent. The inventors surprisingly found that by fermenting with Pichia yeast, the ethanol content of the acerola cherry beverages remained low, but the fermentation enhanced the aroma and complexity, and the sourness and astringency were less obvious. The fermented beverages do not have buttery and fatty flavors that can be seen as off-flavors in fermentation. DETAILED DESCRIPTION

[0011] Thus, the present invention relates in part to a beverage composition comprising (e.g., consisting of) fermented acerola cherry or a fermented acerola cherry extract, 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 150, such as greater than 200, such as greater than 250, such as greater than 500, such as greater than 750, such as greater than 1000, such as greater than 1250, further such as greater than 1400. This ratio can be calculated based on the weight concentration in parts per million as follows:

[0012]

[0013] The compound 2,3-butanedione is also known as diacetyl. It provides a buttery or fatty note. 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 in fermentation. The enhanced fruity aroma serves to mask bitterness, sourness, and astringency.

[0014] The acerola plant (Malpighia emarginata) is a tropical fruiting shrub or small tree in the Malpighiaceae family. Common names include acerola, Guarani cherry, Barbados cherry, acerola, and wild crape cherry. Acerola berries are high in vitamin C and also contain vitamins A, B1, B2, and B3, as well as carotenoids and bioflavonoids.

[0015] In the present invention, unless otherwise specified, the term "acerola" refers to berries or processed forms of berries from the acerola plant.

[0016] 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 of preserving and fortifying food.

[0017] The term "fermented acerola" refers to acerola cherries that have been subjected to a process in which the activity of a microorganism, such as a yeast, causes a chemical change, typically a desired change, in the organic components of the acerola cherries. The fermented acerola cherries may be fermented in an aqueous medium, for example with yeast. The fermented acerola cherries may be dried acerola cherries that are added to an aqueous fermentation medium and fermented and then separated from the aqueous fermentation medium and dried. The fermented acerola cherries may be fermented in a "solid state" fermentation, for example by adding a starter culture of yeast to the acerola cherries, for example to fresh acerola berries or to pulp comprising acerola berry pulp.

[0018] The term "acerola extract" refers to a substance extracted from acerola, e.g., an aqueous extract of acerola, such as acerola juice, or an acerola powder reconstituted with water. The term "fermented acerola extract" refers to an acerola extract in which the acerola extract has been fermented, e.g., an aqueous acerola extract in which the aqueous extract has been fermented (such as by yeast). The fermented acerola extract may be a dried fermented acerola extract.

[0019] In one embodiment, the weight ratio of the sum of 2-phenylethyl acetate and 2-phenylethanol to 2-phenylacetaldehyde of the beverage composition (e.g., beverage) is greater than 3000, such as greater than 4000, such as greater than 6000, such as greater than 8000, and further such as greater than 8500.

[0020] The ratio can be calculated based on the weight concentration in parts per million as follows:

[0021]

[0022] Higher esters such as 2-phenylethyl acetate are initially produced during the fermentation of sugars with Pichia, but as the fermentation proceeds, higher alcohols such as 2-phenylethanol are produced. A high ratio of the sum of 2-phenylethyl acetate and 2-phenylethanol to 2-phenylacetaldehyde corresponds to a pleasant flavor balance in the fermented beverage.

[0023] In one embodiment, the beverage composition comprises less than 10 g / kg acetic acid on a dry basis, such as less than 1 g / kg acetic acid on a dry basis, further such as less than 0.5 g / kg acetic acid on a dry basis. A sour "note" in the flavor profile of beverages such as kombucha or kefir is not always desired by consumers seeking a milder flavor profile.

[0024] Many consumers seek beverages with complex, fresh tastes as an alternative to alcoholic beverages. In one embodiment, the beverage composition has an ethanol content of less than 1.2 wt%, such as less than 0.5 wt%, such as less than 0.2 wt%, further such as less than 0.05 wt%.

[0025] The beverage composition may 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.

[0026] The ready-to-drink beverage can be a fermented acerola infusion or water flavored with fermented acerola. The ready-to-drink beverage can additionally include other components, such as flavoring agents or stabilizers. The ready-to-drink beverage can be carbonated. The ready-to-drink beverage can additionally include fruit or juice, such as strawberry, orange, lemon, lime, peach, pomegranate, watermelon, blackberry or cranberry. The ready-to-drink beverage can additionally include herbs, such as mint or basil. The ready-to-drink beverage can additionally include flower extracts, such as hibiscus or rose. The ready-to-drink beverage can include added vitamins and minerals. The ready-to-drink beverage can include replicating or non-replicating beneficial microorganisms. The beneficial microorganisms can be, for example, probiotics.

[0027] Beverage preparation devices (e.g., beverage preparation machines) that hold extractable portioned ingredients provide a convenient method for preparing beverages. Such portioned ingredients are typically packaged in containers that are configured as, for example, pods, pads, bags, pouches, capsules, etc. One aspect of the present invention provides a container for use in a beverage preparation device that contains a beverage composition of the present invention. The container is used to prepare a beverage when inserted into the beverage preparation device. The container can be, for example, a beverage capsule, as well as other configurations. In one embodiment, the container contains a beverage composition of the present invention. For example, the container can contain dried fermented acerola cherries or dried fermented acerola extract. The container can contain dried fermented acerola cherries and / or dried fermented acerola extract, which can be combined with other dry ingredients (such as tea, herbal tea, dried fruit, soluble coffee, or roasted and ground coffee).

[0028] One aspect of the present invention provides a method of preparing a beverage composition comprising a fermented aqueous acerola extract, wherein the fermentation is performed by a yeast comprising Pichia pastoris.

[0029] The aqueous acerola extract may be, for example, acerola juice, acerola pulp, or acerola powder reconstituted with water.

[0030] 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%, 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 further for example, substantially all of the microbial colony forming units present during fermentation may be Pichia pastoris.

[0031] The beverage composition prepared by the method of the present invention can be an acerola beverage, such as a beverage comprising fermented acerola cherries or fermented acerola extract. The beverage composition prepared by the method of the present invention can have 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, further such as 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.

[0032] Aqueous acerola extract can be obtained by soaking acerola berries, such as acerola pulp, in water. Acerola cherries can be dried and / or ground before soaking. The term soaking refers to immersing and dipping a substance in a liquid. Acerola cherries can be soaked in water for at least 2 minutes, such as at least 3 minutes, such as at least 4 minutes, such as at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such as at least 30 minutes, such as at least 60 minutes, further such as at least 120 minutes. During the soaking process, components of acerola cherries are extracted into the water. The soaking can be carried out at a temperature between 4°C and 98°C, such as between 20°C and 95°C, such as between 60°C and 95°C.

[0033] Although acerola contains some fermentable sugars, additional fermentable sugars, such as sucrose or glucose, may be added to the aqueous acerola extract. The fermentable sugar may be fructose or glucose. The fermentable sugar may be in the form of honey. For example, the fermentable sugar may be added to the aqueous acerola extract at a level of between 2% and 10% by weight, such as between 3% and 7% by weight.

[0034] During fermentation, the ratio of acerola to water on a dry basis may be between 1 :1 and 1 :100 by weight, such as between 1 :2 and 1 :95 by weight, such as between 1 :10 and 1 :90 by weight, such as between 1 :30 and 1 :70 by weight, further such as between 1.3 and 1 :6.

[0035] Prior to fermentation, the temperature of the aqueous acerola extract is adjusted to a temperature suitable for yeast growth, such as between 20°C and 37°C, such as between 25°C and 35°C, such as 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 acerola extract to start fermentation. Fermenting the aqueous acerola extract can be carried out at a temperature between 25°C and 35°C, such as between 28°C and 32°C. Fermenting the aqueous acerola extract can be carried out for at least 2 hours, such as at least 6 hours, such as at least 12 hours, such as at least 24 hours, and further such as at least 48 hours.

[0036] The fermentation may be carried out at a pH between 3 and 8, such as between 4 and 6.

[0037] The fermentation may be carried out in the presence of a nitrogen source such as yeast extract or yeast peptone. The fermentation may 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 acerola extract.

[0038] Fermentation can be carried out under anaerobic, microaerobic 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%.

[0039] Fermentation may be carried out in the presence or absence of acerola water-insoluble components such as acerola skin and / or seeds.

[0040] Preferably, the acerola cherries have not been subjected to chemical processing such as hydrolysis prior to fermentation.

[0041] 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 for example at least 10 7 CFU / g of initial combined yeast level is performed by adding one or more yeasts (eg, Pichia pastoris).

[0042] 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 may be obtained with kefir or kombucha fermentation. In one embodiment, the fermentation is carried out in the absence of lactic acid bacteria.

[0043] 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 that may not be desirable, Saccharomyces cerevisiae produces "fermented", wine, yeasty flavor notes rather than fresh fruity notes.

[0044] In one embodiment, the fermentation is carried out 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 commonly found in kombucha "tea fungus" cultures.

[0045] The inventors have found that fermenting acerola cherries with Pichia kluyveri gives particularly good results. Beverage compositions comprising acerola cherries or acerola cherry extract fermented with Pichia kluyveri have an enhanced aroma sensation and complexity, and have less pronounced sourness and astringency.

[0046] Pichia kluyveri is naturally present throughout the world, for example, on olives, grapes and coffee. In one embodiment, the Pichia 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.

[0047] Pichia kluyveri NCYC 246 (or CBS188) is a model strain of Pichia kluyveri and is publicly available from, for example, the National Collection of Yeast Cultures, Quadram Institute Bioscience, Norwich, UK.

[0048] In one embodiment, the Pichia 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.

[0049] 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.

[0050] In one embodiment, the Pichia 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.

[0051] Pichia kluyveri is widely distributed in nature, for example, found in fruits such as coffee cherries and olives. Pichia kluyveri NYSC5485, collected and isolated from coffee fermentation in Nicaragua, was deposited on June 23, 2020 at the National Collection of Microorganisms (CNCM), 25 rue du Docteur Roux, F-75724 PARIS Cedex 15, France, with the deposit number CNCM I-5525.

[0052] In one embodiment, the yeast is Pichia kluyveri NYSC 5485 (CNCM I-5525). For example, more than 50% of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM I-5525). For example, more than 50% of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM I-5525). For example, substantially all of the yeast colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM I-5525), and further for example, substantially all of the microbial colony forming units present during fermentation can be Pichia kluyveri NYSC 5485 (CNCM I-5525).

[0053] The inventors have found that particularly good results are obtained with the fermentation of acerola cherries with Pichia kluyveri NYSC 5485. The fermentation of acerola cherries with Pichia kluyveri NYSC 5485 results in a higher weight ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate and 2-phenylethanol to 2,3-butanedione, which corresponds to enhanced fruity and floral notes without buttery and fatty notes that can be perceived as off-flavors in fermentation.

[0054] 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 for example at least 10 7 An initial combined yeast level of 100 CFU / g is performed by adding Pichia kluyveri, such as Pichia kluyveri NYSC 5485 (CNCM 1-5525).

[0055] The genetic sequence of Pichia kluyveri NYSC 5485 (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 DNA fragments can be individual chromosomes; 11 chromosomes and 1 mitochondrial DNA (SEQ ID NO:4). The genetic sequence is submitted electronically as SEQ ID NO:1-SEQ ID NO:12.

[0056] In one embodiment of the method 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 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 to SEQ ID NO: 10, a genetic sequence that is at least 99% identical to SEQ ID NO: 11, a genetic sequence that is at least 99% identical to SEQ ID NO: 12, a genetic sequence that is 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 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.

[0057] In one embodiment of the method 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 to SEQ ID NO: 10, a genetic sequence that is at least 99% identical to SEQ ID NO: 11, a genetic sequence that is at least 99% identical to SEQ ID NO: 12, a genetic sequence that is at least 99% identical to SEQ ID NO: 13, a genetic sequence that is at least 99% identical to SEQ ID NO: 14, a genetic sequence that is 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.

[0058] In one embodiment of the methods of the invention, the Pichia 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.

[0059] In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 1. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 2. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 3. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 5. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 6. In one embodiment, Pichia comprises a genetic sequence that is at least 99% identical to SEQ ID NO: 7. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 8. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 9. In one embodiment, Pichia comprises a genetic sequence that is at least 99% identical to SEQ ID NO: 10. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 11. In one embodiment, Pichia comprises a genetic sequence that is at least 98% (e.g., at least 99%) identical to SEQ ID NO: 12.

[0060] The name of yeast may change over time. It is to be understood that the yeast according to the method of the invention may comprise the genetic sequence identity described in the preceding paragraphs of the Pichia pastoris of the invention, regardless of the name applied to the yeast.

[0061] One aspect of the invention provides a method of preparing a beverage composition comprising fermenting an aqueous acerola extract, wherein the fermentation is performed by a yeast comprising a genetic sequence having at least 98% (eg, at least 99%) overall nucleotide identity to a combination of SEQ ID NOs: 1-12.

[0062] In one embodiment, fermentation is performed in the presence of water-insoluble components of acerola, and the aqueous acerola extract is separated from the water-insoluble components after fermentation. For example, a filtration system can be used to separate the acerola water-insoluble components from the aqueous acerola extract. After fermentation, the aqueous acerola extract becomes a fermented acerola extract.

[0063] The fermented acerola extract can be packaged as a ready-to-drink beverage, for example, in a bottle or can. The fermented acerola extract can have a concentration of total dissolved solids between 2% and 60% by weight, for example, a concentration of 2% to 25% by weight total dissolved solids. Before the fermented acerola extract is packaged as a ready-to-drink beverage, additional ingredients such as flavorings and preservatives may be added thereto. The fermented acerola extract may be heat treated before packaging to inactivate or kill yeast and any spoilage organisms. The fermented acerola extract may be passed through a filter before packaging to remove yeast and any spoilage organisms.

[0064] In one embodiment, the aqueous acerola extract is concentrated after fermentation, e.g., packaged as a liquid concentrate suitable for dilution by a consumer prior to consumption. The aqueous acerola extract can be concentrated as an intermediate product for use, e.g., in the manufacture of ready-to-drink products at a different location. The fermented acerola extract can have a concentration of 25% to 60% by weight total dissolved solids, further e.g., 50% to 60% by weight total dissolved solids. Dissolved solids can be measured, e.g., 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 dissolved solids.

[0065] The aqueous acerola extract (e.g., fermented acerola extract) after fermentation can be concentrated to form a powder, such as a soluble beverage powder. For example, the aqueous acerola extract (fermented acerola extract) after fermentation can be dried by spray drying or freeze drying to form a soluble beverage powder. For example, the aqueous acerola extract after fermentation can be spray dried with a carrier (such as maltodextrin) to form a soluble beverage powder. The soluble beverage powder can be packaged and sold as is, or it can be used as an intermediate product for manufacturing ready-to-drink products, such as in different locations. The soluble beverage powder can be filled into a container for a beverage preparation device. The soluble beverage powder can be combined with other ingredients (e.g., dry ingredients, such as tea, herbal tea, dried fruit, soluble coffee, or roasted and ground coffee). For example, the soluble beverage powder can be combined with other ingredients and filled into a container for a beverage preparation device. The soluble beverage powder can be combined with milk powder or beverage creamer powder and optional sugar to form a beverage mixture. After the aqueous acerola extract is fermented in the presence of the water-insoluble components of acerola, the water-insoluble components of acerola become fermented acerola. In one embodiment, the water-insoluble components of acerola present during the fermentation of the aqueous acerola extract are separated from the aqueous acerola extract after fermentation and then dried, for example, using a rotary dryer. The dried water-insoluble components of acerola can be filled into a container for a beverage preparation device, or into a permeable bag (such as a "tea bag"). The water-insoluble components of dried acerola 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 tea refers to the dried leaves, leaf buds, young branches or stems of the plant 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 an infusion made from the dried flowers, fruits, leaves, seeds or roots of plants other than the camellia sinensis plant.

[0066] In one embodiment, the water-insoluble components of acerola cherry present during the fermentation of the aqueous acerola extract are dried together with the aqueous acerola extract after fermentation, for example by freeze drying or spray drying. The dried water-insoluble components of acerola cherry (e.g., dried fermented acerola cherry) can be filled into a container for use in a beverage preparation device together with the aqueous acerola extract dried after fermentation (e.g., dried fermented acerola extract) or into a permeable bag such as a "tea bag". The dried water-insoluble components of acerola cherry and the aqueous acerola extract dried after fermentation can be combined with other dry ingredients such as tea, herbal tea, dried fruit, soluble coffee, or roast and ground coffee.

[0067] One aspect of the present invention provides use of acerola cherry fermented with Pichia kluyveri NYSC 5485 (CNCM I-5525) for producing a beverage.

[0068] Fermented acerola cherries according to the invention may be obtained in a "solid state" fermentation, for example by adding a starter culture of Pichia to acerola cherries (e.g. fresh acerola berries or berries that have been crushed into a pulp). The term "solid state" fermentation refers to a fermentation performed on a solid material. In solid state fermentation liquid water is present, but the term is used in contrast to liquid fermentation, which may occur in an aqueous solution in a fermentation vessel.

[0069] One aspect of the present invention provides a method for preparing a beverage composition, the method comprising:

[0070] A starter culture of yeast is added to the acerola cherries, wherein the starter culture provides at least 10 4 CFU (e.g. at least 10 5 CFU, further for example at least 10 6 CFU) of yeast / g acerola;

[0071] fermented acerola cherries;

[0072] Drying the fermented acerola cherries to form fermented acerola cherries; and any of the following;

[0073] Extracting fermented acerola cherries with water to form a ready-to-drink beverage, or

[0074] The fermented acerola cherries are filled into a container, such as a container for preparing a beverage when inserted into a beverage preparation device.

[0075] The yeast starter culture may be added to the acerola cherries as a liquid or powder. When the yeast starter culture is added, the acerola cherries (e.g., acerola pulp) may have a moisture content of 45% to 55% by weight. The acerola cherries (e.g., acerola pulp) may be treated with steam prior to fermentation to reduce the amount of environmental microorganisms prior to adding the yeast. The fermentation may be performed at a solids content of greater than 40% by weight, such as greater than 45% by weight, further such as greater than 50% by weight.

[0076] While some fermentation is continuing, drying of the fermented acerola cherries may begin. For example, the acerola cherries (e.g., acerola pulp) may be placed in a container (such as a jar), a starter culture may be added, and fermentation may then proceed for 1 to 4 days, after which the fermented acerola cherries may be removed from the container and dried. Further, for example, a starter culture may be added to the acerola cherries (e.g., acerola pulp) before the acerola cherries (e.g., acerola pulp) are slowly dried, for example, on a rack or hanging tray. Once fermentation begins, the acerola cherries may be slowly dried while fermentation continues. For example, the fermented acerola cherries may be dried to a moisture content of less than 20% by weight (e.g., less than 15% by weight) over a period of between 1 day and 30 days, for example, between 4 days and 20 days. The drying temperature may be less than about 40° C., for example, between about 25° C. and 35° C. A fan may be used to blow dry air onto the material until the acerola cherries (e.g., acerola pulp) is dry. The fermented acerola cherries (e.g., acerola pulp) may be dried in the sun. Acerola pulp may contain acerola skin and seeds.

[0077] The yeast may be Pichia pastoris, such as Pichia kluyveri, further such as Pichia kluyveri NYSC 5485. The yeast may comprise a genetic sequence having at least 98% (eg, at least 99%) full-length nucleotide identity to the combination of SEQ ID NOs: 1-12.

[0078] The container for preparing a beverage may be in the form of a capsule, a pad, a sachet, a pouch, a capsule or the like when inserted into a beverage preparation device.

[0079] The ready-to-drink beverage can be filled into bottles or cans. The fermented acerola cherry 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 a ready-to-drink beverage.

[0080] When inserted into a beverage preparation device, the fermented acerola cherries may be combined with tea, herbal tea, dried fruit, soluble coffee or roast and ground coffee in a container for preparing a beverage.

[0081] Those skilled in the art will appreciate that they are free to incorporate all features of the present invention disclosed herein. Specifically, features described for product of the present invention may be combined with method 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 included, as are clearly mentioned in this specification.

[0082] Further advantages and features of the invention will become apparent upon reference to the accompanying drawings and non-limiting examples.

[0083] Example

[0084] Example 1: Method for fermenting acerola cherries

[0085] An infusion was prepared using organic acerola soluble powder (Raab Vitalfood, Germany) as starting material. A hot infusion was first performed by adding 20 g of the dry starting material in 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 any additional debris. The filtrate (aqueous acerola extract) was equally divided as a control (Ref) or used for different fermentation variants.

[0086] Fermented acerola 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, 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 SCOBY) were grown in food grade yeast-peptone broth from glycerol vials and inoculated in a reference acerola wine with an optical density (OD at 600 nm) of 0.5. All inoculated infusions were stored at 30°C for 24 h under microaerobic conditions. After incubation, each fermented infusion was filter sterilized (0.2 μm).

[0087] Example 2: Sensory analysis of acerola cherries fermented with different yeasts

[0088] The sensory profiles of different variations of acerola infusions were tasted. The control tasted astringent and bitter. Acerola infusion fermented with S. cerevisiae had a fruity beer-like character. Acerola fermented with SCOBY had very strong acidity. Acerola infusions fermented with two different Pichia yeasts were fruity and had less bitter, sour, and astringent notes than the control. No buttery and fatty off-flavors were identified in acerola fermented with Pichia. Acerola fermented with Pichia kluyveri NYSC 5485 was found to have a cleaner flavor profile than acerola fermented with Pichia kluyveri NCYC 246.

[0089] Example 3: Analysis of flavor profiles focused on the aromatic compounds present in acerola cherries fermented with different yeasts thing .

[0090] Quantitative volatile profiling of acerola infusions was performed by headspace / solid phase microextraction coupled to chromatography and mass spectrometry (HS / SPME-GC-MS). The instrument used was a gas chromatograph (Agilent Technologies 8890 GC system) coupled to a mass spectrometer (Agilent Technology 7010B TQ).

[0091] Initially, 1.0 ml of acerola infusion sample was incubated in a 10-mL screw cap headspace vial at 30°C for 10 minutes, followed by extraction at 30°C for 10 minutes using a SPME fiber (PDMS / DVB, Chromatography Department (Supelco)). 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 with 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 maintained 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.

[0092] The following table lists the levels of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate, 2-phenylethanol, 2,3-butanedione, 2-phenylacetaldehyde, ethanol and acetic acid in parts per million after 24 hours.

[0093]

[0094]

[0095] The sample fermented with the SCOBY had high levels of acetic acid, corresponding to 222 g / kg on a dry basis.

[0096] The weight ratios of the aroma components A and B are in the table below.

[0097]

[0098]

[0099]

[0100] Samples fermented with Pichia (NYSC5485, NCYC2781 and NCYC246) showed higher ratios A and B than the control and samples fermented with S. cerevisiae or SCOBY. Pichia kluyveri NYSC 5485 provided higher values ​​for ratios A and B than the model strains Pichia kluyveri NCYC 246 or Pichia kluyveri NCYC 2781.

[0101] Example 4: Genetic sequence of Pichia kluyveri NYSC 5485 and model strain Pichia kluyveri NCYC Comparison of 246 .

[0102] 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 submitted in electronic form as SEQ ID NO: 1–SEQ ID NO: 12, and the genetic sequence of Pichia kluyveri NCYC 246 is submitted as SEQ ID NO: 13–SEQ ID NO: 27. The mitochondrial DNA of Pichia kluyveri NYSC 5485 is SEQ ID NO: 4, and the mitochondrial DNA of Pichia kluyveri NCYC 246 is SEQ ID NO: 20.

[0103] The software OrthoANIu was used to compare the genetic sequence of Pichia kluyveri NYSC 5485 (CNCM I-5525) with that of Pichia kluyveri NCYC 246. The overall average nucleotide identity was found to be 97.91%.

[0104] 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.

[0105]

[0106] 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 acerola cherry or a fermented acerola cherry extract, 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 150.

2. The beverage composition according to claim 1, wherein the weight ratio of the sum of 2-phenylethyl acetate and 2-phenylethanol to 2-phenylacetaldehyde is greater than 3000.

3. A beverage composition according to claim 1 or claim 2, having an ethanol content of less than 1.2% by weight.

4. The beverage composition according to any one of claims 1 to 3, 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.

5. A container for use in a beverage preparation device, the container containing a beverage composition according to any one of claims 1 to 4.

6. A method of preparing a beverage composition, the method comprising fermenting an aqueous acerola extract, wherein the fermentation is performed by a yeast comprising Pichia.

7. The method according to claim 6, wherein the Pichia pastoris is Pichia kluyveri.

8. The method according to claim 7, wherein the Pichia pastoris is Pichia kluyveri NYSC 5485 (CNCMI-5525).

9. The method of any one of claims 6 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 6 to 9, wherein the fermentation is carried out in the absence of Saccharomyces cerevisiae.

11. A method according to any one of claims 6 to 9, comprising separating the aqueous acerola extract from the steeped acerola cherries after fermentation.

12. The method of any one of claims 6 to 9, wherein the steeped acerola cherries present during the fermentation of the aqueous acerola extract are dried together with the aqueous acerola extract after fermentation.

13. The method according to any one of claims 6 to 12, wherein the beverage composition is according to any one of claims 1 to 4.

14. Use of acerola cherry fermented with Pichia kluyveri NYSC 5485 (CNCMI-5525) for producing a beverage.

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