Coffee fermentation by using pichia kluyveri and plant lactobacillus

Abstract

The invention belongs to the technical field of food. The present invention relates to a method for producing coffee beans, coffee beverages and coffee products, characterized in that the fermentation is carried out by inoculating coffee cherries with Pichia kluyveri (Pichia kluyveri) and Phytobacterium plantarum (Lactobacillus plantarum). The present invention relates to a method for producing coffee beans, coffee beverages and coffee products, and to a method for producing coffee beverages, coffee beverages and coffee products, characterized in that the fermentation is carried out by inoculating the coffee cherries with Pichia kluyveri (Pichia kluyveri) and Phytobacterium plantarum. The present invention provides leavening agents and their use for fermenting coffee plant material, as well as fermented coffee beans prepared according to the methods described herein.

Description

Technical Field

[0001] The present invention relates to the field of coffee fermentation, and in particular to a method for fermenting coffee using microorganisms and a coffee product obtained thereby. Background of the Invention

[0003] Coffee is a brewed beverage with a distinctive aroma and flavor, made from the roasted seeds / beans of the Coffea plant, a member of the Rubiaceae family.

[0004] Several species of the genus Coffea can be grown for their beans. Coffea arabica accounts for 75%-80% of the world's coffee production, while Coffea canephora accounts for about 20%.

[0005] When the coffee cherries are ripe, they are usually picked by hand, using either "selective picking" (picking only the ripe cherries) or "branch picking" (picking all the cherries from the branches). Because a tree can have both ripe and unripe berries, a crop must be picked multiple times, making harvesting the most labor-intensive procedure in coffee bean production. Machine harvesting can also be used, which is common in countries where labor is scarce and / or expensive. A cost-benefit assessment needs to be made when deciding which harvesting method to use.

[0006] Coffee cherries can be processed by a variety of methods, including wet, dry, and semi-dry processing. The treatment of the plant material (such as cleaning, sorting, and pulping) and the length of time required vary depending on the type of processing.

[0007] Wet processing: The ripe coffee cherries go through a flotation process to remove debris and remove suspended matter. The cherries are then pulped, in some cases through an underwater tank fermentation process, and then washed and dried, for example, in the sun, or in a dryer in the case of sub-specialty coffee commodity manufacturers. This process is also called washed.

[0008] Dry Processing: Whole coffee cherries are dried on platforms and / or floors in the sun without prior pulping. In some cases, the cherries are washed immediately after harvesting and floating material is separated. In other cases, sorting is done after drying. Most coffee production worldwide is dry processed. The process is also called "natural processing".

[0009] Semi-dry processing: The peel is removed within 24 hours of harvesting and the mucilage is left on the seeds as the coffee dries. This process is also known as the honey process or natural pulping.

[0010] To meet the growing demand for unique and premium specialty coffees, alternative methods are becoming increasingly popular. Examples here are variations in pre-treatment, where freshly harvested coffee cherries are fermented in a fermentation vessel (tank, barrel, box, pouch) before entering the traditional washed, natural or pulped natural process. Pre-treatment can occur with or without sorting, pulping, water addition and / or CO addition. 2 Common names for pre-treated coffee include carbonic maceration (CM process) and anaerobic fermentation / process / natural / pulped natural.

[0011] After processing, green coffee beans are obtained which can be stored and / or transported.

[0012] Spontaneous fermentation in traditional methods does not require the addition of fermentation agents. However, spontaneous fermentation is very heterogeneous and has a large variation in microbial populations and species composition, even when fermentation occurs at the same location. The variation depends on many factors, including country, farm, weather events, maturity, post-harvest handling, coffee type, fermentation method, batch size, stirring or not, fermentation time, etc., which makes coffee fermentation difficult to control and therefore fermentation reproducibility almost impossible. A major challenge is the lack of control over the growth and development of microorganisms and metabolites produced during the fermentation process, which leads to variable quality of the finished coffee beans.

[0013] Attempts have been made in the prior art to control the fermentation conditions for coffee bean fermentation. WO2014177666 discloses the use of a Pichia strain to ferment coffee berries. It discloses that Pichia kluyveri is more suitable for fermentation than Pichia anomala. However, there is no mention of the use of Lactiplantibacillus plantarum.

[0014] The fermentation process should be controlled to ensure the development of beneficial microorganisms, thereby producing a high-quality beverage with a good aroma. When fermentation fails, it leads to the development of spoilage microorganisms, which adversely affect the aroma and flavor of the coffee.

[0015] Therefore, being able to modulate the flavor of coffee beans, combined with high-quality coffee beans, will allow the development of new technologies that will enable coffee producers to obtain high-quality coffee beans. Therefore, such technology will have important commercial value.

[0016] Therefore, there is a need for continuous process improvements to improve the quality of coffee beans and coffee products prepared therefrom. SUMMARY OF THE INVENTION

[0018] In a first aspect, the present invention provides a method for producing coffee beans, comprising:

[0019] a) providing plant material consisting of coffee cherries,

[0020] b) inoculating the plant material with a Pichia kluyveri starter culture,

[0021] c) inoculating the plant material with a Lactiplantibacillus plantarum starter culture,

[0022] d) fermenting the plant material to obtain fermented coffee material, and

[0023] e) processing the fermented coffee material to obtain coffee beans,

[0024] Wherein steps b) and c) are carried out simultaneously or separately.

[0025] In a second aspect, the present invention provides coffee beans obtained by the method disclosed herein.

[0026] In a third aspect, the present invention provides coffee beans fermented with Pichia kluyveri and Lactobacillus plantarum.

[0027] In a fourth aspect, the present invention provides the use of Pichia kluyveri and Lactobacillus plantarum starter cultures to ferment coffee cherries.

[0028] It has been found that fermenting coffee material with Pichia kluyveri and Lactobacillus plantarum results in an improvement in flavor characteristics. For example, it has been found that Pichia kluyveri and Lactobacillus plantarum can be used to reduce the level of off-flavor compounds including acetic acid.

[0029] Coffee producers can improve the fruity flavor of freshly harvested coffee cherry batches by co-inoculating Pichia kluyveri and Lactobacillus plantarum simultaneously or at different times.

[0030] Additionally, controlled fermentation minimizes the risk of spoilage that can occur during natural fermentation.

[0031] The present method allows the development of desired properties such as flavor compounds and other quality advantages of fermented coffee beans and coffee products prepared therefrom.

[0032] Another aspect of the present invention relates to roasted coffee beans, coffee beverages or coffee products prepared from the coffee beans produced by the present invention.

[0033] Another aspect relates to the use of a starter composition comprising Pichia kluyveri and Lactobacillus plantarum to ferment coffee material.

[0034] definition

[0035] The term "plant material" includes anything that is or has been a living plant, in particular plants and any part thereof.

[0036] The term "fermentation" refers broadly to any activity or process involving the enzymatic breakdown (digestion) of organic material by microorganisms. The term "fermentation" includes anaerobic and aerobic processes, as well as combined or continuous processes comprising one or more anaerobic and / or aerobic stages.

[0037] As used herein, "spontaneous fermentation" refers to fermentation using microorganisms naturally present in the fermenting organic material and / or inadvertently introduced into the fermenting organic material at the beginning of the fermentation or during the fermentation. In contrast, the present invention provides "controlled fermentation" which includes the intentional addition of microbial strains as described herein.

[0038] The term "coffee product" herein refers to a product prepared using fermented coffee beans. These products refer to products prepared starting from coffee beans that have been fermented as described herein. Coffee-based products may be in liquid form or in a dried or freeze-dried form, such as granules, pellets or powder. For example, an example of a coffee-based product may be coffee itself. For example, another example may be a coffee cake.

[0039] "Coffee cherries" are the fruits of the coffee plant. They have a berry-like appearance and are initially green, small, and unripe. In the art, it is sometimes referred to as coffee berry.

[0040] The "coffee bean" is the seed of the coffee plant and the source of coffee. It is the kernel inside the red or purple coffee fruit. Although they are seeds, they are often called "beans" due to their resemblance to true beans.

[0041] The term "plant material comprising coffee cherries" is the starting material for producing coffee beans. Depending on the processing method used (e.g. "wet processing" or "dry processing", etc.), the skin and pulp of the cherries are removed before the fermentation process begins.

[0042] The term "starter" refers to a composition comprising live microorganisms capable of initiating or affecting fermentation (also referred to herein as a "starter composition"). Starter as used herein refers to a high-density culture of microorganisms.

[0043] The term "co-inoculating" or "co-inoculation" of Pichia kluyveri and Lactobacillus plantarum is to be understood as not requiring that the inoculation of each microorganism be carried out simultaneously. It may occur sequentially.

[0044] In this context, the term "mutant" is understood to be a strain derived from the strain of the present invention by, for example, genetic engineering, radiation and / or chemical treatment. Preferably, the mutant is a functionally equivalent mutant, for example, a mutant having substantially the same or improved properties as the parent strain (for example, with respect to enhancing the desired coffee flavor compounds). Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain subjected to any conventionally used mutagenic treatment of the strain of the present invention, including strains obtained by treatment with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or spontaneously generated mutants. The mutant may have undergone several mutagenic treatments (a single treatment is understood to be a mutagenic step followed by a screening / selection step), but it is currently preferred to perform no more than 20 or no more than 10 or no more than 5 treatments (or screening / selection steps). In currently preferred mutants, less than 5%, or less than 1%, or even less than 0.1% of the nucleotides in the yeast genome are replaced by another nucleotide, or are deleted, compared to the parent strain.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1

[0047] Figure 1 Schematic diagram of the process for preparing sample 1 (control) and sample 2 (inoculated with both Pichia kluyveri and Lactobacillus plantarum) in Example 1.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials equivalent to or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. The detailed information provided for using the present invention is also relevant to the methods of the present invention.

[0050] Coffee bean production is generally known. The relevant steps, such as cleaning, pulping, drying and roasting of the coffee beans, can be carried out according to standard known processes for making coffee. Therefore, it is not necessary to describe such standard processes in great detail herein.

[0051] A first aspect of the present invention relates to a method for producing coffee beans, comprising the following steps

[0052] - providing plant material including coffee cherries,

[0053] - inoculating the plant material with a Pichia kluyveri starter culture,

[0054] - inoculating the plant material with a Lactobacillus plantarum starter,

[0055] - fermenting the plant material to obtain fermented coffee material, and

[0056] -processing fermented coffee material to obtain coffee beans,

[0057] - wherein the inoculation of Pichia kluyveri (P. kluyveri) and Lactobacillus plantarum (L. plantarum) is carried out simultaneously or separately.

[0058] The present application is characterized in that Pichia kluyveri and Lactobacillus plantarum are used in combination. It has been found that this combination is very advantageous compared to the use of any one microorganism alone.

[0059] To practice the present invention, plant material including coffee cherries is provided. The plant material is co-inoculated with Pichia kluyveri and Lactobacillus plantarum using a starter culture of these microorganisms. These microorganisms are added to the plant material in an amount sufficient to initiate and maintain fermentation. Generally, a skilled artisan will be able to determine the appropriate inoculum concentration using conventional methods and in light of the present description and examples.

[0060] Preferably, the plant material is inoculated at a concentration of at least 10 2 CFU / g, such as at least 10 3 CFU / g, at least 10 4 CFU / g or at least 10 5 CFU / g of plant material.

[0061] Preferably, the plant material is inoculated at a concentration of at least 10 2 CFU / g, such as at least 10 3 CFU / g, at least 10 4 CFU / g or at least 10 5 CFU / g plant material of Lactobacillus plantarum ferment.

[0062] The plant material preferably consists essentially of coffee cherries, which may be whole cherries or pulped cherries. Preferably, the amount of plant material is at least 50 kg, more preferably at least 100 kg, such as at least 200 kg, such as at least 500 kg or at least 1000 kg.

[0063] A preferred embodiment of the invention relates to "wet processing" of coffee cherries. In this method, the plant material is soaked in water and a fermentation agent is applied.

[0064] Another preferred embodiment of the present invention relates to "dry processing" of coffee cherries, wherein the plant material is not soaked in water prior to inoculation.

[0065] Furthermore, in a preferred embodiment, when a "natural processing" method is used, inoculation of the starter culture may be preferably accomplished by spraying the starter onto the spread dry plant material.

[0066] The inoculation with Pichia kluyveri and Lactobacillus plantarum starter cultures can be carried out simultaneously or separately. For example, Pichia kluyveri can be inoculated before Lactobacillus plantarum inoculation, or vice versa.

[0067] Inoculation may be performed using any conventional method known in the art. Preferably, the starter culture is applied by spraying a suspension of the starter culture.

[0068] In a preferred embodiment, the concentration of living cells in the suspension is 10 3 cfu to 10 12 The range of cfu (colony forming units) includes at least 10 per ml of composition. 4 cfu, for example at least 10 5 cfu / ml, for example at least 10 6 cfu / ml, for example at least 10 7 cfu / ml, for example at least 10 8 cfu / ml, for example at least 10 9 cfu / ml, for example at least 10 10 cfu / ml, for example at least 10 11 cfu / ml, for example at least 10 12 cfu / ml.

[0069] In a preferred embodiment, the present application provides a method for producing coffee beans, comprising the following steps:

[0070] a) providing plant material comprising coffee cherries,

[0071] b) At least 10 2 The concentration of CFU / g plant material was determined by inoculating plant material with a Pichia kluyveri starter culture.

[0072] c) At least 10 2 The concentration of CFU / g plant material was inoculated with the plant material using the Lactobacillus plantarum starter culture.

[0073] d) fermenting the plant material to obtain fermented coffee material, and

[0074] e) processing the fermented coffee material to obtain coffee beans,

[0075] Wherein steps b) and c) are carried out simultaneously or separately.

[0076] In another preferred embodiment, the present application provides a method for producing coffee beans, comprising the following steps:

[0077] a) providing plant material comprising coffee cherries,

[0078] b) At least 10 3 CFU / g plant material concentration using Pichia kluyveri starter culture inoculation

[0079] Plant materials

[0080] c) At least 10 3 CFU / g plant material concentration inoculated with Lactobacillus plantarum starter

[0081] Material

[0082] d) fermenting the plant material to obtain fermented coffee material, and

[0083] e) processing the fermented coffee material to obtain coffee beans,

[0084] Wherein steps b) and c) are carried out simultaneously or separately.

[0085] The inoculated plant material is then placed under suitable conditions, and the fermentation process begins and continues for a period of time. One of ordinary skill in the art knows how to select suitable process conditions. Preferably, the fermentation is for at least 12 hours, e.g., at least 12 hours overnight, e.g., at least 1 day, e.g., at least 2 days, at least 3 days, including up to 5 days-10 days.

[0086] Fermentation can be carried out at ambient temperature, which may include temperatures of 5° C. to 40° C. Preferably, fermentation can be carried out at a temperature of 10° C. to 30° C. In a more preferred embodiment, fermentation can be carried out at a temperature of 20° C. to 30° C. Fermentation can be carried out in an open or closed container, or in the open air above ground.

[0087] During the fermentation process, a person skilled in the art is able to adjust other fermentation parameters known to him in order to obtain the desired end product.

[0088] After fermentation, fermented coffee material can be obtained and then subjected to further processing steps to obtain coffee beans.

[0089] In a preferred embodiment, the Pichia kluyveri starter culture is a high density culture comprising at least 10 7 CFU / g of Pichia kluyveri cells, for example at least 10 8 CFU / g, at least 10 9 CFU / g, at least 10 10 CFU / g, at least 10 11 CFU / g and at least 10 12 CFU / g. More than one strain may be present.

[0090] In a preferred embodiment, the Pichia kluyveri cells can be hydrated or dehydrated. Examples of hydrated cells include baker's yeast cake, compressed yeast and yeast paste. Examples of dehydrated cells include instant high active dry yeast, active dry yeast (ADY), freeze-dried yeast and partially dried compressed yeast.

[0091] The term "compressed yeast" herein refers to yeast having a dry matter content of 35% to 90% (w / w), typically produced by growing the yeast in a fermenter, then concentrating, filtering, squeezing and optionally partially drying in a dryer such as a fluidized bed dryer. In some embodiments, the dry matter content is 30% to 45%, for example 30% to 40% or 35% to 45%.

[0092] The term "yeast extract" refers herein to liquid yeast having a dry matter content of less than 28% (w / w), usually produced by propagating the yeast in a fermenter and then concentrating it by centrifugation.

[0093] The term "active dry yeast" or "ADY" refers herein to yeast having a dry matter content exceeding 90% (w / w), typically produced by growing the yeast in a fermenter and then concentrating, filtering, pressing and drying on a fluid bed dryer.

[0094] The term "partially dried compressed yeast" herein refers to yeast with a dry matter content of 45% to 90% (w / w) produced by propagating the yeast in a fermenter and then concentrating, filtering, pressing and partially drying on a dryer such as a fluidized bed dryer.

[0095] The term "lyophilized yeast" refers to a freeze-dried culture in powder or pellet form. In this form, the starter can be shipped without refrigeration.

[0096] The leavening agent composition of the application can also include cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, spices or their mixtures. Compositions can be in any form, including liquid (e.g., slurries), freezing or dry forms (e.g., freeze-dried or spray-dried). Compositions preferably include one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants, and most preferably cryoprotectants or lyoprotectants, or both. The purposes of protective agents such as cryoprotectants and lyoprotectants are well known to those skilled in the art. Suitable cryoprotectants or lyophilization protectants include monosaccharides, disaccharides, trisaccharides and polysaccharides (e.g. glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia), etc.), polyols (e.g. erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (e.g. proline, glutamic acid), complex substances (e.g. skim milk, peptone, gelatin, yeast extract) and inorganic compounds (e.g. sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (e.g. vitamin B group, vitamin C). The composition may optionally include additional substances, including emulsifiers (e.g. sorbitan monostearate), fillers (e.g. lactose, maltodextrin) and / or spices.

[0097] In a preferred embodiment, the Lactobacillus plantarum starter culture is a high-density culture comprising at least 10 7 CFU / g Lactobacillus plantarum bacterial cells, for example at least 10 8 CFU / g, at least 10 9 CFU / g, at least 10 10 CFU / g, at least 10 11 CFU / g and at least 10 12 CFU / g. More than one strain may be present.

[0098] In a preferred embodiment, the starter may be in frozen, liquid or dried form, including, for example, freeze-dried forms and spray / fluid bed dried forms, or a frozen or freeze-dried concentrate.The starter may comprise more than one yeast strain or bacterial strain.

[0099] Any suitable Lactobacillus plantarum may be used. In a preferred embodiment, Lactobacillus plantarum is harvest LB-1 (available from Chr. Hansen A / S) or DSM27565 as described in WO2015 / 110484. For the purposes of the present application, mutants having similar properties may also be used.

[0100] Any suitable Pichia kluyveri can be used. In a preferred embodiment, Pichia kluyveri is PK-KR1 or PK-KR2, which is deposited in the National Measurement Institute (541-65 Clarke Street, South Melbourne, Victoria 3205, Australia) by the School of Biological Sciences, University of Auckland (Auckland 1142, New Zealand), and is given accession numbers V06 / 022711 and V06 / 022712, respectively, as described in WO2009 / 110807. In another preferred embodiment, Pichia kluyveri is Pichia kluyveri DSM 28484 described in WO 2020 / 035268. For the purposes of this application, mutants with similar properties can also be used. In another preferred embodiment, Pichia kluyveri is Pichia kluyveri DSM 34278 or DSM 34279.

[0101] Preferably, the method as described herein further comprises the step of drying the fermented coffee material to obtain dry beans. The dry beans can be stored and transported to other destinations. Preferably, the method further comprises the step of roasting the dry beans using any method known in the art.

[0102] Preferably, during the fermentation process the plant material is preferably completely submerged in water.

[0103] In a preferred embodiment, the coffee plant material is Coffea arabica, Coffea canephora, Coffea eugenioides or Coffea liberica.

[0104] Thus, a second aspect of the invention relates to coffee beans obtained or obtainable by the method described herein.

[0105] A third aspect of the present invention relates to coffee beans fermented with Lactobacillus plantarum and Pichia kluyveri.

[0106] In a preferred embodiment, the coffee beans are fermented with Lactobacillus plantarum (such as Harvest LB-1 or DSM 27565) and Pichia kluyveri (such as DSM 34278, DSM 34279, DSM 28484, PK-KR1 or PK-KR2).

[0107] In a fourth aspect, the present application provides the use of Pichia kluyveri and Lactobacillus plantarum to ferment coffee material. These microorganisms can be used to inoculate plant material by the method described in the present application.

[0108] In a preferred embodiment, the plant material is fermented with the microorganism for at least 12 hours, such as at least 24 hours, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days, such as at least 7 days, such as at least 8 days, such as at least 9 days, such as at least 10 days.

[0109] Pichia kluyveri and Lactobacillus plantarum can be in the same or different starter compositions. In some embodiments, the starter can be a frozen or lyophilized starter, a so-called "direct-dip starter" (DVS) culture, intended to be directly inoculated into a fermentation vessel or cylinder. In addition, the composition can also be a liquid obtained by suspending a frozen, dried or lyophilized cell concentrate in a liquid medium (such as water or PBS buffer).

[0110] In a preferred embodiment, the present application provides the use of Pichia kluyveri and Lactobacillus plantarum for fermenting coffee material, wherein the use is carried out by a method comprising the following steps:

[0111] a) providing plant material comprising coffee cherries,

[0112] b) At least 10 2 The concentration of CFU / g plant material was determined by inoculating plant material with a Pichia kluyveri starter culture.

[0113] c) At least 10 2 The concentration of CFU / g plant material was inoculated with the plant material using the Lactobacillus plantarum starter culture.

[0114] d) fermenting the plant material to obtain fermented coffee material, and

[0115] e) processing the fermented coffee material to obtain coffee beans,

[0116] Wherein steps b) and c) are carried out simultaneously or separately.

[0117] In another preferred embodiment, the present application provides the use of Pichia kluyveri and Lactobacillus plantarum for fermenting coffee material, wherein the use is carried out by a method comprising the following steps:

[0118] a) providing plant material comprising coffee cherries,

[0119] b) At least 10 3 The concentration of CFU / g plant material was determined by inoculating plant material with a Pichia kluyveri starter culture.

[0120] c) At least 10 3 The concentration of CFU / g plant material was inoculated with the plant material using the Lactobacillus plantarum starter culture.

[0121] d) fermenting the plant material to obtain fermented coffee material, and

[0122] e) processing the fermented coffee material to obtain coffee beans,

[0123] Wherein steps b) and c) are carried out simultaneously or separately.

[0124] The inventors have found that the synergistic effect between Pichia kluyveri and Lactobacillus plantarum has a beneficial effect on the flavor profile of coffee products compared to the use of Pichia kluyveri or Lactobacillus plantarum alone.

[0125] Preferably, the methods described herein are particularly suitable for commercially relevant large-scale coffee production.Thus, the present invention can help reduce batch-to-batch quality inconsistencies.

[0126] Fermentation with Pichia kluyveri and Lactobacillus plantarum can enhance the flavor of coffee due to the presence of increased amounts of desirable flavor compounds.

[0127] The methods described herein can also be implemented to reduce off-flavors (e.g., vinegar off-flavors). In addition, the lack of fruity flavor can be overcome and the acidic characteristics can be improved.

[0128] As discussed above, the results of Example 2 herein show that coffee beverages prepared from roasted coffee beans fermented with Pichia kluyveri and Lactobacillus plantarum result in a satisfactory flavor improvement compared to the control. The sensory improvement can be determined by the cupping analysis described in Example 2 or other known methods, such as according to the widely used Specialty Coffee Association (SCA) cupping protocol (Lingle, TR (2011). Systematic guide to the sensory evaluation of Coffee's flavor. In Specialty Coffee Association of America (Ed.), The coffeecupper's handbook (pp. 1–66). Specialty Coffee Association of America). The level of volatile compounds can be determined by headspace gas chromatography-flame ionization detector as described in Example 3 herein or by any other method known to the skilled person.

[0129] ***

[0130] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a, an" and "said / the" and similar references should be interpreted as covering the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including, but not limited to"). Unless otherwise indicated herein, the description of the numerical range herein is intended only as a shorthand method for referring to each individual numerical value within the range individually, and each individual value is incorporated into the specification as if it were described separately herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise required. Any language in the specification should not be interpreted as indicating that any unrequired element is essential to the practice of the present invention.

[0131] Taxonomy

[0132] Lactobacillus plantarum is now called Lactiplantibacillus plantarum as described in Zheng et al., Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107.

[0133] Preservation and Expert Solutions

[0134] The applicant requests that, in accordance with existing regulations of industrial property offices of contracting states of the Budapest Treaty, samples of the deposited microorganisms described below may only be provided to experts until the date of grant of the patent.

[0135] The applicant deposited DSM 34278 on May 31, 2022 at the Leibniz DSMZ Institute - German Collection of Microorganisms GmbH (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, and received the accession number: DSM 34278.

[0136] The applicant deposited DSM 34279 on May 31, 2022 at the Leibniz DSMZ Institute - German Collection of Microorganisms GmbH (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, and received the accession number: DSM 34279.

[0137] The applicant deposited DSM 28484 on March 5, 2014 at the Leibniz DSMZ Institute - German Collection of Microorganisms GmbH (DSMZ), Inhofenstrasse 7B, D-38124 Brunswick, and received the accession number: DSM 28484.

[0138] The applicant deposited Lactobacillus plantarum DSM 27565 on August 1, 2013 at the Leibniz DSMZ Institute - German Collection of Microorganisms GmbH (DSMZ), Inhofenstrasse 7B, D-38124 Brunswick, and received the accession number: DSM27565. Example

[0139] Example 1 Processing of coffee beans

[0140] The trials were conducted on coffee cherries using a natural processing method, where pre-treatment of the cherries was performed by fermenting the cherries in a container without adding water. For both the cupping analysis (Example 2) and the volatile aroma analysis (Example 3) of the resulting coffees, a non-inoculated control batch was included. Apart from the inoculation, the two batches were prepared in the same way.

[0141] In detail, Arabica coffee cherries from the same harvest day and harvest batch were sorted to remove over-ripe and under-ripe cherries, and then divided into two closed fermentation containers. The whole cherries were fermented in these two containers for 12 days.

[0142] Batch 1 (control): no inoculation (spontaneous fermentation)

[0143] Batch 2: Inoculation with Lactobacillus plantarum and Pichia kluyveri

[0144] Fermentation Agents

[0145] 1. Freeze-dried Lactobacillus plantarum starter culture DSM 27565

[0146] Vaccination rate: 1x 10 7 CFU / g coffee fruit

[0147] 2. Frozen Pichia kluyveri starter culture PK-KR1

[0148] Vaccination rate: 1x 10 6 CFU / g coffee fruit

[0149] Vaccination regimen

[0150] Preparation and inoculation of Lactobacillus plantarum:

[0151] • At room temperature, mix the lyophilized starter pellets with 10 times the amount of water (non-chlorinated).

[0152] Stir the mixture well with a clean spoon and wait for 5 minutes.

[0153] Pour the bacterial suspension into a clean spray bottle and spray an equal amount of the coffee cherries in each container.

[0154] Preparation and inoculation of Pichia kluyveri:

[0155] Transfer the frozen starter from the refrigerator to a warm water bath at about 30°C for 1.5 hours.

[0156] Spray the liquid yeast as evenly as possible over the coffee cherries in the tank.

[0157] Fermentation took place over 12 days. The fermented whole fruit was transferred to elevated beds for drying.

[0158] Both batches were dried to the same moisture content before being ground and packaged as green coffee beans.

[0159] Example 2 Sensory Evaluation

[0160] 2.1 Materials and methods

[0161] Green coffee beans were roasted in the same roasting mode and fresh coffee was prepared from the first and second batches (designated as Sample 1 and Sample 2, respectively) by grinding 12 g of coffee and using a coffee to water ratio of 1:17 at 94°C-95°C. The husk was broken and crushed after 4 minutes and then cupped.

[0162] In the sensory analysis, the attributes of each sample were evaluated by cupping analysis according to the CoE (Coffee of Excellence) methodology (http: / / cupofexcellence.org / rules-protocols / ). The following attributes were evaluated: clarity, sweetness, acidity, mouthfeel, flavor, aftertaste, balance and overall. In this method, each sample has a starting score of 36 points, and the scores of each attribute (0-8) will be combined with this score to get the final score.

[0163] 2.2 Results

[0164] Table 1 Cupping grading results:

[0165]

[0166] Table 2 Overall cupping description:

[0167] Cupping Instructions Sample 1 Fluffy, flat, murky flavor, herbal, damp leaves Sample 2 Hibiscus, red apple, dried spices, raspberry, sour cherry, grapefruit

[0168] 2.3 Conclusion

[0169] As shown herein, the combined use of Pichia kluyveri and Lactobacillus plantarum advantageously improves the quality grade of coffee and, therefore, the commercial value of green beans. Scores above 85 points can be considered to be of excellent quality. High quality scores have a direct impact on determining the price of specialty coffee.

[0170] Example 3 VOC Analysis

[0171] 3.1 Materials and methods

[0172] Two batches of coffee beans were roasted and brewed in the same manner as in Example 2. Afterwards, 1 L of coffee from each sample was filtered and frozen at the Laboratorio de Análisis del Aroma y Enología (LAAE) at the University of Zaragoza, Spain, for analysis of volatile compounds.

[0173] 3.1.1 Analysis of main volatile compounds

[0174] The main compounds were determined using a method developed for the main volatile compounds based on gas chromatography-flame ionisation detection analysis of dichloromethane microextracts according to Ortega, Catalina et al. "Fast analysis of important wine volatile compounds: Development and validation of a new method based on gaschromatographic–flame ionisation detection analysis of dichloromethane microextracts." Journal of Chromatography A923.1-2 (2001): 205-214.

[0175] 3.1.2 Analysis of trace and minor volatile compounds

[0176] Minor and trace volatile compounds were determined by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) according to Lopez, Ricardo et al. "Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with massspectrometric detection." Journal of Chromatography A966.1-2 (2002): 167-177.

[0177] 3.2 Results

[0178] Table 3 Levels (ppm) of selected compounds detected in Sample 1 and Sample 2.

[0179]

[0180]

[0181] 3.3 Conclusion

[0182] The combined use of Pichia kluyveri and Lactobacillus plantarum resulted in an increase in aroma compounds responsible for positive fruity and fermented aroma descriptors such as acetaldehyde, ethyl acetate and alpha-terpineol. In addition, the solution resulted in a decrease in acetic acid content.

[0183] Example 4 Processing of coffee beans

[0184] The test was conducted on coffee cherries using a washed processing method, in which a soaking step was performed on de-sizing parchment with minimal water added to a container. For cupping analysis of the resulting coffees (Example 5), an uninoculated control batch and batches inoculated with either Lactobacillus plantarum or Pichia kluyveri alone were included. The six batches were prepared identically, except for the inoculation.

[0185] In detail, Arabica coffee cherries from the same harvest day and harvest batch are sorted before mechanical depulping to remove over-ripe and under-ripe cherries. The parchments are fermented / macerated in plastic boxes for 48 hours before being washed and dried.

[0186] Batch 1 (control): no inoculation (spontaneous fermentation)

[0187] Batch 2: Inoculation with Lactobacillus plantarum DSM 27565

[0188] Batch 3: Inoculation with Pichia kluyveri PK-KR1

[0189] Batch 4: Inoculation with Pichia kluyveri DSM 28484

[0190] Batch 5: Inoculation with Lactobacillus plantarum and Pichia kluyveri PK-KR1

[0191] Batch 6: Inoculation with Lactobacillus plantarum and Pichia kluyveri DSM 28484

[0192] Fermentation Agents

[0193] 1. Freeze-dried Lactobacillus plantarum starter culture DSM 27565

[0194] Vaccination rate: 1x 10 7 CFU / g coffee fruit

[0195] 2. Frozen Pichia kluyveri starter culture PK-KR1

[0196] Vaccination rate: 1x 10 6 CFU / g coffee fruit

[0197] 3. Frozen Pichia kluyveri starter culture DSM 28484

[0198] Vaccination rate: 1x 10 6 CFU / g coffee fruit

[0199] Vaccination regimen

[0200] Preparation and inoculation of Lactobacillus plantarum:

[0201] • Inoculation of the Lactobacillus plantarum starter culture was performed by adding the freeze-dried pellets directly to a mixture of desized parchment paper and water.

[0202] Preparation and inoculation of Pichia kluyveri:

[0203] Transfer the frozen starter from the refrigerator to a warm water bath at about 30°C for 1.5 hours.

[0204] Spray the thawed liquid yeast as evenly as possible over the coffee cherries in the tank.

[0205] All 6 batches were produced simultaneously. All batches underwent a mixing step immediately after inoculation.

[0206] Fermentation takes place for 48 hours, after which all batches are washed and dried to the same moisture content before being ground and packaged as green coffee beans.

[0207] Example 5 Sensory Evaluation

[0208] 5.1 Materials and methods

[0209] In the sensory analysis, coffees were prepared from batches 1-6 (designated samples 1-6) in Example 4 and evaluated by cupping analysis according to the SCAA protocol (Specialty Coffee Association of America protocol 2015) https: / / www.scaa.org / PDF / resources / cupping-protocols.pdf.

[0210] 5.2 Results

[0211] Table 4 Cupping grading results:

[0212]

[0213]

[0214] 5.3 Conclusion

[0215] As demonstrated herein, the combined use of Pichia kluyveri and Lactobacillus plantarum advantageously improves the quality grade of coffee compared to their use alone.

[0216] Example 6 Processing of coffee beans

[0217] The trial was conducted on depulped coffee cherries using a processing method known as the Honey Process, in which the cherries are depulped after sorting and then inoculated by spraying directly onto depulped kraft paper on a drying bed. For cupping analysis of the resulting coffees (Example 7), an uninoculated control batch and batches inoculated with either Lactobacillus plantarum or Pichia kluyveri alone were included. The four batches were prepared identically, except for the inoculation.

[0218] Specifically, Arabica coffee cherries from the same harvest day and harvest batch were sorted to remove overripe and underripe cherries, then mechanically depulped and divided into four smaller batches of equal size and spread on raised drying beds. The parchment was then dried to a moisture content of 11%.

[0219] Batch 1 (control): no inoculation (spontaneous fermentation)

[0220] Batch 2: Inoculation with Lactobacillus plantarum harvest LB-1

[0221] Batch 3: Inoculation with Lactobacillus plantarum and Pichia kluyveri PK-KR1

[0222] Batch 4: Inoculation with Lactobacillus plantarum and Pichia kluyveri DSM 28484

[0223] Fermentation Agents

[0224] 1. Freeze-dried Lactobacillus plantarum fermentation culture harvest LB-1

[0225] Vaccination rate: 1x10 7 CFU / g coffee fruit

[0226] 2. Frozen Pichia kluyveri starter culture PK-KR1

[0227] Vaccination rate: 1x 10 6 CFU / g coffee fruit

[0228] 3. Frozen Pichia kluyveri starter culture DSM 28484

[0229] Vaccination rate: 1x 10 6 CFU / g coffee fruit

[0230] Vaccination regimen

[0231] Preparation and inoculation of Lactobacillus plantarum:

[0232] • Mix the lyophilized starter pellets with 10 times the amount of water (non-chlorinated) at room temperature.

[0233] Mix the mixture well with a clean spoon and wait for 5 minutes.

[0234] Pour the bacterial suspension into a clean spray bottle and spray directly onto the coffee cherries on the raised drying beds, allocating an equal amount of bacterial suspension to each batch of coffee to be inoculated.

[0235] Preparation and inoculation of Pichia kluyveri:

[0236] Transfer the frozen starter from the refrigerator to a warm water bath at about 30°C for 1.5 hours.

[0237] Pour the thawed liquid yeast into a clean spray bottle and spray directly onto the coffee cherries on the elevated drying beds, allocating an equal amount of yeast to each batch of coffee to be inoculated.

[0238] Batches of coffee are fermented in the early stages of the drying process while the water activity still allows microbial activity. Four batches are dried to the same degree of moisture content (10%), which takes about 10 days. After this, all the coffee is ground and packaged as green coffee beans.

[0239] Example 7 Sensory Evaluation

[0240] 7.1 Materials and methods

[0241] Coffees were prepared from batches 1-4 (designated samples 1-4) of Example 6 and evaluated as described in Example 2.1.

[0242] 7.2 Results

[0243] Table 5: Cupping grading results:

[0244]

[0245]

[0246] Table 6 Overall cupping description:

[0247] Cupping Instructions Sample 1 Citrus, apricot, jelly-like Sample 2 Husky, phenol, hay, brown sugar Sample 3 Citrus, sugar cane, candy-like, apple Sample 4 Strong citrus, candy-like flavor

[0248] 7.3 Conclusion

[0249] As demonstrated herein, the combined use of Pichia kluyveri and Lactobacillus plantarum advantageously increases the yield of very high quality coffees that can be obtained compared to the use of either microorganism alone.

Claims

1. A method for producing coffee beans, comprising the following steps: a) providing plant material comprising coffee cherries, b) inoculating the plant material with a Pichia kluyveri starter culture, c) inoculating the plant material with a Lactiplantibacillus plantarum starter culture, d) fermenting the plant material to obtain fermented coffee material, and e) processing the fermented coffee material to obtain coffee beans, Wherein steps b) and c) are carried out simultaneously or separately.

2. The method according to any one of the preceding claims, wherein at least 10 2 The concentration of CFU / g plant material was inoculated with Pichia kluyveri or Lactobacillus plantarum.

3. The method according to any one of the preceding claims, wherein the inoculation steps b) and c) comprise spraying the starter culture.

4. The method according to any one of the preceding claims, wherein the fermentation of step d) of claim 1 is a fermentation of at least 12 hours.

5. The method according to any one of the preceding claims, wherein the Pichia kluyveri starter culture comprises 10 3 CFU / g to 10 12 CFU / g of yeast cells.

6. The method according to any one of the preceding claims, wherein the plant lactobacillus fermentation culture has a concentration of 10 3 CFU / g to 10 12 CFU / g of bacterial cells.

7. The method according to any one of the preceding claims, wherein the Lactobacillus plantarum is harvest LB-1, DSM27565 or a mutant thereof.

8. A method according to any one of the preceding claims, wherein: The Pichia kluyveri is DSM 34278, DSM 34279, DSM 28484, PK-KR1, PK-KR2 or a mutant thereof.

9. The method according to any one of the preceding claims, step e) comprising drying the fermented coffee material of step d) to obtain dried coffee beans, and optionally roasting the dried coffee beans.

10. A method according to any one of the preceding claims, wherein in step d) the plant material is immersed.

11. A method according to any one of the preceding claims, wherein the coffee plant material is from Coffea arabica, Coffea canephora, Coffea eugenioides or Coffea liberica.

12. Coffee beans obtained by the method according to any one of the preceding claims.

13. Coffee beans fermented with Pichia kluyveri and Lactobacillus plantarum.

14. Use of coffee materials fermented by Pichia kluyveri and Lactobacillus plantarum.

15. Use of a starter composition comprising Pichia kluyveri and Lactobacillus plantarum to ferment coffee material.

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