Inhibition of Saccharomyces by Pichia kudriavzevii

Abstract

This patent relates to the use of one or more strains of Pichia kudriavzevii for inhibiting or delaying the growth of Saccharomyces genus bacteria in beverages, preferably beverages with a low alcohol content. Also provided herein are Pichia kudriavzevii deposited as DSM 34278, Pichia kudriavzevii and its variants deposited as DSM 34279, and compositions comprising said Pichia kudriavzevii.

Description

Technical Field

[0001] The present invention belongs to the field of microbiology, and particularly relates to a method for inhibiting or delaying the growth of Saccharomyces bacteria in the production of beverages with a low alcohol content, particularly fermented beverages such as beer with a low alcohol content.

Background Art

[0002] Due to the increasing demand for healthier foods and beverages, the reduction of ethanol in alcoholic beverages, particularly beer and wine, has attracted significant commercial interest. In particular, non-alcoholic beer, which is based on malt, has reduced calories, and has various nutritionally physiologically favorable characteristics, is becoming increasingly popular.

[0003] Compared to standard beer, which usually contains 4% (vol / vol) or more alcohol, low-alcohol beer or non-alcoholic beer (also called light beer, non-alcoholic, non-alcoholic beer, small beer, small ale, near beer) is a beer with a low alcohol content that aims to reproduce the full flavor of standard beer.

[0004] Various methods are known and already established for the production of beer with a reduced alcohol content, low-alcohol beer, and non-alcoholic beer. Generally, these methods can be classified into two groups. On the one hand, there are physical methods that selectively remove ethanol as much as possible from a normal beer or other alcoholic beverage matrix such as wine and cider, mainly by heat or membrane processes. On the other hand, there are biological methods based on limited ethanol production, such as the use of a modified mashing process, the use of a limited fermentation process such as stopped fermentation, or the use of special yeast.

[0005] The conversion from wort to alcoholic beer is the result of a fermentation process by yeast of the genus Saccharomyces spp., resulting in the production of alcohol and flavor compounds derived from fermentation such as esters and higher alcohols.

[0006] However, the microenvironment of a brewery or brewing tank typically has a very high presence of yeasts of the genus Saccharomyces, such as S. cerevisiae or S. pastorianus used in breweries, and other Saccharomyces contaminants such as Saccharomyces diastaticus. It is difficult to completely remove these from brewing tanks through cleaning and disinfection. This can pose a problem in the production of low-alcohol or non-alcoholic beer. When brewing this type of beer, Saccharomyces contamination can result in the production of ethanol, not only exceeding the target alcohol concentration but also potentially causing off-flavors and other quality degradation. The spoilage of Saccharomyces in beer is described, for example, in "Spoilage Yeasts in Beer and Beer Products" by Suiker et al., Current Opinion in Food Science 44 (2022): 100815. The lower the residual alcohol tolerated in the beer, the more important the inhibition of Saccharomyces in the process becomes. Therefore, when using biological methods for the production of alcohol-reduced beer, it is essential to suppress or completely inhibit the growth of Saccharomyces that may be present in the fermentation tank.

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, when manufacturing beverages, especially those susceptible to Saccharomyces contamination, there is a need for an improved method to inhibit the growth of Saccharomyces species. Specifically, it is desirable to effectively inhibit Saccharomyces species during beverage production while providing a beverage with good sensory or organoleptic properties, such as a good aroma or flavor profile.

Means for Solving the Problem

[0008] This problem has been solved by using one or more Pichia kluyveri strains to inhibit or delay the growth of Saccharomyces species in beverages.

[0009] The use of Pichia kluyveri in the production of low-alcohol beer is known from, for example, International Publication No. WO2014 / 135673, which discloses the use of Pichia kluyveri strains PK-KR1 and PK-KR2, but does not disclose the inhibition of Saccharomyces species.

[0010] One or more Pichia kluyveri strains in the present invention are characterized by the ability to inhibit the growth of S. cerevisiae or S. pastorianus by at least 40%, such as at least 50%, at least 60%, at least 70%, or at least 80% when inoculated with one or more Pichia kluyveri strains simultaneously with S. cerevisiae or S. pastorianus, compared to other identical samples that do not contain one or more Pichia kluyveri strains.

[0011] The inventors of the present invention have surprisingly discovered a new method for managing Saccharomyces species contamination by using Pichia kluyveri. Therefore, the present invention contributes to providing an effective solution for managing the growth of Saccharomyces species by biological means and can avoid the use of chemical bactericides.

[0012] The uses and methods of the present invention are particularly suitable for the fermentation of low-alcohol or non-alcohol beverages. Saccharomyces species are commonly present in the microenvironment of breweries. Since these yeasts are difficult to completely remove from the brewing tank by washing and disinfection, it is highly advantageous to use Pichia kudriavzevii strains to efficiently inhibit the growth of Saccharomyces contaminants present in the system. By the uses and methods of the present invention, the growth of Saccharomyces present in the fermentation tank can be inhibited, and preferably, there will be no inhibition in the next production batch in the same fermentation tank used for normal beer brewing. This has the advantage that breweries do not need to have dedicated tanks for non-alcohol beer production.

[0013] In particular, the inventors have identified two novel Pichia kudriavzevii strains (DSM 34278 and DSM 34279) that are particularly suitable for the uses and methods of the present invention because they can effectively inhibit Saccharomyces species. Furthermore, these strains also provide a very good flavor profile when used in the fermentation of non-alcohol beverages.

[0014] To address the problem of microbial contamination, according to a first aspect, the present invention provides the use of one or more Pichia kudriavzevii strains for inhibiting or delaying the growth of Saccharomyces species in beverages, preferably fermented beverages. The one or more Pichia kudriavzevii strains are characterized in that when inoculated simultaneously with one or more Pichia kudriavzevii strains, they can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% compared to other identical samples that do not contain the one or more Pichia kudriavzevii strains. Preferably, Pichia kudriavzevii is added to the beverage at a concentration of at least 10 3 CFU / ml, for example at least 10 4 CFU / ml, for example at least 10 5 CFU / ml, for example 10 3 ~10 9 CFU / ml, preferably 10 4 ~10 8 CFU / ml, most preferably 10 7 CFU / ml.

[0015] According to a second aspect, the present invention provides a method for inhibiting or delaying the growth of Saccharomyces genus bacteria in a beverage, preferably a fermented beverage, more preferably a beverage having a low alcohol content, for example, an alcohol content of less than 4.2% by volume, the method comprising the step of adding an effective amount of one or more Pichia kluyveri strains. The one or more Pichia kluyveri strains, when inoculated simultaneously with the one or more Pichia kluyveri strains, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40%, for example, at least 50%, at least 60%, at least 70%, or at least 80% compared to other identical samples that do not contain the one or more Pichia kluyveri strains. Preferably, Pichia kluyveri is at least 10 3 CFU / ml, for example, at least 10 4 CFU / ml, for example, at least 10 5 CFU / ml, for example, 10 3 ~10 9 CFU / ml, preferably 10 4 ~10 8 CFU / ml, most preferably 10 7 CFU / ml is added to the beverage at a concentration of.

[0016] According to a third aspect, the present invention provides a yeast of the Pichia kluyveri species deposited as DSM 34278, or a variant of Pichia kluyveri obtained from the deposited yeast. This variant, when inoculated simultaneously with a Saccharomyces and a Pichia kluyveri strain, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40%, for example, at least 50%, at least 60%, at least 70%, or at least 80% as measured using an assay, for example, Assay I, compared to other identical samples that do not contain the Pichia kluyveri strain.

[0017] According to a fourth aspect, the present invention provides a yeast of the species Pichia kudriavzevii deposited as DSM 34279, or a variant of Pichia kudriavzevii obtained from the deposited yeast. This variant, when Saccharomyces is co-inoculated with the Pichia kudriavzevii strain, inhibits the growth of S. cerevisiae or S. pastorianus, measured using an assay, such as Assay I, by at least 40%, for example at least 50%, at least 60%, at least 70%, or at least 80% compared to other identical samples not containing the Pichia kudriavzevii strain.

[0018] According to a further aspect, the present invention provides a composition or beverage comprising the yeast of the third and / or fourth aspect of the present invention, particularly a beverage with an alcohol content of less than 4.2% by volume.

Brief Description of the Drawings

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Figure 5

DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, representative methods and materials are described herein. The detailed information provided for the use of the present invention is also relevant to the methods of the present invention.

[0025] According to a first aspect, the present invention provides the use of one or more Pichia kudriavzevii strains for inhibiting or delaying the growth of Saccharomyces spp. in beverages, wherein the Pichia kudriavzevii strain can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% compared to other identical samples without the Pichia kudriavzevii strain when Saccharomyces is inoculated simultaneously with the Pichia kudriavzevii strain.

[0026] As used herein, the terms "to inhibit" and "inhibiting" with respect to Saccharomyces spp. mean that the growth, number, or concentration of Saccharomyces spp. is decreased. Inhibition can be observed by comparing the growth, number, or concentration of Saccharomyces in a product containing Pichia kudriavzevii strains with a control. The control may be an identical composition except that it does not contain Pichia kudriavzevii strains. Yeast growth can be measured by various methods known to those skilled in the art. Methods for determining yeast growth inhibition or delay are known to those skilled in the art. For example, yeast growth can be measured by using a real-time PCR (qPCR) assay to quantify the amount of yeast in a sample after a certain time and comparing the determined amount with a negative control.

[0027] The term "to delay" generally means an act of stopping, postponing, interfering with, or causing something to occur later than normal. As used herein, "delaying the growth of Saccharomyces spp." refers to the act of delaying the growth of Saccharomyces spp. This can be observed by comparing the time required for Saccharomyces spp. to grow to a certain level in two identical beverages, one containing a Pichia kudriavzevii strain and the other not.

[0028] According to some embodiments, "delaying growth" refers to delaying by 1 day, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 days, etc.

[0029] According to a preferred embodiment, the growth of Saccharomyces spp. in a beverage is inhibited by at least 40%, preferably at least 50% or 75%, compared to another identical sample that does not contain one or more Pichia kudriavzevii strains. This is shown, for example, in Examples 1 and 2. Saccharomyces cerevisiae can be used as the target microorganism.

[0030] Inhibition can be determined by Assay I: Assay I: - Wort (8°P and 13 IBU) was inoculated with or without Pichia kudriavzevii at an initial concentration of 10 5 CFU / ml, and - The wort was fermented at 18°C and 120 rpm for 3 days, - The growth of S. cerevisiae in the wort medium with or without Pichia kudriavzevii was compared by qPCR to determine the inhibition rate.

[0031] The inventors of the present invention surprisingly discovered that Saccharomyces spp. can be inhibited by an effective amount of Pichia kudriavzevii. Strains of Pichia kudriavzevii that can be used in the present invention include Pichia kudriavzevii DSM 34278, Pichia kudriavzevii DSM 34279, Pichia kudriavzevii DSM 28484, Pichia kudriavzevii CBS188, their mutants, and mixtures thereof. These mutants are functionally equivalent mutants with the same or improved ability to inhibit or delay the growth of Saccharomyces spp. compared to their respective parental strains. This ability can be determined using an assay such as Assay I.

[0032] In the present context, the term "mutant" should be understood as a strain derived from the strain of the invention, for example by 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 deposited strain, in particular with regard to the growth inhibitory effect on Saccharomyces. Each mutant represents an embodiment of the invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenesis treatment, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or a spontaneously occurring mutant. The mutant may have been subjected to multiple mutagenesis treatments (a single treatment being understood as one mutagenesis step followed by a screening / selection step), although currently it is preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps) are carried out. In currently preferred mutants, less than 5%, or less than 1%, or less than 0.1% of the nucleotides in the bacterial genome have been shifted to another nucleotide or deleted compared to the parent strain.

[0033] According to a preferred embodiment, the Pichia kluyveri strain used herein is not PK-KR1 or PK-KR2.

[0034] According to a particularly preferred embodiment, the Pichia kluyveri strain is selected from the group consisting of the strains deposited under DSM 34278 and DSM 34279 or mutants thereof.

[0035] Pichia kluyveri strains useful in the present invention may be characterized by an ability to inhibit the growth of S. cerevisiae or S. pastorianus by at least 40%, e.g., at least 50%, at least 60%, at least 70%, or at least 80%, when Saccharomyces is inoculated simultaneously with the Pichia kluyveri strain, relative to a negative control (i.e., an otherwise identical sample not containing the Pichia kluyveri strain).

[0036] According to a preferred embodiment, when the Pichia kudriavzevii strain is inoculated simultaneously with the Pichia kudriavzevii strain in wort, it is characterized in that it inhibits the growth of S. cerevisiae or S. pastorianus by at least 40% as compared with other identical worts not containing the Pichia kudriavzevii strain.

[0037] According to a preferred embodiment, when Saccharomyces is simultaneously inoculated with 10 5 CFU / ml of the Pichia kudriavzevii strain, the Pichia kudriavzevii strain can inhibit the growth of Saccharomyces genus bacteria at 10 3 CFU / ml by at least 40% as compared with other identical samples not containing the Pichia kudriavzevii strain.

[0038] According to a preferred embodiment, when one or more Pichia kudriavzevii strains are inoculated into wort simultaneously with 10 5 CFU / ml of the Pichia kudriavzevii strain for 3 days, the growth of Saccharomyces genus bacteria at 10 + CFU / ml can be inhibited by at least 40% as compared with other identical worts not containing the Pichia kudriavzevii strain.

[0039] According to a preferred embodiment, the Pichia kudriavzevii strain is preferably added to the fermented beverage at a concentration of at least 10 3 CFU / ml, for example at least 10 4 CFU / ml, for example at least 10 5 CFU / ml, for example 10 3 ~10 9 CFU / ml, preferably 10 4 ~10 8 CFU / ml, most preferably 10 7 CFU / ml at the start or during fermentation.

[0040] According to a preferred embodiment, the growth of one or more of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces eubayanus, Saccharomyces bayanus, Saccharomyces uvarum, Saccharomyce kudriavzevii and / or Saccharomyces pastorianus is inhibited.

[0041] Beverage Beverages in which the growth of Saccharomyces spp. is suppressed or delayed can be any beverage such as wine, cider, tea, fruit beverages, beer, etc., preferably beverages obtained by fermentation, most preferably beverages obtained by fermentation of wort, including wort-based beverages such as beer. Beverages with a low alcohol content are preferred, for example, those with an alcohol content of less than 4.2% by volume.

[0042] As used herein, the term "beer" refers to at least the types of beer obtained from brewing malt obtained from malted grains, as well as brewing malt obtained from unmalted grains, and types of beer obtained from brewing malt obtained from a mixture of malted grains and unmalted grains.

[0043] As used herein, the term "wort" has its conventional meaning in the art and refers to a sugary liquid extracted from the mashing process of beer brewing.

[0044] As used herein, the term "fermentation" refers to a metabolic process that includes chemical changes in the wort substrate by the action of a culture. This includes aerobic and anaerobic processes.

[0045] The beverage is a low-alcohol-content beverage, which may be a beverage with a reduced alcohol content, a low-alcohol beverage, an alcohol-free beverage, or a non-alcoholic beverage. The meanings of these terms may vary depending on national regulations. However, as used herein, in the current context, the term "beverage with a reduced alcohol content" in this specification refers to a beverage with an alcohol content of 1.2 to 4.2% by volume. Here, the term "low-alcohol beverage" is defined as a drinking liquid with an alcohol content exceeding 0.5% by volume and not exceeding 1.2% by volume. Here, the term "non-alcoholic beverage" is defined as a drinking liquid with an alcohol content not exceeding 0.5% by volume. Here, the term "alcohol-free beverage" is defined as a drinking liquid with an alcohol content of less than 0.05% by volume. According to one embodiment, the beverage is a low-alcohol wine.

[0046] Beer According to a preferred embodiment, the beverage is beer with a reduced alcohol content, low alcohol beer, non-alcoholic beer, or alcohol-free beer. "Reduced" means having a lower alcohol content compared to regular beer. In this context, the term "beer with a reduced alcohol content" as used herein refers to beer having an alcohol content of 1.2 to 4.2% by volume. The term "low alcohol beer" as used herein refers to beer having an alcohol content of 0.5 to 1.2% by volume. The term "non-alcoholic beer" as used herein refers to beer having an alcohol content of less than 0.5% by volume. Finally, the term "alcohol-free beer" as used herein refers to beer having an alcohol content of less than 0.05% by volume.

[0047] Fruit beverage According to another preferred embodiment, the beverage is a fruit beverage. In this context, the term "fruit beverage" refers to a beverage containing fruit juice, fruit concentrate, and / or fruit puree. The term "fruit beverage" includes "fruit juice", "fruit drink", and "fruit nectar". The "fruit beverage" may be either one containing pulp or one from which the pulp has been removed by an operation such as centrifugation. The fruit beverage may further contain, for example, oats, soybeans, almonds, whey, and / or non-fermented milk (for example, in the form of powdered milk).

[0048] Examples of fruit beverages suitable for use in the present invention include, for example, fruit juice, concentrated fruit juice, fruit drink, fruit smoothie, and fruit nectar optionally containing fruit puree and / or water.

[0049] The term "fruit juice" refers to the liquid naturally contained in fruits made by mechanically squeezing or macerating fresh fruits without using heat or solvents. "Fruit juice" is composed of the juice of one type of fruit or a mixture of two or more types of fruits.

[0050] The term "fruit drink" as used herein refers to a beverage with a fruit juice content of 0 to 29% by volume.

[0051] The term "fruit nectar" in this context refers to a beverage with a fruit juice content of 30 to 99% by volume.

[0052] The term "fruit puree" as used herein refers to a product obtained by grinding, compressing, and filtering fruits without using heat or solvents to form a thick liquid or soft paste. "Puree" is made from 100% fruit, not just from the juice of the fruit.

[0053] The total content of fruit juice and / or fruit puree in fruit beverages is generally about 20% to about 99.99% by weight, preferably about 30% to 95% by weight, more preferably about 40% to 90% by weight, still more preferably about 50% to 80% by weight, and most preferably 60% to 70% by weight.

[0054] The method of the present invention According to a second aspect, the present invention provides a method for inhibiting or delaying the growth of Saccharomyces spp. in a beverage with a low alcohol content, for example, an alcohol content of less than 4.2% by volume. This method includes the step of adding an effective amount of one or more strains of Pichia kluyveri to the beverage, and the one or more strains of Pichia kluyveri can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% when compared with other identical samples without the one or more strains of Pichia kluyveri when inoculated simultaneously with Saccharomyces.

[0055] Pichia kudriavzevii can be added in any form, including in the form of liquid, frozen or dried (such as freeze-dried or spray-dried) compositions. The composition preferably contains one or more of a cryoprotectant, a freeze-drying protectant, an antioxidant and / or a nutrient, more preferably a cryoprotectant, a freeze-drying protectant and / or an antioxidant, most preferably a cryoprotectant or a freeze-drying protectant, or both. The use of protectants such as cryoprotectants and freeze-drying protectants is known to those skilled in the art. Suitable cryoprotectants or freeze-drying protectants (including monosaccharides, disaccharides, trisaccharides and polysaccharides) are polyols, amino acids, complex substances and inorganic compounds. The composition can optionally contain further substances such as an emulsifier (such as sorbitan monostearate), a filler and / or a flavoring.

[0056] According to a preferred embodiment, the composition contains at least 10 7 CFU / g, 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 contains at least 10 6 CFU / g of Pichia kudriavzevii.

[0057] Methods for preparing high-density yeast compositions are known in the art and are described, for example, in International Publication No. WO2011 / 134952.

[0058] In a further aspect, the present invention provides a method for producing a beverage having a low alcohol content, for example, an alcohol content of less than 4.2% by volume, the method comprising the step of adding an effective amount of one or more Pichia kudriavzevii strains to inhibit or delay the growth of Saccharomyces genus bacteria in the beverage, wherein the one or more Pichia kudriavzevii strains, when Saccharomyces is inoculated simultaneously with the one or more Pichia kudriavzevii strains, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% compared to other identical samples not containing the one or more Pichia kudriavzevii strains.

[0059] When using yeast strains in the method for producing a beverage, those skilled in the art can adjust various parameters such as pH, temperature, and the amount of yeast in consideration of the examples provided by the present invention and the characteristics of the produced beverage to achieve the desired results.

[0060] The method disclosed herein is particularly useful for inhibiting or delaying the growth of yeast in fermentation processes such as the production of beer with reduced alcohol content, low-alcohol beer, non-alcohol beer, or alcohol-free beer.

[0061] Yeasts and compositions of the present invention According to a third aspect, the present invention provides a yeast of the Pichia kudriavzevii species deposited as DSM 34278, or a variant of Pichia kudriavzevii obtained from the deposited yeast, which variant, when Pichia kudriavzevii is inoculated simultaneously with a Pichia kudriavzevii strain, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40%, for example, at least 50%, at least 60%, at least 70%, or at least 80% compared to other identical samples not containing the Pichia kudriavzevii strain.

[0062] According to a fourth aspect, the present invention provides a yeast of the species Pichia kudriavzevii deposited as DSM 34279, or a variant of Pichia kudriavzevii obtained from the deposited yeast, which variant, when Pichia kudriavzevii is co-inoculated with a Pichia kudriavzevii strain, inhibits the growth of S. cerevisiae or S. pastorianus by at least 40%, such as at least 50%, at least 60%, at least 70%, or at least 80% compared to other identical samples that do not contain the Pichia kudriavzevii strain.

[0063] According to a preferred embodiment, the inhibition is 10 5 CFU / ml of the starting concentration of Pichia kudriavzevii and 10 3 CFU / ml of the starting concentration of S. cerevisiae or S. pastorianus. The inhibition is preferably tested in wort medium as described in the examples.

[0064] According to a preferred embodiment, the inhibition is tested in wort after 3 days using a starting concentration of 10 5 CFU / ml of Pichia kudriavzevii and a starting concentration of 10 3 CFU / ml of S. cerevisiae or S. pastorianus.

[0065] According to a fifth aspect, the present invention provides a composition comprising the yeast of the third and / or fourth aspects.

[0066] The composition of the present invention may further contain a cryoprotectant, a lyoprotectant, an antioxidant, a nutrient, a filler, a flavoring agent, or a mixture thereof. The composition may be in any form including a liquid (such as a slurry), a frozen or dried form (such as lyophilized or spray-dried). The composition preferably contains one or more of a cryoprotectant, a lyoprotectant, an antioxidant and / or a nutrient, more preferably contains a cryoprotectant, a lyoprotectant and / or an antioxidant, and most preferably contains a cryoprotectant or a lyoprotectant, or both. The use of protectants such as cryoprotectants and lyoprotectants is known to those skilled in the art. Suitable cryoprotectants or lyoprotectants include monosaccharides, disaccharides, trisaccharides and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, starch, gum arabic (acacia), etc.), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (such as proline, glutamic acid, etc.), complex substances (such as skim milk, peptone, gelatin, yeast extract, etc.), inorganic compounds (such as sodium tripolyphosphate, etc.). 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 (such as vitamin B group, vitamin C, etc.). The composition may optionally contain additional substances including an emulsifier (such as sorbitan monostearate), a filler (such as lactose) and / or a fragrance.

[0067] According to a preferred embodiment, the composition contains at least one cryoprotectant or cryoprotectants. According to another embodiment, the composition contains sorbitan monostearate. According to a preferred embodiment, the composition is in a liquid or lyophilized form.

[0068] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the following claims) shall be construed to cover both the singular and plural forms, unless otherwise indicated herein or unless clearly contradicted by the context. The terms "consisting," "possessing," "including," and "containing" shall be construed as non-limiting terms (i.e., meaning "including but not limited to") unless otherwise indicated.

Example

[0069] Example The invention described and claimed herein is not limited in scope by the specific aspects disclosed herein, which are intended as illustrations of some aspects of the invention. All equivalent aspects are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples. Such modifications are also within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

[0070] Example 1: Inhibition of Saccharomyces cerevisiae 1.1 Preparation of Samples Saccharomyces cerevisiae (Safale US-05, Fermentis) was cultured in a malt medium (8°P, Barlex 7215, prepared from Harboes Bryggeri A / S, Denmark, pH = 4.4) at 25°C and 150 rpm for 48 hours before inoculation.

[0071] Subsequently, wort with 8°P and 13 IBU was prepared from malt extract (Barlex 7215) containing hops (Magnum 12.9% AA), and the pH was adjusted to 4.4. Each sample was divided into 200 mL portions and placed in blue cap bottles. The samples were inoculated with Saccharomyces cerevisiae (Safale US-05, Fermentis) simultaneously, with or without various Pichia kluyveri strains. Refer to Table 1 for an overview of the inoculation. The origin of the strains is shown in Table 2. Fermentation was carried out at 18°C and 120 rpm for 10 days. 10 mL samples were collected on the 1st, 3rd, and 6th days after inoculation and then frozen at -50°C.

[0072]

Table 1

[0073]

Table 2

[0074] 1.2 Analysis of yeast growth 1.2.1 DNA Extraction from Fermentation Samples First, yeast cells were separated by centrifuging 12 mL of the fermentation product on the 1st day and 5 mL of the fermentation products on the 3rd and 6th days at 5,000 × g for 10 minutes. The supernatant was discarded, and the cells were resuspended and washed in 500 μl of buffer (1.2 M sorbitol, 100 mM TRIS, 100 mM CaCl2, 4H2O). The fermentation pellet was centrifuged again at 5,000 × g for 10 minutes, and the supernatant was discarded. Subsequently, 200 μl of lysis buffer (1.2 M sorbitol, 100 mM TRIS, 100 mM CaCl2, 4H2O, 200 U of liquid lyticase) was added to the pellet and mixed vigorously by pipetting. Finally, the samples were loaded onto a QIAcube connect device (Qiagen, Hilden, Germany), and DNA was purified using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) with a final elution volume of 125 μl according to the manufacturer's instructions.

[0075] 1.2.2 Real-time PCR (qPCR) assay Primer specificity was evaluated using primer blast comparison, in silico primer targeting (CLC Genomics Workbench v.20 Qiagen), and real-time PCR experimental validation using pure DNA from each strain during fermentation. Primers specific to the Saccharomyces sensu stricto complex in the fermentation samples were used to target and quantify only Saccharomyces cerevisiae (Safale US-05, Fermentis) and S. pastorianus (SmartBev, Chr. Hansen). Primers SI4F: 5’-ATTGCTGGCCTTTTCATTG-3’ (SEQ ID NO: 1), SI7R: 5’-CGCCTAGACGCTCTTCTTAT-3’ (SEQ ID NO: 2) (Chang et al. "Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces." Journal of microbiological methods 71.3 (2007): 191-201) and S probe: FAM-5’-GCCGCAGTTGGTAAAACCTA-3’-BHQ (SEQ ID NO: 3) were designed to anneal within the ITS1-5.8S-ITS2 rDNA region and amplify a 163 bp product. Real-time PCR amplification and detection were performed on a QuantStudio™ 12K Flex real-time PCR system (Applied Biosystems, Foster City, CA, USA).The probe-based reaction mix consisted of 6.25 μL of 1 × qPCR Master Mix Plus - Low ROX (Eurogentec, Seraing, Belgium), 0.3 μM nmol each of forward and reverse primers, 0.6 μM of probe, 2.5 μl of 10-fold and 100-fold diluted template DNA (to avoid inhibitory effects), and molecular grade H2O (Invitrogen, Life Technologies, Paisley, UK), with a final volume of 12.5 μL. The amplification program consisted of one cycle of 2 min at 50°C, one cycle of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The standard curve was generated using DNA from a pure culture of S. pastorianus (SmartBev, Chr. Hansen) and was generated in parallel with the amplification of fermentation samples every time qPCR analysis was performed. Serial dilutions (10-fold) of the standard were performed starting from 5 ng / μL to 10. 8 ~10 1 to obtain a standard solution in the range of pure DNA of 10. All samples were run in triplicate, and in the control reaction, molecular grade H2O was substituted for the template.

[0076] 1.3 Results In total, 18 samples belonging to different fermentations and controls, taken on days 1, 3, and 6, were analyzed. To quantify S. cerevisiae in the experiments conducted in fermentation, the qPCR primers SI4F / SI7R and the S probe were used to amplify the appropriate product size in the ITS rRNA gene. The specificity of the primers and probe was verified by qPCR using genomic DNA of all the yeasts listed in Table 1. This assay showed excellent efficiency (95% amplification efficiency, R2 0.998) and specificity for S. cerevisiae, although various Pichia kluyveri strains were below the reliable limit of detection.

[0077] Figure 1 shows the total concentration of S. cerevisiae detected in fermentation after 1 day, 3 days, and 6 days, expressed as gene copies / mL. A decrease in the growth of Saccharomyces was observed between day 3 and day 6 compared to the control batch (without Pichia kluyveri). On the other hand, when fermentation was carried out with P. kluyveri PK-KR1, no growth inhibition of Saccharomyces occurred.

[0078] These results indicate that various Pichia kluyveri strains effectively inhibit Saccharomyces cerevisiae inoculated at a very high level (much higher than when present as a contaminant). Therefore, an even better inhibitory effect is expected when the genus Saccharomyces is present only as a contaminant.

[0079] Example 2: Inhibition of Saccharomyces pastorianus 2.1 Sample preparation Saccharomyces pastorianus (SmartBev, Chr. Hansen A / S, Denmark) was cultured in a malt medium (8°P, Barlex 7215, prepared from Harboes Bryggeri A / S, Denmark, pH = 4.4) at 25°C and 150 rpm for 48 hours before inoculation.

[0080] Wort with 8°P and 13 IBU was prepared from malt extract (Barlex 7215) containing hops (Magnum 12.9% AA), and the pH was adjusted to 4.4. Each sample was divided into 200 mL portions and placed in blue cap bottles. The samples were inoculated with S. pastorianus (SmartBev, Chr. Hansen A / S, Denmark) simultaneously with or without various Pichia kluyveri strains. Refer to Table 3 for an overview of the inoculation. Fermentation was carried out at 18°C and 120 rpm for 10 days. 10 mL samples were taken on days 1, 3, and 6 after inoculation and then frozen at -50°C.

[0081] [Table 3]

[0082] 2.2 Analysis of yeast growth The analysis of yeast growth was carried out as described in 1.2.

[0083] 2.3 Results In total, 12 samples belonging to different fermentations and controls, taken on days 1, 3, and 6, were analyzed. To quantify S. pastorianus in the experiments carried out in fermentation, the appropriate product size in the ITS rRNA gene was amplified using the aforementioned qPCR primers SI4F / SI7R and S probe. The specificity of the primers and probe was verified by qPCR using the genomic DNA of all the yeasts listed in Table 3. This assay showed excellent efficiency (95% amplification efficiency, R2 0.998) and specificity for S. pastorianus, while the different Pichia kudriavzevii strains were below the reliable minimum detection limit.

[0084] Figure 2 shows the total concentration of S. pastorianus detected after 1, 3, and 6 days of fermentation, expressed as gene copies / mL. A decrease in the growth of Saccharomyces pastorianus was observed between days 3 and 6 with the different Pichia kudriavzevii strains, compared to the control batch (without Pichia kudriavzevii). These results indicate that the different Pichia kudriavzevii strains are effective in inhibiting the yeast S. pastorianus.

[0085] Example 3: Production of non-alcoholic beer using Pichia kudriavzevii strains DSM34278, DSM34279, and DSM28484 2.1 Production of non-alcoholic beer 6.5 kg of pilsner malt was added to a mash kettle containing 19.5 L of water at 68 °C and held at this temperature for 30 minutes. The mash (malt and water) was heated to 72 °C, held at this temperature for 10 minutes, then heated to 78 °C and held at this temperature for 5 minutes. Next, the mash was filtered, sparged with 40.7 L of water, and the liquid was transferred to a boil kettle and heated until boiling. 22 g of hops with an alpha acid content of 12.8% was added. Boiling continued for 60 minutes. The resulting wort was transferred to a 5 L plastic jerrican and stored in a refrigerator (< 5 °C).

[0086] 5 L of wort was adjusted to pH 5.3, and 400 ml of the pH-adjusted wort was placed into eight 500 ml blue-cap bottles. The blue-cap bottles containing the pH-adjusted wort were placed in a 90 °C water bath for 30 minutes for pasteurization and then cooled to room temperature (about 20 °C). After cooling, DSM 34278, DSM 34279, or DSM 28484 (duplicate) was inoculated to a cell count of 1E+05 cells / ml. Two of the blue-cap bottles were not inoculated and used as negative controls. The eight blue-cap bottles were transferred to a shaking incubator set at 18 °C and 100 rpm and stored for 5 days. The alcohol content of the non-alcoholic beer fermented with yeast was all 0.2 - 0.3% by volume.

[0087] 3.2 Analysis of volatile compounds The samples were analyzed by gas chromatography-mass spectrometry (GC-MS) at the Laboratorio de Analisis del Aroma y Enologia (LAAE) of the University of Zaragoza in Spain for the main volatile compounds and free volatile sulfur by GC-MS.

[0088] Figures 3 to 5 show the results of ethyl acetate (which gives a sweet, fruity, tropical aroma) and the off-odor compounds hydrogen sulfide (which smells like rotten eggs) and methional (which smells like sulfurous soup or meat). The dotted lines indicate the odor detection thresholds of each compound in alcoholic beer (the lowest concentration of a specific odor compound that can be perceived by the human sense of smell). The values for ethyl acetate and hydrogen sulfide were obtained from www.aroxa.com as of March 18, 2022. The value for methional was provided by LAAE.

[0089] These results indicate that Pichia kudriavzevii strains DSM 34278 and DSM 34279 produce more ethyl acetate, less hydrogen sulfide and methional (below the odor threshold), and exhibit a better flavor profile than DSM 28484.

[0090] Deposit and Expert Resolution The Applicant requests that, until the date of patent grant, samples of the following deposited microorganisms be provided only to experts in accordance with the regulations determined by the industrial property offices of the Contracting States of the Budapest Treaty.

[0091] [Table 4]

Claims

1. The use of one or more Pichia kluyveri strains to inhibit or delay the growth of Saccharomyces sp. in beverages with an alcohol content of less than 4.2% by volume, wherein the Pichia kluyveri strains, when inoculated simultaneously with the Pichia kluyveri strains, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% compared to other identical samples that do not contain the Pichia kluyveri strains, wherein the Pichia kluyveri strains are not PK-KR1.

2. A method for inhibiting or delaying the growth of Saccharomyces species in a beverage with an alcohol content of less than 4.2% by volume, comprising the step of adding an effective amount of one or more Pichia crevelii strains, wherein the Pichia crevelii strains, when inoculated simultaneously with the Pichia crevelii strains, can inhibit the growth of S. cerevisiae or S. pastorianus by at least 40% compared to other identical samples that do not contain the Pichia crevelii strains, wherein the Pichia crevelii strains are not PK-KR1.

3. The use or method according to either claim 1 or 2, wherein the beverage is a beverage with an alcohol content of less than 1.2%.

4. The use or method according to claim 3, wherein the beverage is a beverage with an alcohol content of less than 0.5%.

5. The use or method according to claim 4, wherein the beverage is a beverage with an alcohol content of less than 0.05%.

6. The aforementioned Pikia Kleiber is added to the wort in a 10 ５ When inoculated simultaneously with Pichia kruiberi strain at CFU / ml for 3 days, compared to the same wort without Pichia kruiberi strain, 10 ３ The use or method according to claim 1 or 2, which can suppress the growth of Saccharomyces species at CFU / ml by at least 40%.

7. The use or method according to claim 1 or 2, wherein the Pichia kruiberi strain is selected from the group consisting of the strains deposited as DSM34278 and DSM34279 and their variants.

8. The use or method according to claim 1 or 2, wherein the growth of one or more of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces eubayanus, Saccharomyces bayanus, Saccharomyces uvarum, Saccharomyces kudriavzevii, and / or Saccharomyces pastorianus is inhibited.

9. One or more Pichia cruiberi strains, at least 10 ３ The use or method according to claim 1 or 2, wherein the beverage is added at a concentration of CFU / ml.

10. The use or method according to claim 9, wherein one or more Pichia kruiberi strains are added to a beverage at a concentration of at least 10⁵ CFU / ml.

11. The use or method according to claim 10, wherein one or more Pichia kruiberi strains are added to a beverage at a concentration of at least 10⁷ CFU / ml.

12. The use or method according to either claim 1 or 2, wherein the beverage is wine, cider, tea, fruit beverage, or beer having an alcohol content of less than 4.2% by volume.

13. The use or method according to claim 12, wherein the beverage is wine, cider, tea, fruit beverage, or beer having an alcohol content of less than 1.2% by volume.

14. The use or method according to claim 13, wherein the beverage is wine, cider, tea, fruit beverage, or beer having an alcohol content of less than 0.5% by volume.

15. The use or method according to claim 14, wherein the beverage is wine, cider, tea, fruit beverage, or beer having an alcohol content of less than 0.05% by volume.

16. The use or method according to claim 1 or 2, wherein the growth of Saccharomyces species is inhibited by at least 50%.

17. The use or method according to claim 16, wherein the growth of Saccharomyces species is inhibited by at least 60% or 75%.

18. The aforementioned Pichia kruiberi strain is 10 ３ Saccharomyces species at CFU / ml 10 ５ The use or method according to claim 1 or 2, characterized in that, when inoculated simultaneously with Pichia cruiberi strain at CFU / ml, it can inhibit the growth of Saccharomyces species by at least 40% compared to other identical samples that do not contain Pichia cruiberi strain.

19. Yeast of the Pichia kruiberi species deposited as DSM34278, or a functionally equivalent mutant Pichia kruiberi obtained from the deposited yeast.

20. Yeast of the Pichia kruiberi species deposited as DSM34279, or a functionally equivalent mutant Pichia kruiberi obtained from the deposited yeast.

21. A composition comprising the yeast of the Pichia kruiberi species according to claim 19 or 20, wherein the Pichia kruiberi is present in the composition in a quantity of at least 10 ６ A composition optionally present at a concentration of CFU / g, optionally in liquid, freeze-dried, or dry form, or optionally further comprising one or more freeze-protecting agents, freeze-drying protectants, antioxidants, nutrients, fillers, flavorings, or mixtures thereof.

22. A beverage containing yeast of the Pichia kruiberi species according to claim 19 or 20, preferably a beverage with an alcohol content of less than 4.2%.

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