Kluyveromyces marxianus LRCC 8279 and LRCC 8325 suitable for non-alcoholic alcoholic beverages and fermented beverages

Abstract

The present invention relates to Kluyveromyces marxianus LRCC 8325 strain and Kluyveromyces marxianus LRCC 8279 strain suitable for producing non-alcoholic alcoholic beverages and fermented root vegetable beverages, and uses thereof.

Description

[Technical Field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2023-0126551, filed with the Korean Intellectual Property Office on September 21, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to Kluyveromyces marxianus LRCC 8325 strain and Kluyveromyces marxianus LRCC 8279 strain, which are suitable for producing non-alcoholic alcoholic beverages and fermented root vegetable beverages, and uses thereof. [Background technology]

[0003] In recent years, as the beverage market has become more specific and segmented, products tailored to consumer needs have been released onto the market, and in particular, alcohol-free and low-alcohol beverages have become increasingly popular in the alcoholic beverage market. In particular, with the recent spread of infectious diseases such as COVID-19 restricting people from going out and visiting liquor stores, consumers have been enjoying alcohol alone at home, so-called so-called solo drinking or at-home drinking, and the popularity of such beverages has increased even further.

[0004] Meanwhile, as drinking at home has become a global trend, the average amount of alcohol consumed per occasion has decreased, but the number of times people drink has increased. In particular, alcohol consumption among women has increased as women turn to alcohol to relieve stress due to the increased amount of housework and childcare responsibilities. In addition, as people are increasingly drinking at home with their families or alone, low-alcohol alcohol products such as fruit liquor, low-alcohol drinks, and cocktails are becoming very popular, and the related market is expanding significantly as young people are also showing interest in these products.

[0005] In the Korean alcoholic beverage market, alcohol-free beverages are those with an alcohol content of less than 1% that are not considered alcoholic beverages and are produced using a non-fermentation process that does not use yeast. Low-alcohol beverages are those with a low alcohol content of approximately 1% to 3% and are produced by selectively removing only the alcohol using a separation method in the final filtration stage of the existing beer production process, or by completing the fermentation process in a short period of time to prevent the production of alcohol.

[0006] However, this manufacturing process can impair the inherent aroma and flavor of fermented beer, so flavoring ingredients are artificially added to satisfy consumer needs. To overcome this, a method using yeast that has low maltose utilization has been proposed, but because this method relies on genetically modified organisms (GMOs), it is still not enough to alleviate consumer concerns. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been devised to solve the above problems, and aims to provide a non-genetically modified Kluyveromyces marxianus LRCC 8325 strain that is suitable for producing non-alcoholic alcoholic beverages and fermented root vegetable beverages, and uses thereof.

[0008] Another object of the present invention is to provide a non-genetically modified Kluyveromyces marxianus LRCC 8279 strain and uses thereof.

[0009] The objectives of the present invention are not limited to the above-mentioned objectives, but will become clearer from the following description and will be realized by the means and combinations thereof set forth in the claims. [Means for solving the problem]

[0010] The present invention will now be described in more detail.

[0011] One embodiment of the present invention relates to Kluyveromyces marxianus strain LRCC 8325, deposited as KCCM 13373P.

[0012] Another embodiment of the present invention relates to a composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus strain LRCC 8325 deposited under KCCM 13373P, a culture of the strain, and a combination thereof.

[0013] Another embodiment of the present invention relates to an ethanol-containing food product comprising at least one of the Kluyveromyces marxianus strain LRCC 8325 deposited under KCCM 13373P, a culture of said strain, and combinations thereof.

[0014] The food may include at least one of drinking water, tea, a health drink, makgeolli, soju, whiskey, wine, beer, and combinations thereof.

[0015] Another embodiment of the present invention relates to a method for producing an ethanol-containing food product, comprising culturing Kluyveromyces marxianus strain LRCC 8325, deposited under KCCM 13373P, in the presence of a substrate.

[0016] The substrate may include at least one of wort, arrowroot, burdock, ginger, bellflower, and combinations thereof.

[0017] The sugar content of the substrate may be 10 plato to 11 plato.

[0018] The step of culturing the strain may be carried out at 5°C to 35°C for 12 to 72 hours.

[0019] In the step of culturing the strain, the initial inoculation concentration of the strain may be 10 5 CFU / ml to 10 7 CFU / ml.

[0020] One embodiment of the present invention relates to Kluyveromyces marxianus strain LRCC 8279, deposited as KCCM 13372P.

[0021] Another embodiment of the present invention relates to a composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus strain LRCC 8279 deposited under accession number KCCM 13372P, a culture of the strain, and a combination thereof.

[0022] Another embodiment of the present invention relates to an ethanol-containing food product comprising at least one of the Kluyveromyces marxianus strain LRCC 8279 deposited under KCCM 13372P, a culture of the strain, and combinations thereof.

[0023] The food may include at least one of drinking water, tea, a health drink, makgeolli, soju, whiskey, wine, beer, and combinations thereof.

[0024] Another embodiment of the present invention relates to a method for producing an ethanol-containing food product, comprising culturing Kluyveromyces marxianus strain LRCC 8279, deposited under KCCM 13372P, in the presence of a substrate.

[0025] The substrate may include at least one of wort, arrowroot, burdock, ginger, bellflower, and combinations thereof.

[0026] The sugar content of the substrate may be 10 plato to 11 plato.

[0027] The step of culturing the strain may be carried out at 5°C to 35°C for 12 to 72 hours.

[0028] In the step of culturing the strain, the initial inoculation concentration of the strain may be 10 5 CFU / ml to 10 7 CFU / ml. [Effects of the Invention]

[0029] By using the novel strains isolated and identified according to the present invention, it is possible to produce non-alcoholic alcoholic beverages and fermented root vegetable beverages with low ethanol contents of approximately 1% to 3% without a separate ethanol removal process or the use of genetically modified organisms (GMOs).

[0030] In addition, the non-alcoholic alcoholic beverages and fermented root vegetable beverages produced according to the present invention have a strong floral aroma and weak fatty and oily aromas, and therefore can embody a fragrant yet refreshing taste.

[0031] Furthermore, by using the strain of the present invention, the efficiency of the production process of non-alcoholic alcoholic beverages and fermented root vegetable beverages can be improved, costs can be reduced, and fermented beverages can be produced using various root vegetables.

[0032] The effects of the present invention are not limited to those mentioned above, and should be understood to include all effects that can be inferred from the following description. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a diagram showing a phylogenetic tree of Kluyveromyces marxianus LRCC 8325 strain (hereinafter referred to as LRCC 8325 strain) according to one embodiment of the present invention. [Figure 2] FIG. 1 is a diagram showing a phylogenetic tree of the Kluyveromyces marxianus LRCC 8279 strain (hereinafter referred to as the LRCC 8279 strain) according to one example of the present invention. [Figure 3] 1 shows photographs confirming the non-glycosyltransferase activity of the LRCC 8325 and LRCC 8279 strains. [Figure 4] 1 is a graph showing the analysis of the alcohol production ability of yeast in a medium containing a single sugar. [Figure 5] 1 is a graph showing an analysis of the alcohol production ability of yeast in a medium containing a mixed sugar. [Figure 6] 1 is a graph showing an analysis of the alcohol production ability and growth of yeast depending on the culture time at a culture temperature of 10° C. [Figure 7] 1 is a graph showing an analysis of the alcohol production ability and growth of yeast depending on the culture time at a culture temperature of 15° C. [Figure 8] 1 is a graph showing an analysis of the alcohol production ability and growth of yeast depending on the culture time at a culture temperature of 20° C. [Figure 9] 1 is a graph showing an analysis of the alcohol production ability and growth of yeast depending on the incubation time at an incubation temperature of 25° C. [Figure 10] 1 is a graph showing an analysis of the alcohol production ability and growth of yeast depending on the incubation time at an incubation temperature of 30° C. [Figure 11] 1 is a graph showing an analysis of alcohol production when wort is fermented using yeast according to an example of the present invention. [Figure 12] 1 is a graph showing the calculation of RE (Real Extract) when wort is fermented using yeast according to an example of the present invention. [Figure 13] 1 is a graph showing the calculated RDF (real degree of fermentation) when wort is fermented using yeast according to an example of the present invention. [Figure 14] 1 is a graph showing a comparative analysis of aroma components of fermented wort using gas chromatography / mass spectrometry (GC-MS) according to an example of the present invention. [Figure 15] 1 shows an analysis of the similarity of aroma patterns of fermented wort according to an experimental example of the present invention. [Figure 16] 1 is a graph showing an analysis of the amount of alcohol produced and the number of live bacteria when a root vegetable concentrate is fermented. [Figure 17] 1 is a graph showing a comparative analysis of aroma components of kudzu by GC-MS according to an example of the present invention. [Figure 18] This is an analysis of the similarity of the scent patterns of kudzu according to an experimental example of the present invention. [Figure 19] 1 is a graph showing a comparative analysis of aroma components of burdock by GC-MS according to an example of the present invention. [Figure 20] This is an analysis of the similarity of burdock scent patterns in an experimental example of the present invention. [Figure 21] 1 is a graph showing a comparative analysis of aroma components of ginger by GC-MS according to an example of the present invention. [Figure 22] The similarity of ginger scent patterns was analyzed according to an experimental example of the present invention. [Figure 23] 1 is a graph showing comparative analysis of aroma components of Platycodon grandiflorum by GC-MS according to an example of the present invention. [Figure 24] This is an analysis of the similarity of the fragrance patterns of bellflowers according to an experimental example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] Kluyveromyces marxianus strain LRCC 8325, deposited as KCCM 13373P.

[0035] Kluyveromyces marxianus strain LRCC 8279, deposited as KCCM 13372P. [Example]

[0036] The above and other objects, features, and advantages of the present invention will be readily understood from the following preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed content will be thorough and complete, and so that the concept of the present invention will be fully conveyed to those skilled in the art.

[0037] In the description of each figure, like reference numerals are used for like components. In the accompanying drawings, the dimensions of structures are exaggerated for clarity of the present invention. Terms such as "first," "second," etc. may be used to describe various components, but these components should not be limited by such terms. These terms are used only for the purpose of distinguishing one component from another. For example, a first component may be designated as a "second component," and similarly, a second component may be designated as a "first component," without departing from the scope of the present invention. A singular term includes a plural term unless otherwise specified in the context.

[0038] As used herein, terms such as "comprise" or "have" are intended to specify the presence of a stated feature, numeral, step, operation, component, part, or combination thereof, but should be understood not to preclude the possible presence or addition of one or more other features, numerals, steps, operations, components, parts, or combinations thereof. Furthermore, when a layer, film, region, substrate, or other part is described as being "on" another part, this includes not only the case where it is "directly on" the other part, but also the case where there is another part between them. Similarly, when a layer, film, region, substrate, or other part is described as being "under" another part, this includes not only the case where it is "directly under" the other part, but also the case where there is another part between them.

[0039] Unless otherwise specified, all numbers, values, and / or expressions used herein expressing ingredients, reaction conditions, and amounts of ingredients should be understood in all instances to be modified by the term "about" in that these numbers are approximations that reflect various uncertainties of measurement that arise in deriving such values ​​from disparate materials. Also, when numerical ranges are disclosed herein, such ranges are continuous and include every value from the minimum value to the maximum value, inclusive, unless otherwise specified. Furthermore, when such a range denotes integers, all integers from the minimum value to the maximum value, inclusive, are included unless otherwise specified.

[0040] When a range is recited herein for a variable, the variable will be understood to include all values ​​within the stated range, including the recited endpoints of the range. For example, the range "5 to 10" can be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any value between the integers that fall within the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, a range of "10% to 30%" will be understood to include not only values ​​such as 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, but also any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between any reasonable integers within the stated range, such as 10.5%, 15.5%, 25.5%, etc.

[0041] An example of the present invention relates to Kluyveromyces marxianus strain LRCC 8325, deposited under accession number KCCM 13373P.

[0042] In the present invention, the Kluyveromyces marxianus LRCC 8325 strain may be the Kluyveromyces marxianus LRCC 8325 strain deposited at the Korea Microorganism Collection on July 26, 2023 under accession number KCCM 13373P.

[0043] The 26S rRNA base sequence of the Kluyveromyces marxianus strain LRCC 8325 may comprise the base sequence of SEQ ID NO:1.

[0044] The Kluyveromyces marxianus LRCC 8325 strain may be derived from malt or rice koji.

[0045] The Kluyveromyces marxianus LRCC 8325 strain may have the ability to decompose at least one of glucose (D-glucose), xylose (D-xylose), xylitol (Xylitol), galactose (D-galactose), sorbitol (D-sorbitol), lactose (D-lactose), sucrose (D-saccharose), raffinose (D-raffinose), and combinations thereof.

[0046] Another example of the present invention relates to a composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus strain LRCC 8325 deposited under accession number KCCM 13373P, a culture of the strain, and a combination thereof.

[0047] As used herein, the term "culture" refers to a liquid corresponding to a culture medium containing a bacterial strain after culturing the strain, a isolate separated from the culture medium of the bacterial strain, a fraction thereof, a culture filtrate, a fermentation broth, or a supernatant separated therefrom, or a dried product obtained by freeze-drying the supernatant, but is not limited thereto.

[0048] In the present invention, the compositions may be prepared in the form of, for example, but not limited to, directly sprayable solutions, powders and suspensions, or highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, fly ash, or granules.

[0049] Another example of the present invention relates to an ethanol-containing food product containing at least one of the Kluyveromyces marxianus strain LRCC 8325 deposited under accession number KCCM 13373P, a culture of the strain, and a combination thereof.

[0050] The ethanol-containing food may include at least one of drinking water, tea, a health drink, makgeolli, soju, whiskey, wine, beer, and a combination thereof, and may also include any health functional food in the usual sense as long as it is a drinkable food, but is not particularly limited thereto.

[0051] The ethanol-containing food may contain various natural carbohydrates or sweeteners as additional ingredients, as in conventional foods, including beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. The ethanol-containing food of the present invention may contain, for example, about 0.00001 g to 10 g of the natural carbohydrates or sweeteners per 100 ml, but is not limited thereto.

[0052] In the present invention, the ethanol-containing food product may contain additives such as various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, and carbonation agents used in carbonated beverages. In addition, the food product may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of such additives in the ethanol-containing food product of the present invention may be, for example, 0.001 to 10 parts by weight per 100 parts by weight, but is not limited thereto.

[0053] In the present invention, the ethanol-containing food may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, the ethanol-containing food may contain fruit pulp for producing natural fruit juices, fruit juice drinks, and vegetable drinks, but is not particularly limited thereto.

[0054] Another example of the present invention relates to a method for producing an ethanol-containing food product, comprising culturing Kluyveromyces marxianus LRCC 8325 strain, deposited as KCCM 13373P, in the presence of a substrate.

[0055] The substrate may include at least one of wort, kudzu, burdock, ginger, bellflower, and combinations thereof, and the substrate may be pretreated on a solution.

[0056] The sugar content of the substrate may be, but is not limited to, 10 to 11 plato, 10 to 10.8 plato, or 10 to 10.6 plato, and may be selected within an appropriate range taking into account the sugar content of the ethanol-containing food.

[0057] The step of culturing the strain may be carried out at a temperature of 5°C to 35°C, 5°C to 30°C, 10°C to 35°C, or 10°C to 30°C.

[0058] The step of culturing the strain may be carried out for 12 to 72 hours, or 24 to 72 hours.

[0059] In the step of culturing the bacterial strain, the initial inoculation concentration of the bacterial strain may be 10 5 CFU / ml to 10 7 CFU / ml, 10 6 CFU / ml to 10 7 CFU / ml, or 10 6 CFU / ml.

[0060] As used herein, "cultivation" can mean growing microorganisms under appropriate artificially controlled environmental conditions.

[0061] The culture can be cultured in a continuous manner, including but not limited to a batch process, a fed batch, or a repeated fed batch process.

[0062] The method for producing the ethanol-containing food product may also include a step of concentrating the ethanol produced from the strain or its culture. The ethanol can be concentrated by a method well known in the art, such as distillation.

[0063] The method for producing an ethanol-containing food may include substantially the same content as the content of the Kluyveromyces marxianus LRCC 8325 strain deposited under KCCM 13373P, a composition containing the strain and a culture thereof, and an ethanol-containing food, and therefore a description of the overlapping parts will be omitted.

[0064] Another example of the present invention relates to the Kluyveromyces marxianus strain LRCC 8279 deposited under accession number KCCM 13372P.

[0065] In the present invention, the Kluyveromyces marxianus LRCC 8279 strain may be the Kluyveromyces marxianus LRCC 8279 strain deposited at the Korea Microorganism Collection on July 26, 2023 under accession number KCCM 13372P.

[0066] The 26S rRNA base sequence of the Kluyveromyces marxianus strain LRCC 8279 may comprise the base sequence of SEQ ID NO:2.

[0067] The Kluyveromyces marxianus LRCC 8279 strain may be derived from malt or rice koji.

[0068] The Kluyveromyces marxianus LRCC 8279 strain may have the ability to decompose at least one of glucose (D-glucose), xylose (D-xylose), xylitol (Xylitol), galactose (D-galactose), sorbitol (D-sorbitol), lactose (D-lactose), sucrose (D-saccharose), raffinose (D-raffinose), and combinations thereof.

[0069] Another example of the present invention relates to a composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus strain LRCC 8325 deposited under accession number KCCM 13372P, a culture of the strain, and a combination thereof.

[0070] Another example of the present invention relates to an ethanol-containing food product containing at least one of the Kluyveromyces marxianus strain LRCC 8279 deposited as KCCM 13372P, a culture of the strain, and a combination thereof.

[0071] Another example of the present invention relates to a method for producing an ethanol-containing food product, comprising culturing Kluyveromyces marxianus LRCC 8279 strain deposited under accession number KCCM 13372P in the presence of a substrate.

[0072] The composition for ethanol production, the ethanol-containing food, and the method for producing the same, related to the Kluyveromyces marxianus LRCC 8279 strain deposited as KCCM 13372P, may include content that substantially overlaps with the content of the composition for ethanol production, the ethanol-containing food, and the method for producing the same, related to the Kluyveromyces marxianus LRCC 8325 strain deposited as KCCM 13373P, and therefore a description of the overlapping parts will be omitted.

[0073] Other embodiments of the present invention will be described in more detail below using experimental examples. The following experimental examples are merely illustrative examples to aid in understanding the present invention, and are not intended to limit the scope of the present invention.

[0074] Experimental example 1. Isolation and identification of yeast Malt and rice koji from Mangwon Traditional Market (Seoul Mapo, Korea) were enriched in YPD broth medium for 24 hours, then serially diluted and cultured in YPD agar medium for 48 hours. Pure colonies were then subcultured up to three times to isolate the yeast.

[0075] The 26S rRNA sequences of the isolated yeasts were amplified using the universal primers 785F and 907R, and the sequence similarity to Kluyveromyces marxianus was analyzed using the NCBI GenBank database. As a result, two novel yeast strains were isolated and identified, and named K. marxianus LRCC 8325 (hereinafter referred to as the LRCC 8325 strain) and K. marxianus LRCC 8279 (hereinafter referred to as the LRCC 8279 strain). The K. marxianus LRCC 8325 strain was deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on July 26, 2023, and assigned the accession number KCCM 13373P. The K. marxianus LRCC 8279 strain was deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on July 26, 2023, and assigned the accession number KCCM 13372P.

[0076] The 26S rRNA base sequence of the Kluyveromyces marxianus LRCC 8325 strain may include the base sequence of SEQ ID NO: 1, and the 26S rRNA base sequence of the Kluyveromyces marxianus LRCC 8279 strain may include the base sequence of SEQ ID NO: 2, which are shown in Table 1 below.

[0077] [Table 1] TIFF2025534144000003.tif144164

[0078] The phylogenetic relationships of the isolated and identified strains with other strains belonging to the genus Kluyveromyces are shown in Figures 1 and 2.

[0079] 2. Confirmation of yeast sugar utilization and non-glycosyltransferase activity 2-1. Sugar utilization The sugar utilization of the LRCC 8325 and LRCC 8279 strains was analyzed using the API 20C AUX kit (Biomerieux, France). This kit tests and identifies the biochemical ability of yeast strains to utilize 19 different sugars. Diluted strains were dispensed onto strips containing 19 different sugar substrates and cultured for 48 to 72 hours or more. The fermentation results of the sugar substrates by each strain were evaluated, and the results are shown in Tables 2 and 3.

[0080] [Table 2]

[0081] [Table 3]

[0082] (Where, CON: No sugar substrate added, GLU: D-Glucose, GLY: Glycerol, 2KG: Ca2-keto-Gluconate, ARA: L-Arabinose, XYL: D-Xylose, ADO: Adonitol, XLT: Xylitol, GAL: D-Galactose, INO: Inositol, SOR: D-Sorbitol, MDG :Methyl-αD-Glusopyranoside, NAG: N-Acetyl-Glucosamine, CEL: D-Cellobiose, LAC: D-Lactose, MAL: D-Maltose, SAC: D-Saccharose, TRE: D-Trehalose, MLZ: D-Melezitose, RAF: D-Raffinose).

[0083] As can be seen from Tables 2 and 3, the LRCC 8325 and LRCC 8279 strains were able to use glucose (D-glucose), xylose (D-xylose), xylitol (Xylitol), galactose (D-galactose), sorbitol (D-sorbitol), lactose (D-lactose), sucrose (D-saccharose, sucrose), and raffinose (D-raffinose) as carbon sources.

[0084] 2-2. Non-glycosyltransferase activity The LRCC 8325 and LRCC 8279 strains were dispensed onto paper discs in Bile Esculin Agar medium, and the black rings that appeared when esculin was decomposed were observed. The results are shown in Figure 3.

[0085] Here, the +control was set as a control group by selecting a strain of the genus Saccharomyces (Saccharomyces cerevisiae) with high non-glycosidic activity, and the -control was set as a control group by selecting a strain of the genus Saccharomyces with low non-glycosidic activity.

[0086] Referring to FIG. 3, it can be seen that LRCC 8279 and LRCC 8325 have excellent β-glucosidase activity, and therefore can produce a large amount of non-glycosides that increase bioavailability while fermenting root vegetables.

[0087] 3. Confirmation of yeast alcohol production ability and growth at different temperatures 3-1. Alcohol production potential depending on the type of sugar The initial inoculation concentration of the LRCC 8325 strain, the LRCC 8279 strain, and the control Saccharomyces cerevisiae strain was 10 CFU / ml. They were cultured in five media (YP, YPD, YPF, YPM, and YPS) at 30°C for 48 hours to determine the alcohol production ability depending on the type of sugar.

[0088] All five media contain 1% by weight of yeast extract and 2% by weight of peptone, while YPD medium further contains 2% by weight of glucose, YPF medium 2% by weight of fructose, YPM medium 2% by weight of maltose, and YPS medium 2% by weight of sucrose. The weight percentages are based on the total weight of the medium.

[0089] The results of confirming the alcohol production ability of the yeast are shown in FIG.

[0090] [Table 4]

[0091] 3-2. Alcohol production potential depending on the mixed sugar content The initial inoculation concentration of the LRCC 8325 strain, the LRCC 8279 strain, and the control Saccharomyces cerevisiae strain was 10 CFU / ml. They were cultured at 30°C for 5 days using four types of mixed carbon source media, and the alcohol production ability depending on the mixed sugar content was confirmed.

[0092] The mixed carbon source media all contain 1% by weight of yeast extract and 2% by weight of peptone, while YPDM0.8 medium contains 0.8% by weight each of glucose and maltose, YPDM1 medium contains 1% by weight of each sugar, YPDM1.5 medium contains 1.5% by weight of each sugar, and YPDM2 medium contains 2% by weight of each sugar. The weight percentages are based on the total weight of the medium.

[0093] The results of confirming the alcohol-producing ability of the yeast are shown in FIG. 5 and Table 5.

[0094] [Table 5]

[0095] Referring to Figure 5 and Table 5 together with Figure 4 and Table 4, it was confirmed that when exposed to glucose and maltose, the control strain produced ethanol using both glucose and maltose, whereas the LRCC 8325 and LRCC 8279 strains had low maltose utilization and primarily consumed glucose to produce small amounts of ethanol.

[0096] 3-3. Alcohol production and yeast growth depending on the incubation temperature and incubation time The LRCC 8325 strain, the LRCC 8279 strain, and S. cerevisiae 2 (maltose-negative) as a control were prepared.

[0097] The initial inoculation concentration was 10 6 CFU / ml, and the bacteria were cultured in YPD medium (containing 2% by weight of glucose) at 10°C, 15°C, 25°C, and 30°C for 1 to 5 days.

[0098] The alcohol production ability and yeast growth rate were examined according to the culture temperature and culture time, and the results are shown in FIGS.

[0099] [Table 6]

[0100] As can be seen from FIGS. 6 to 10 and Table 6, there was no significant difference in viable cell counts according to the culture temperature and culture time among the control strain, LRCC 8325 strain, and LRCC 8279 strain.

[0101] However, when the culture temperature was 10°C to 15°C, up to about day 5, at 20°C up to about day 3, and at 25°C to 30°C up to about day 1, the LRCC 8325 and LRCC 8279 strains were measured to have similar viable cell counts but significantly lower alcohol production compared to the control group. Therefore, as the culture temperature increases, a shorter culture time is more advantageous for producing low-alcohol beverages, and it was determined that a culture temperature of 15°C and a culture time of 3 days were the culture conditions that produced less than 1% alcohol while maintaining excellent growth potential compared to the initial inoculum amount.

[0102] 4. Wort fermentation using yeast 4-1. Wort fermentation The initial inoculation concentration of the LRCC 8325 strain, LRCC 8279 strain, and the control Saccharomyces cerevisiae strain was 10 CFU / ml, and the wort adjusted to 10.4 Brix (Plato) was fermented at 30°C for 1 to 3 days to prepare the fermented wort.

[0103] The wort was prepared by crushing malt, first adjusting the sugar content through a saccharification process, filtering, and then boiling with hops. The sugar content of the wort was adjusted to 10.4 bp, taking into account the sugar content of the final product after fermentation, which is 1.8 bp.

[0104] The amount of alcohol produced from the fermented wort is shown in Figure 11, RE (Real Extract) in Figure 12, and RDF (Real Degree of Fermentation) in Figure 13, and the experimental values ​​are summarized in Table 7. RDF is an index for measuring the degree of fermentation, and was calculated using the following formula 1 presented in "William Hardwick, (1994). Handbook of Brewing, CRC Press."

[0105]

number

[0106] 0 means original gravity (°P).

[0107] E stands for real extract (°P).

[0108] [Table 7]

[0109] (Here, RE is the residual sugar after fermentation of the wort, and RDF is the amount of sugar in the wort that is fermented into alcohol.) As can be seen from Figures 11, 12, 13, and Table 7, malt accounts for 60% to 70% of the sugar composition in wort. When exposed to glucose and maltose, the control strain produced ethanol using both glucose and maltose, whereas the LRCC 8325 and LRCC 8279 strains had low maltose utilization and primarily consumed glucose to produce small amounts of ethanol.

[0110] 4-2. Sensory evaluation of fermented wort The fermented wort prepared in 4-1 was analyzed for changes in taste and aroma patterns using gas chromatography / mass spectrometry (GC-MS) (GC: Aglient Technologies 7890B, MS: Aglient Technologies 5977A) and an electronic nose, Hercles II (Alpha MOS, Toulouse, France). The results are shown in Figures 14 and 15, and the experimental values ​​for Figure 14 are shown in Table 8.

[0111] The main components of the flavor profile are isoamyl alcohol and ethyl acetate for fermented, ethyl hexanoate, ethyl decanoate, and ethyl butanoate for fruity, 2-phenylethyl acetate and phenylethyl alcohol for floral, caprylic acid for fatty, ethyl octanoate for waxy and oily, and guaiacol for phenolic.

[0112] The aroma components were analyzed using GC-MS (GC: Aglient Technologies 7890B, MS: Aglient Technologies 5977A), and the main components classified by aroma characteristics were as follows:

[0113] Fermented aroma: Isoamyl alcohol and ethyl acetate

[0114] Fruity: Ethyl hexanoate, Ethyl decanoate, and Ethyl butanoate

[0115] Floral: 2-phenylethyl acetate and phenylethyl alcohol

[0116] Fatty: Caprylic acid (Octanoic acid)

[0117] Waxy & Oily: Ethyl octanoate

[0118] Phenolic: Guaiacol

[0119] [Table 8]

[0120] As can be seen from Figure 14 and Table 8, the floral aroma characteristics were relatively strong. Therefore, it was confirmed that the use of the LRCC 8279 and LRCC 8235 strains according to the present invention is advantageous in producing low-alcohol beverages with enhanced floral aromas.

[0121] As can be seen from Figure 15, the PC1 and PC2 values ​​were 93.719 and 5.039, respectively, and the differences between the samples were mainly determined by PC1, which corresponds to the x-axis. The LRCC 8279 and LRCC 8325 strains showed negative values ​​on the x-axis compared to the S. cerevisiae strain, confirming differences in aroma components, while the relatively closer values ​​confirmed similarity in aroma components.

[0122] 5. Fermentation of root vegetable concentrate 5-1. Fermentation of concentrated liquid The initial inoculation concentration of LRCC 8325 strain, LRCC 8279 strain, S. cerevisiae, and S. cerevisiae 2 was 10 CFU / ml, and the root vegetable concentrate was fermented at 15°C for 3 days to prepare the root vegetable fermentation product. The root vegetables used were kudzu, ginger, bellflower, and burdock.

[0123] The root vegetable concentrate may be prepared by preparing refrigerated or frozen root vegetables (kudzu, ginger, bellflower, and burdock), squeezing or heating the root vegetables, and extracting the extract using a filtration and concentration device such as a filter press. The root vegetables may be dried as needed. The root vegetable concentrate may also be subjected to a sterilization process as needed.

[0124] The amount of alcohol produced and the viable cell count of the root vegetable fermented products were measured for each strain, and the results are shown in FIG. 16 and Table 9.

[0125] [Table 9]

[0126] As can be seen from Figure 16 and Table 9, the LRCC 8279 and LRCC 8325 strains tended to produce lower amounts of alcohol than S. cerevisiae strains, despite having similar or superior growth potential. This confirms that the LRCC 8279 and LRCC 8325 strains of the present invention are advantageous for producing low-alcohol beverages.

[0127] 5-2. Sensory evaluation of fermented root vegetables Fermented root vegetables were prepared according to the method described in 5-1 above, and the changes in taste and aroma patterns of each strain of bacteria due to the fermentation were analyzed using GC-MS (GC: Aglient Technologies 7890B, MS: Aglient Technologies 5977A) and an electronic nose, Hercles II (Alpha MOS, Toulouse, France). The results are shown in Figures 17 to 24 and Tables 10 to 13.

[0128] The results of the aroma pattern analysis of the fermented kudzu are shown in Figures 17 and 18 and Table 10.

[0129] [Table 10]

[0130] As can be seen from FIG. 17 and Table 10, the fermented arrowroot product exhibited relatively strong ethereal and fermented aromas, and the main components were ethyl acetate, which has ethereal characteristics, and isoamyl alcohol, which has fermented characteristics.

[0131] As can be seen from Figure 18, for the fermented arrowroot, the PC1 and PC2 values ​​were 96.763 and 2.718, respectively, and differences between the samples were primarily determined by PC1, which corresponds to the x-axis. Compared to the S. cerevisiae strain, the LRCC 8279 and LRCC 8325 strains showed positive values ​​on the x-axis, indicating differences in aroma components, and the relatively closer proximity between the two strains confirmed similar aroma components.

[0132] The results of the aroma pattern analysis of the fermented burdock are shown in Figures 19 and 20 and Table 11.

[0133] [Table 11]

[0134] As can be seen from FIG. 19 and Table 11, the burdock fermented product exhibited relatively strong floral and ethereal aromas, and the main components were methyl salicylate and phenyl alcohol, which have floral characteristics, and ethyl acetate, which has ethereal characteristics.

[0135] As can be seen from Figure 20, in the case of fermented burdock, the PC1 and PC2 values ​​were 92.323 and 7.285, respectively, with differences between the samples being determined primarily by PC1, which corresponds to the x-axis. Compared to the S. cerevisiae strain, the LRCC 8279 and LRCC 8325 strains showed positive values ​​on the x-axis, indicating differences in aroma components, and the relatively closer proximity between the two strains confirmed similar aroma components.

[0136] The results of the aroma pattern analysis of the fermented ginger are shown in Figure 21, Figure 22 and Table 12.

[0137] [Table 12]

[0138] As can be seen from FIG. 21 and Table 12, the ginger fermented product exhibited relatively strong floral and ethereal aromas, and the main components were methyl salicylate and phenyl alcohol, which have floral characteristics, and ethyl acetate, which has ethereal characteristics.

[0139] As can be seen from Figure 22, for the ginger fermentation product, the PC1 and PC2 values ​​were 60.680 and 33.536, respectively, and differences between the samples were mainly determined by PC1, which corresponds to the x-axis. Compared to the S. cerevisiae strain, the LRCC 8279 and LRCC 8325 strains showed positive values ​​on the x-axis, indicating differences in aroma components, and the fact that the aroma components were relatively closer to each other confirmed that the aroma components were similar.

[0140] The results of the aroma pattern analysis of the fermented bellflower are shown in Figures 23 and 24 and Table 13.

[0141] [Table 13]

[0142] As can be seen from Figure 23 and Table 13, the bellflower fermented product exhibited relatively strong floral and ethereal aromas, and the main components were methyl salicylate and phenyl alcohol, which have floral characteristics, and ethyl acetate, which has ethereal characteristics.

[0143] As can be seen from Figure 24, for the ginger fermentation product, the PC1 and PC2 values ​​were 98.257 and 1.393, respectively, and differences between the samples were mainly determined by PC1, which corresponds to the x-axis. Compared to the S. cerevisiae strain, the LRCC 8279 and LRCC 8325 strains showed positive values ​​on the x-axis, indicating differences in aroma components, and the fact that the aroma components were relatively closer to each other confirmed that the aroma components were similar.

[0144] Therefore, it was confirmed that the use of the LRCC 8279 and LRCC 8325 strains according to the present invention is advantageous in producing low-alcohol beverages that have an overall enhanced ethereal aroma and, depending on the type of root vegetable, an enhanced fermented or floral aroma.

[0145] TIFF2025534144000017.tif169164

[0146] TIFF2025534144000018.tif166164 [Industrial Applicability]

[0147] The present invention relates to Kluyveromyces marxianus LRCC 8325 strain and Kluyveromyces marxianus LRCC 8279 strain suitable for producing non-alcoholic alcoholic beverages and fermented root vegetable beverages, and uses thereof.

Claims

1. Kluyveromyces marxianus strain LRCC 8325, deposited as KCCM 13373P.

2. A composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus LRCC 8325 strain deposited under accession number KCCM 13373P, a culture of said strain, and a combination thereof.

3. An ethanol-containing food product comprising at least one of the Kluyveromyces marxianus LRCC 8325 strain deposited as KCCM 13373P, a culture of said strain, and a combination thereof.

4. The ethanol-containing food product according to claim 3 , wherein the food product comprises at least one of drinking water, tea, a health drink, makgeolli, soju, whiskey, wine, beer, and combinations thereof.

5. Cultivating Kluyveromyces marxianus strain LRCC 8325, deposited under KCCM 13373P, in the presence of a substrate; the substrate comprises wort or root vegetable extract; The root vegetables include at least one of kudzu, burdock, ginger, bellflower, and combinations thereof.

6. The method for producing an ethanol-containing food according to claim 5, wherein the step of culturing the strain is carried out at 5°C to 35°C for 12 hours to 72 hours.

7. 6. The method for producing an ethanol-containing food according to claim 5, wherein in the step of culturing the strain, an initial inoculation concentration of the strain is 10 CFU / ml to 10 CFU / ml.

8. Kluyveromyces marxianus strain LRCC 8279, deposited as KCCM 13372P.

9. A composition for producing ethanol, comprising at least one of the Kluyveromyces marxianus LRCC 8279 strain deposited under accession number KCCM 13372P, a culture of said strain, and a combination thereof.

10. An ethanol-containing food product comprising at least one of the Kluyveromyces marxianus LRCC 8279 strain deposited as KCCM 13372P, a culture of said strain, and a combination thereof.

11. The ethanol-containing food product according to claim 10, wherein the food product comprises at least one of drinking water, tea, a health drink, makgeolli, soju, whiskey, wine, beer, and combinations thereof.

12. Cultivating Kluyveromyces marxianus LRCC 8279 strain deposited under KCCM 13372P in the presence of a substrate; the substrate comprises wort or root vegetable extract; The root vegetables include at least one of kudzu, burdock, ginger, bellflower, and combinations thereof.

13. The method for producing an ethanol-containing food product according to claim 12, wherein the step of culturing the strain is carried out at 5°C to 35°C for 12 to 72 hours.

14. The method for producing an ethanol-containing food according to claim 12, wherein in the step of culturing the strain, the initial inoculation concentration of the strain is 10 CFU / ml to 10 CFU / ml.

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