Inhibition of pichia kluyveri on yeast

Abstract

The present patent relates to the use of one or more strains of Pichia kluyveri for inhibiting or delaying the growth of a species of the genus Saccharomyces spp. In a beverage, preferably a beverage with reduced alcohol content. The invention further provides the pichia kluyveri with the preservation number of DSM 34278, the pichia kluyveri with the preservation number of DSM 34279 and a mutant of the pichia kluyveri, and a composition containing the pichia kluyveri.

Description

Field of the Invention

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

[0003] Due to the growing demand for healthier foods and beverages, reducing ethanol in alcoholic beverages, especially beer and wine, has considerable commercial value. Specifically, alcohol-free beer is becoming increasingly popular as a malt-based reduced-calorie beverage with a variety of nutritional and physiological positive properties.

[0004] Low-alcohol or non-alcoholic beer (also known as light beer, no-alcohol beer, nonalcoholic beer, small beer, small ale, near-beer) is beer with a lower alcohol content than standard beer, which typically contains more than 4% (vol / vol) alcohol, and is intended to reproduce the full flavor of standard beer.

[0005] Various processes for producing beer, low-alcohol beer and non-alcohol beer with reduced alcohol content are known and established. In general, these processes can be divided into two groups. On the one hand, there are physical processes, wherein mainly by thermal processes or membrane processes, ethanol is selectively removed as far as possible from common beer or other alcoholic beverage matrices (such as wine or cider). On the other hand, there are biological methods based on ethanol formation that are limited, for example, using improved saccharification processes, using limited fermentation processes (such as stopping fermentation) or using special yeasts.

[0006] The conversion of wort into alcoholic beer is the result of a fermentation process by yeasts of species of the genus Saccharomyces, resulting in the production of alcohol and flavor compounds of fermentation origin, such as esters and higher alcohols.

[0007] However, the microenvironment of breweries and brewing tanks is usually highly dominated by species of the genus Saccharomyces, including Saccharomyces cerevisiae and S. pastorianus used in breweries, as well as other Saccharomyces such as Saccharomyces diastaticus as contaminants. It is difficult to completely eliminate these yeasts from brewing tanks by cleaning and disinfection. This may cause problems for the production of low-alcohol beer and non-alcoholic beer. When brewing this type of beer, any contamination of species of the genus Saccharomyces may lead to ethanol production, and thus exceed the target alcohol concentration, as well as potential off-flavor or other quality degradation. The deterioration of the genus Saccharomyces in beer is described in Suiker et al. "Spoilage yeasts in beer and beer products." Current Opinion in Food Science 44 (2022): 100815. The lower the residual alcohol allowed in beer, the more critical the inhibition of species of the genus Saccharomyces in the process. Therefore, when using biological methods to produce reduced alcohol beer, the growth of Saccharomyces species that may be present in the fermenter must be inhibited or completely suppressed.

[0008] Therefore, there is a need for improved methods of inhibiting the growth of Saccharomyces species when producing beverages, particularly beverages susceptible to contamination by Saccharomyces. In particular, it is desirable to provide effective inhibition of Saccharomyces species during beverage production while preferably providing a beverage with good organoleptic or sensory properties, such as a good aroma or flavor profile. SUMMARY OF THE INVENTION

[0010] This problem has now been solved by using one or more strains of Pichia kluyveri to inhibit or delay the growth of Saccharomyces species in beverages.

[0011] Although the use of Pichia kluyveri for producing low-alcohol beer is known from, for example, WO 2014 / 135673, which discloses the use of Pichia kluyveri strains PK-KR1 and PK-KR2, the inhibition of Saccharomyces species is not disclosed.

[0012] The one or more Pichia kluyveri strains of the present invention are characterized in that, when Saccharomyces cerevisiae or Saccharomyces pastorianus are inoculated simultaneously with the one or more Pichia kluyveri strains, the one or more Pichia kluyveri strains are capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%, compared to an otherwise identical sample not comprising the one or more Pichia kluyveri strains.

[0013] The inventors of the present invention surprisingly found a new method for managing the contamination of Saccharomyces species by using Pichia kluyveri. Thus, the present invention helps to provide an effective solution that manages the growth of Saccharomyces species by biological means and can avoid the use of chemical disinfectants.

[0014] The uses and methods of the present invention are particularly suitable for fermenting low-alcohol beverages or non-alcoholic beverages. Species of the genus Saccharomyces are commonly present in brewery microenvironments. Since these yeasts are difficult to completely eliminate from brewing tanks by cleaning and disinfection, it is highly advantageous to use Pichia kluyveri strains to effectively inhibit the growth of contaminants of species of the genus Saccharomyces present in the system. Through the uses and methods of the present invention, the growth of the genus Saccharomyces present in the fermenter can be inhibited, and preferably, the inhibition is not present in the next batch of production for ordinary beer brewing in the same fermenter. This has the following advantages: the brewery does not need dedicated tanks to produce non-alcoholic beer.

[0015] In particular, the inventors of the present application have identified two novel Pichia kluyveri strains (DSM 34278 and DSM 34279) which are particularly suitable for the use and method of the present invention due to their ability to effectively inhibit species of the genus Saccharomyces. In addition, these strains also provide very good flavor characteristics when used to ferment non-alcoholic beverages.

[0016] In order to solve the problem of microbial contamination, the present invention provides, in a first aspect, the use of one or more Pichia kluyveri strains for inhibiting or delaying the growth of species of the genus Saccharomyces in beverages (preferably fermented beverages). One or more Pichia kluyveri strains may be characterized in that, when Saccharomyces cerevisiae or Saccharomyces pastoris is inoculated simultaneously with one or more Pichia kluyveri strains, the one or more Pichia kluyveri strains are capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, compared to an otherwise identical sample not comprising one or more Pichia kluyveri strains. Preferably, the 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, e.g. 10 3 CFU / ml to 10 9 CFU / ml, preferably 10 4 CFU / ml to 10 8 CFU / ml, most preferably 10 7 CFU / ml concentration was added to the beverage.

[0017] In a second aspect, the present invention provides a method for inhibiting or delaying the growth of a species of the genus Saccharomyces in a beverage, preferably a fermented beverage, more preferably a beverage with a lower alcohol content, such as a beverage with an alcohol content of less than 4.2 vol.-%, comprising the step of adding an effective amount of one or more Pichia kluyveri strains. The one or more Pichia kluyveri strains may be characterized in that, when Saccharomyces cerevisiae or Saccharomyces pastorianus is inoculated simultaneously with the one or more Pichia kluyveri strains, the one or more Pichia kluyveri strains are capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%, compared to an otherwise identical sample not comprising the one or more Pichia kluyveri strains. Preferably, the Pichia kluyveri is inhibited by at least 10 3 CFU / ml, for example at least 10 4 CFU / ml, for example at least 10 5 CFU / ml, e.g. 10 3 CFU / ml to 10 9 CFU / ml, preferably 10 4 CFU / ml to 10 8 CFU / ml, most preferably 10 7 CFU / ml concentration was added to the beverage.

[0018] In a third aspect, the present invention provides a yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under number DSM 34278 or a Pichia kluyveri mutant obtainable from the deposited yeast. The mutant may be characterized by being capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%, as determined using an assay (e.g. Assay I) when Saccharomyces is co-inoculated with the Pichia kluyveri strain, compared to an otherwise identical sample not comprising the Pichia kluyveri strain.

[0019] In a fourth aspect, the present invention provides a yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under number DSM 34279 or a Pichia kluyveri mutant obtainable from the deposited yeast. The mutant may be characterized by being capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%, as determined using an assay (e.g. Assay I) when Saccharomyces is co-inoculated with the Pichia kluyveri strain, compared to an otherwise identical sample not comprising the Pichia kluyveri strain.

[0020] In a further aspect, the present invention provides a composition or beverage comprising the yeast of the third and / or fourth aspect of the invention, in particular a beverage having a lower alcohol content (eg below 4.2 vol.-%).

[0021] Detailed disclosure of the invention

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Typical methods and materials are described, although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure. The detailed information provided for using the present invention also relates to the methods of the present invention.

[0023] In a first aspect, the present invention provides a use of one or more Pichia kluyveri strains for inhibiting or delaying the growth of a species of the genus Saccharomyces in a beverage, wherein the Pichia kluyveri strain is characterized in that the Pichia kluyveri strain is capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40% when the Saccharomyces genus is inoculated simultaneously with the Pichia kluyveri strain, compared to an otherwise identical sample not comprising the Pichia kluyveri strain.

[0024] As used herein, the terms "inhibit" and "inhibiting" in relation to a species of Saccharomyces refer to a reduction in the growth, amount or concentration of a species of Saccharomyces. Inhibition can be observed by comparing the growth, amount or concentration of Saccharomyces in a product having a Pichia kluyveri strain with a control. The control can be an otherwise identical composition but without the Pichia kluyveri strain. Yeast growth can be measured using various methods known to those skilled in the art. Methods for determining inhibition or delay of yeast growth are known to those skilled in the art. For example, yeast growth can be measured by quantifying the amount of yeast in a sample after a given time using a real-time PCR (qPCR) assay and comparing the assayed amount to a negative control.

[0025] The term "delay" generally refers to the act of stopping something, postponing something, hindering something, or causing something to occur more slowly than normal. As used herein, "delaying the growth of a Saccharomyces species" refers to the act of delaying the growth of a Saccharomyces species. This can be observed by comparing the time required for the Saccharomyces species to grow to a given level in two otherwise identical beverages, one of which contains a Pichia kluyveri strain and the other does not.

[0026] In some embodiments, "delaying growth" refers to a delay of 1 day, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days.

[0027] In a preferred embodiment, the growth of Saccharomyces species in the beverage is inhibited by at least 40%, preferably at least 50% or 75%, compared to an otherwise identical sample not comprising one or more Pichia kluyveri strains. This is illustrated for example in Examples 1 and 2. Saccharomyces cerevisiae may be used as target microorganism.

[0028] Inhibition can be determined by assay I:

[0029] Assay I:

[0030] -Inoculation with / without starting concentration of 10 5 CFU / ml of Pichia kluyveri wort (8°P and 13IBU),

[0031] - ferment the wort at 18°C ​​for 3 days at 120 rpm, and

[0032] - The growth of S. cerevisiae in wort medium with / without Pichia kluyveri was compared by qPCR and the percentage of inhibition was determined.

[0033] The inventors of the present invention surprisingly found that species of the genus Saccharomyces can be inhibited by an effective amount of Pichia kluyveri. Pichia kluyveri strains that can be used in the present invention include Pichia kluyveri DSM 34278, Pichia kluyveri DSM 34279, Pichia kluyveri DSM 28484, Pichia kluyveri CBS188, their mutants and mixtures thereof. These mutants are functionally equivalent mutants that have the same or improved ability to inhibit or delay the growth of species of the genus Saccharomyces compared to their respective parent strains. This ability can be determined using an assay (e.g., assay 1).

[0034] In the context of this article, the term "mutant" should be understood as a strain derived from the strain of the present invention (e.g., 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 (particularly a mutant with respect to the effect of inhibiting the growth of species of the genus Saccharomyces). Each mutant represents an embodiment of the present invention. The term "mutant" refers in particular to a strain or a spontaneously generated mutant obtained by subjecting the strain of the present invention to any conventionally used mutagenic treatment (including treatment by chemical mutagens such as ethane methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light). The mutant may have undergone several mutagenic treatments (a single treatment should be understood as a mutagenic step, followed by a screening / selection step), but it is currently preferred to carry out 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 bacterial genome are replaced by another nucleotide, or are deleted, compared to the parent strain.

[0035] In a preferred embodiment, the Pichia kluyveri strain used herein is not PK-KR1 or PK-KR2.

[0036] In a particularly preferred embodiment, the Pichia kluyveri strain is selected from the group consisting of the strain deposited under the number DSM 34278 and the strain deposited under the number DSM 34279 or a mutant thereof.

[0037] A Pichia kluyveri strain useful in the present invention may be characterized in that the Pichia kluyveri strain is capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80% when Saccharomyces is inoculated simultaneously with the Pichia kluyveri strain, compared to a negative control (i.e., an otherwise identical sample not comprising the Pichia kluyveri strain).

[0038] In a preferred embodiment, the Pichia kluyveri strain is characterized in that it inhibits the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40% when Saccharomyces cerevisiae or Saccharomyces pastorianus are inoculated simultaneously with the Pichia kluyveri strain in wort, compared to an otherwise identical wort not comprising said strain.

[0039] In a preferred embodiment, when 10 3 CFU / ml of yeast species and 10 5 When the Pichia kluyveri strain with a CFU / ml is inoculated at the same time, the Pichia kluyveri strain is able to inhibit the growth of the yeast genus by at least 40%.

[0040] In a preferred embodiment, when 10 3 CFU / ml of yeast species and 10 5 When CFU / ml of Pichia kluyveri strains are simultaneously inoculated in wort for three days, one or more Pichia kluyveri strains are capable of inhibiting the growth of said Saccharomyces by at least 40%.

[0041] In a preferred embodiment, the Pichia kluyveri strain is at least 10 3 CFU / ml, for example at least 10 4 CFU / ml, for example at least 10 5 CFU / ml, e.g. 10 3 CFU / ml to 10 9 CFU / ml, preferably 10 4 CFU / ml to 10 8 CFU / ml, most preferably 10 7 The concentration of CFU / ml is preferably added to the fermented beverage at the beginning of fermentation or during fermentation.

[0042] In preferred embodiments, 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.

[0043] drinks

[0044] The beverage in which the growth of a species of the genus Saccharomyces is inhibited or delayed may be any beverage, such as wine, cider, tea, fruit drink or beer, preferably a beverage obtainable by fermentation, and most preferably a beverage obtainable by wort fermentation, including wort-based beverages, such as beer. The beverage preferably has a low alcohol content, such as an alcohol content of less than 4.2 vol.-% or less.

[0045] As used in this specification, the term "beer" refers at least to beer types obtained from brewing malt obtained from malted cereals and brewing malt obtained from unmalted cereals and brewing malt obtained from a mixture of malted cereals and unmalted cereals.

[0046] The term "wort" herein has the conventional meaning in the art and refers to the sugar-containing liquid extracted from the mashing process of beer brewing.

[0047] The term "fermentation" as used herein refers to a metabolic process which involves chemical changes in a wort substrate by the action of a culture. This includes both aerobic and anaerobic processes.

[0048] Beverage is a beverage with low alcohol content, which can be a beverage with reduced alcohol content, a low-alcohol beverage, an alcohol-free beverage or a non-alcoholic beverage. However, as used herein, the meanings of these terms may vary depending on national regulations. In this context, the term "beverage with reduced alcohol content" in this specification refers to a beverage with an alcohol content of 1.2 vol.-% to 4.2 vol.-%. The term "low-alcohol beverage" herein is defined as a drinking liquid with an alcohol content exceeding 0.5 vol.-% and being no more than 1.2 vol.-%. The term "non-alcoholic beverage" herein is defined as a drinking liquid with an alcohol content no more than 0.5 vol.-%. The term "non-alcoholic beverage" herein is defined as a drinking liquid with an alcohol content lower than 0.05 vol.-%. In one embodiment, beverage is a wine with a low alcohol content.

[0049] beer

[0050] In a preferred embodiment, the beverage is a beer, low-alcohol beer, non-alcoholic beer or non-alcoholic beer with reduced alcohol content. Reduced means that the alcohol content is lower than that of ordinary beer. In this context, the term "beer with reduced alcohol content" in this specification refers to a beer with an alcohol content of 1.2 vol.-% to 4.2 vol.-%. The term "low-alcohol beer" as used herein refers to a beer with an alcohol content of 0.5 vol.-% to 1.2 vol.-%. The term "non-alcoholic beer" in this specification refers to a beer with an alcohol content lower than 0.5 vol.-%. Finally, the term "non-alcoholic beer" herein refers to a beer with an alcohol content lower than 0.05 vol.-%.

[0051] Fruit Drinks

[0052] In another preferred embodiment, the beverage is a fruit drink. In this context, the term "fruit drink" refers to a beverage comprising fruit juice, fruit juice concentrate and / or fruit puree. The term "fruit drink" encompasses "fruit juice", "fruit juice drink" and "fruit pulp drink". A "fruit drink" can be a beverage containing fruit pulp or a beverage from which the fruit pulp has been removed by an operation such as centrifugation. The fruit drink can further contain, for example, oats, soy, almonds, whey and / or non-fermented milk, for example in the form of milk powder.

[0053] Fruit beverages suitable for use in the present invention may be, for example, fruit juice, fruit juice from concentrate, fruit drinks, fruit smoothies and fruit drinks (optionally containing fruit puree and / or water).

[0054] The term "juice" refers to the liquid naturally contained in fruit prepared by mechanical pressing or maceration of fresh fruit in the absence of heat and solvents. "Juice" may consist of the juice from one fruit or a mixture of more than one fruit.

[0055] In the present context the term "juice drink" refers to a beverage having a juice content of 0 vol.-% to 29 vol.-%.

[0056] In the present context, the term "nectar" refers to a beverage having a juice content of 30 vol.-% to 99 vol.-% juice.

[0057] In this context, the term "puree" refers to fruit reduced to the consistency of a thick liquid or soft paste by grinding, pressing and / or filtering in the absence of heat and solvents. "Puree" is made from 100% fruit, not just fruit juice.

[0058] The total content of juice and / or puree in the fruit beverage is generally from about 20% to about 99.99% by weight, preferably from about 30% to 95% by weight, more preferably from about 40% to 90% by weight, still more preferably from about 50% to 80% by weight, and most preferably from 60% to 70% by weight.

[0059] Method of the present invention

[0060] In a second aspect, the present invention provides a method for inhibiting or delaying the growth of species of the genus Saccharomyces in a beverage having a lower alcohol content (e.g. an alcohol content below 4.2 vol.-%), the method comprising the step of adding to the beverage an effective amount of one or more Pichia kluyveri strains, wherein the one or more Pichia kluyveri strains are characterized in that, when the Saccharomyces genus is inoculated simultaneously with the one or more Pichia kluyveri strains, the one or more Pichia kluyveri strains are capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40%, compared to an otherwise identical sample not comprising the one or more Pichia kluyveri strains.

[0061] The Pichia kluyveri strain can be added in any form, including liquid, frozen or dry (e.g., freeze-dried or spray-dried) compositions. Compositions preferably include one or more cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants, and most preferably cryoprotectants or lyoprotectants, or both. Protective agents such as cryoprotectants and lyoprotectants are well known to those skilled in the art. Suitable cryoprotectants or lyoprotectants (including monosaccharides, disaccharides, trisaccharides and polysaccharides), polyols, amino acids, complex substances and inorganic compounds. Compositions can optionally include other substances, including emulsifiers (e.g., sorbitan monostearate), fillers and / or spices.

[0062] In a preferred embodiment, the composition comprises at least 10 6 CFU / g, including 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 Pichia kluyveri.

[0063] Methods for preparing high density yeast compositions are known in the art and are described, for example, in WO 2011 / 134952.

[0064] In a further aspect, the present invention provides a method for producing a beverage with a lower alcohol content (e.g., an alcohol content below 4.2 vol.-%), the method comprising the step of adding an effective amount of one or more Pichia kluyveri strains to inhibit or delay the growth of Saccharomyces species in the beverage, wherein the one or more Pichia kluyveri strains are characterized in that, when Saccharomyces genus is inoculated simultaneously with the one or more Pichia kluyveri strains, the one or more Pichia kluyveri strains are capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40%, compared to an otherwise identical sample not comprising the one or more Pichia kluyveri strains.

[0065] When using the yeast strains in a method for producing a beverage, the skilled person, taking into account the examples provided in the present invention and the nature of the beverage to be produced, is able to adjust various parameters, such as pH, temperature and amount of yeast, to achieve the desired result.

[0066] The methods disclosed herein are particularly useful for inhibiting or delaying yeast growth in fermentation processes, such as producing reduced alcohol beer, low alcohol beer, non-alcoholic beer, or alcohol-free beer.

[0067] Yeast and composition of the present invention

[0068] In a third aspect, the present invention provides a yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under number DSM 34278 or a Pichia kluyveri mutant obtainable from the deposited yeast, wherein the mutant is characterized in that, when the Saccharomyces genus is inoculated simultaneously with the Pichia kluyveri strain, the mutant is capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%, compared to an otherwise identical sample not comprising the Pichia kluyveri strain.

[0069] In a fourth aspect, the present invention provides a yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under number DSM 34279 or a Pichia kluyveri mutant obtainable from the deposited yeast, wherein the mutant is characterized in that, compared to an otherwise identical sample not comprising the Pichia kluyveri strain, when the Saccharomyces genus is inoculated simultaneously with the Pichia kluyveri strain, the mutant is capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastoris by at least 40%, such as at least 50%, at least 60%, at least 70% or at least 80%.

[0070] In a preferred embodiment, the inhibitory concentration used is 10 5 CFU / ml of Pichia kluyveri and the starting concentration was 10 3 The inhibition is preferably tested in wort medium, as described in the examples.

[0071] In a preferred embodiment, the inhibitory concentration used is 10 5 CFU / ml of Pichia kluyveri and the starting concentration was 10 3 CFU / ml of Saccharomyces cerevisiae or Saccharomyces pastoris in the wort was tested after three days.

[0072] In a fifth aspect, the present invention provides a composition comprising the yeast of the third aspect and / or the fourth aspect.

[0073] Compositions of the present invention may additionally include cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, spices or their mixtures. Compositions may be in any form, including liquid (e.g., slurry), freezing or dry forms (e.g., freeze-dried or spray-dried). Compositions preferably include one or more cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants, and most preferably cryoprotectants or lyoprotectants, or both. The use of protective agents such as cryoprotectants and lyoprotectants is 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, starch and gum arabic (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, gallic acid esters, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include carbohydrates, 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) and / or spices.

[0074] In a preferred embodiment, the composition comprises at least one cryoprotectant or lyoprotectant. In another embodiment, the composition comprises sorbitan monostearate. In a preferred embodiment, the composition is liquid or lyophilized.

[0075] In the context of describing the invention (especially in the context of the appended claims), the use of the terms "a", "an", "the" and similar references should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising", "having", "including" and "comprising" should be construed as non-limiting terms (i.e., meaning "including but not limited to")

[0076] picture

[0077] Figure 1 Shown is the total concentration of S. cerevisiae found in the fermentation at different time points (day 1, day 3, and day 6) expressed as gene copies / mL.

[0078] Figure 2The total concentration of S. pastoris found in the fermentation at different time points (day 1, day 3 and day 6) is shown, expressed as gene copies / mL.

[0079] Figure 3 Shown are the concentrations of ethyl acetate in beer samples prepared with Pichia kluyveri DSM 34278, DSM 34279 and DSM 28484 measured by GC-MS.

[0080] Figure 4 Shown are the concentrations of the off-flavor compound hydrogen sulfide in beer samples prepared with Pichia kluyveri DSM 34278, DSM 34279 and DSM 28484 measured by GC-MS.

[0081] Figure 5 Shown are the concentrations of the off-flavor compound methylthiopropionaldehyde in beer samples prepared with Pichia kluyveri DSM 34278, DSM 34279 and DSM 28484 measured by GC-MS. Example

[0082] The scope of the invention described and claimed herein is not limited by the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to fall within the scope of the invention. In fact, various modifications of the invention, except those shown and described herein, will become apparent to those skilled in the art based on the foregoing description and the following examples. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure (including definitions) shall prevail.

[0083] Example 1: Inhibition of Saccharomyces cerevisiae

[0084] 1.1 Sample preparation

[0085] Prior to inoculation, Saccharomyces cerevisiae (Safale US-05, Fermentis) was cultivated in malt medium (8°P, prepared by Barlex 7215 from Harboes Bryggeri A / S, Denmark; pH = 4.4) at 25°C and 150 rpm for 48 hours.

[0086] Then, a wort of 8°P, 13IBU was prepared from a malt extract (Barlex 7215) with hops (Magnum 12.9% AA), and the pH was adjusted to 4.4. It was divided into 200 mL volumes / sample in blue-capped bottles. The samples were inoculated with or without various Pichia kluyveri strains simultaneously with Saccharomyces cerevisiae (Safale US-05, Fermentis). An overview of the inoculation is shown in Table 1. The sources of the strains are shown in Table 2. The fermentation was incubated at 18°C ​​at 120 rpm for 10 days. 10 mL samples were collected on the 1st, 3rd and 6th days after inoculation and subsequently frozen at -50°C.

[0087] Table 1 – Overview of fermentation and inoculation protocols with Pichia kluyveri and Saccharomyces cerevisiae

[0088]

[0089] Table 2 - Source of strains

[0090] microorganism strain source Pichia kluyveri DSM 28484 Grape Pichia kluyveri DSM 34278 cocoa Pichia kluyveri DSM 34279 cocoa Pichia kluyveri PK-KR1 Grape Juice Pichia kluyveri type strains CBS188 Olives

[0091] 1.2 Analysis of yeast growth

[0092] 1.2.1 DNA extraction from fermentation samples

[0093] Yeast cells were first isolated from 12 mL of day 1 fermentation and 5 mL of day 3 and day 6 fermentation by centrifugation at 5.000 × g for 10 min. 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 precipitate was centrifuged again at 5.000 × g for 10 min, and the supernatant was discarded. Subsequently, 200 μl of lysis buffer (1.2 M sorbitol; 100 mM TRIS; 100 mM CaCl2, 4H2O, 200 U liquid lysing enzyme (Lyticase)) was added to the precipitate and mixed thoroughly by pipetting. Finally, samples were loaded onto a QIAcube Connect instrument (Qiagen, Hilden, Germany) and DNA was purified using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions in a final elution volume of 125 μl.

[0094] 1.2.2 Real-time PCR (qPCR) assay

[0095] Primer specificity was assessed using Primer-blast comparisons, in silico primer-targets (CLC Genomics Workbench v.20 Qiagen) and real-time PCR validation using pure DNA from each strain in fermentation. Specific primers for the Saccharomyces sensu stricto complex were used to target and quantify Saccharomyces cerevisiae (Safale US-05, Fermentis) and Saccharomyces pasteurianus (SmartBev, Chr. Hansen) in fermentation samples. Primers SI4F: 5'-ATTGCTGGCCTTTTCATTG-3' (SEQ ID NO: 1), SI7R: 5'-CGCCTAGACGCTCTCTTCTTAT-3' (SEQ ID NO: 2) (Chang et al. "Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and theSaccharomyces 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 in the ITS1-5.8S-ITS2 rDNA region and amplify a product of 163 bp in length. Real-time PCR amplification and detection were performed in a QuantStudioTM 12K Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). The probe-based reaction mixture includes 6.25 μL 1× qPCR TMMaster Mix Plus–Low ROX (Eurogentec, Seraing, Belgium), 0.3 μM nmol of each forward and reverse primer, 0.6 μM probe, 2.5 μL of 10× and 100× diluted template DNA (to avoid inhibitor effects), and molecular grade HO (Invitrogen, Life Technologies, Paisley, UK) in a final volume of 12.5 μL. The amplification program consisted of 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, then 40 cycles at 95°C for 15 s and 60°C for 1 min. A standard curve was constructed using DNA from a pure culture of Saccharomyces cerevisiae (SmartBev, Chr. Hansen) and was generated for each qPCR analysis in parallel with the fermentation sample amplification. The standards were serially diluted (10-fold) to obtain 10 starting at 5 ng / μL. 8 –10 1 All samples were run in triplicate and in control reactions the template was replaced by molecular grade H2O.

[0096] 1.3 Results

[0097] A total of 18 samples belonging to different fermentations and controls collected on day 1, day 3 and day 6 were analyzed. To quantify S. cerevisiae in experiments performed on fermentations, qPCR primers SI4F / SI7R and S-probe were used to amplify the appropriate product size located on the ITS rRNA gene. The specificity of the primers and probe was verified by qPCR using genomic DNA of all yeasts listed in Table 1. The assay showed excellent efficiency for S. cerevisiae (95% amplification efficiency, R 2 0.998) and specificity, while various Pichia kluyveri strains were still below the minimum reliable detection limit.

[0098] Figure 1 The total concentration of Saccharomyces cerevisiae found in the fermentation after 1, 3 and 6 days is shown as gene copies / mL. Compared to the control batch (without Pichia kluyveri), a reduction in the growth of Saccharomyces was observed from day 3 to day 6. On the other hand, when the fermentation was carried out in the presence of Pichia kluyveri PK-KR1, no growth inhibition of the yeast occurred.

[0099] These results show the effective use of various Pichia kluyveri strains to inhibit brewer's yeast, Saccharomyces cerevisiae, inoculated at very high levels (much higher than the level as a contaminant). Therefore, even better inhibition is expected when Saccharomyces species are present only as a contaminant.

[0100] Example 2: Inhibition of Pasteurella

[0101] 2.1 Sample preparation

[0102] Prior to inoculation, Pasteurella (SmartBev, Chr. Hansen A / S, Denmark) was cultivated in malt medium (8°P, prepared by Barlex 7215 from Harboes Bryggeri A / S, Denmark; pH = 4.4) at 25°C and 150 rpm for 48 hours.

[0103] 8°P, 13 IBU wort was prepared from malt extract (Barlex 7215) with hops (Magnum 12.9% AA) and the pH was adjusted to 4.4. It was divided into 200 mL volumes / sample in blue-capped bottles. The samples were inoculated with or without various Pichia kluyveri strains simultaneously with Pasteurella (SmartBev, Chr. Hansen A / S, Denmark). See Table 3 for an overview of the inoculation. The fermentation was incubated at 18°C ​​at 120 rpm for 10 days. 10 mL samples were collected on the 1st, 3rd and 6th day after inoculation and subsequently frozen at -50°C.

[0104] Table 3 – Overview of fermentation and inoculation protocols for Pichia kluyveri and Saccharomyces pastoris

[0105]

[0106]

[0107] 2.2 Analysis of yeast growth

[0108] Yeast growth assay was performed as described in 1.2.

[0109] 2.3 Results

[0110] A total of 12 samples belonging to different fermentations and controls collected on day 1, day 3 and day 6 were analyzed. To quantify S. pastoris in experiments performed on fermentations, qPCR primers SI4F / SI7R and S-probe as described previously were used to amplify the appropriate product size located on the ITS rRNA gene. The specificity of the primers and probe was verified by qPCR using genomic DNA of all yeasts listed in Table 3. The assay showed excellent efficiency for S. pastoris (95% amplification efficiency, R 2 0.998) and specificity, while various Pichia kluyveri strains were still below the minimum reliable detection limit.

[0111] Figure 2The total concentration of Pasteurella in gene copies / mL found in the fermentation after 1, 3, and 6 days is shown. Compared to the control batch (without Pichia kluyveri), a reduction in Pasteurella growth from day 3 to day 6 was observed for various Pichia kluyveri strains. These results show the effective use of various Pichia kluyveri strains to inhibit Pasteurella.

[0112] Example 3: Preparation of non-alcoholic beer using Pichia kluyveri strains DSM 34278, DSM 34279 and DSM 28484

[0113] 2.1 Preparation of non-alcoholic beer

[0114] 6.5 kg of Pilsner malt was added to a mash kettle containing 19.5 L of 68°C water and kept at this temperature for 30 min. The mash (malt and water) was heated to 72°C and kept at this temperature for 10 min, then heated to 78°C and kept at this temperature for 5 minutes. The mash was then filtered and sparged with 40.7 L of water, the liquid was transferred to a boil kettle and heated to boiling. 22 g of hops with an alpha acid content of 12.8% were added. Boiling continued for 60 min. The resulting wort was transferred to 5 L plastic jerry cans and stored in a cold room (<5°C).

[0115] 5 liters of wort are adjusted to pH 5.3, and 8 500ml blue-capped bottles are filled with 400ml of pH-regulated wort. The blue-capped bottles equipped with pH-regulated wort are placed in 90 ℃ of water baths for 30 minutes and pasteurized, then cooled to room temperature (about 20 ℃). After cooling, they are inoculated with DSM34278, DSM 34279 or DSM 28484 (repetitions), to a cell count of 1E+05 cells / ml. Wherein two blue-capped bottles are not inoculated, and as negative controls. Eight blue-capped bottles are transferred to an oscillating incubator that is set to 18 ℃ and 100rpm, and kept there for 5 days. The non-alcoholic beer fermented with yeast all has an alcohol content range of 0.2-0.3%vol.-%.

[0116] 3.2 Analysis of volatile compounds

[0117] The samples were analyzed by gas chromatography-mass spectrometry (GC-MS) at the Laboratory of Aroma Analysis and Enology (LAAE) of the University of Zaragoza, Spain, where the main volatile compounds and free volatile sulfur were analyzed by GC-MS.

[0118] Figure 3-Figure 5Results are shown for ethyl acetate (which imparts a sweet, fruity, and tropical flavor) and the off-flavor compounds hydrogen sulfide (rotten egg odor) and methylthiopropionaldehyde (brothy, meaty, sulfurous flavor). The dashed lines indicate the odor detection thresholds (the lowest concentration of a certain odor compound that can be perceived by the human sense of smell) of the corresponding compounds in alcoholic beer. The values ​​for ethyl acetate and hydrogen sulfide were retrieved from www.aroxa.com (as of March 18, 2022). The values ​​for methylthiopropionaldehyde were provided by LAAE.

[0119] These results show that Pichia kluyveri strains DSM 34278 and DSM 34279 produced higher ethyl acetate and less hydrogen sulfide and methylthiopropionaldehyde (below the odor threshold) compared to DSM 28484, indicating a better flavor profile.

[0120] Preservation and Expert Solutions

[0121] At the applicant's request, only samples of the deposited microorganisms described below may be provided to the expert, subject to the applicable regulations administered by the industrial property office of a Contracting State to the Budapest Treaty until the date of grant of the patent.

[0122] Table 4: The applicant has made the following deposits with a depository institution which has acquired the status of an international depository institution under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, 38124 Braunschweig, Germany.

[0123] strain Accession number Date of deposit Pichia kluyveri DSM 28484 DSM 28484 2014.03.05 Pichia kluyveri DSM 34278 DSM 34278 2022.05.31 Pichia kluyveri DSM 34279 DSM 34279 2022.05.31

Claims

1. Use of one or more Pichia kluyveri strains for inhibiting or delaying the growth of Saccharomyces spp. in a beverage having an alcohol content of less than 4.2 vol.-%, wherein the Pichia kluyveri strain is characterized in that, when S. cerevisiae or S. pastorianus is inoculated simultaneously with the Pichia kluyveri strain, the Pichia kluyveri strain is capable of inhibiting the growth of Saccharomyces cerevisiae or S. pastorianus by at least 40% compared to an otherwise identical sample not comprising the Pichia kluyveri strain, wherein the Pichia kluyveri strain is not PK-KR1.

2. A method for inhibiting or delaying the growth of a species of the genus Saccharomyces in a beverage having an alcohol content of less than 4.2 vol.-%, the method comprising the step of adding an effective amount of one or more Pichia kluyveri strains, wherein the Pichia kluyveri strain is characterized in that, when Saccharomyces cerevisiae or Saccharomyces pastorianus are inoculated simultaneously with the Pichia kluyveri strain, the Pichia kluyveri strain is capable of inhibiting the growth of Saccharomyces cerevisiae or Saccharomyces pastorianus by at least 40% compared to an otherwise identical sample not comprising the Pichia kluyveri strain, wherein the Pichia kluyveri strain is not PK-KR1.

3. Use or method according to any one of the preceding claims, wherein the beverage is a beverage having an alcohol content of less than 1.2 vol.-%.

4. Use or method according to any of the preceding claims, wherein the beverage is a beverage having an alcohol content below 0.5 vol.-% and most preferably below 0.05 vol.-%.

5. The use or method according to any one of the preceding claims, wherein the amount of 10 3 CFU / ml of yeast species and 10 5 CFU / ml of the Pichia kluyveri strain were simultaneously inoculated in wort for three days, said Pichia kluyveri was able to inhibit the growth of said Saccharomyces species by at least 40%.

6. Use or method according to any one of the preceding claims, wherein the Pichia kluyveri strain is selected from the group consisting of the strain deposited under the number DSM 34278 and the strain deposited under the number DSM 34279 and mutants thereof.

7. The use or method of any preceding claim, 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.

8. The use or method according to any one of the preceding claims, wherein the one or more Pichia kluyveri strains are present in an amount of at least 10 3 CFU / ml, preferably at least 10 5 CFU / ml, more preferably at least 10 7 The concentration of CFU / ml was added to the beverage.

9. Use or method according to any of the preceding claims, wherein the beverage is a wine, cider, tea, fruit drink or beer having an alcohol content of less than 4.2 vol.-%, preferably less than 1.2 vol.-%, more preferably less than 0.5 vol.-% and most preferably less than 0.05 vol.-%.

10. Use or method according to any one of the preceding claims, wherein the growth of the Saccharomyces species is inhibited by at least 50%, preferably at least 60% or 75%.

11. The use or method according to any one of the preceding claims, wherein the Pichia kluyveri strain is characterized in that, compared to an otherwise identical sample not comprising the Pichia kluyveri strain, when 10 3 CFU / ml of yeast species and 10 5 When a Pichia kluyveri strain with a CFU / ml is inoculated simultaneously, the Pichia kluyveri strain is capable of inhibiting the growth of Saccharomyces species by at least 40%.

12. A yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under the number DSM 34278 or a functionally equivalent mutant of Pichia kluyveri obtainable from the deposited yeast.

13. A yeast of the species Pichia kluyveri, which is Pichia kluyveri deposited under the number DSM 34279 or a functionally equivalent mutant of Pichia kluyveri obtainable from the deposited yeast.

14. A composition comprising yeast of the species Pichia kluyveri according to any one of claims 11 to 12, wherein the Pichia kluyveri is optionally present in an amount of at least 10 6 The concentration of CFU / g is present in the composition, wherein the composition is optionally in liquid, frozen or dried form, or wherein the composition optionally further comprises one or more cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavors or mixtures thereof.

15. A beverage comprising a yeast of the species Pichia kluyveri according to any one of claims 11 to 12, preferably a beverage having an alcohol content of less than 4.2 vol.-%.

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