FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No. 2022901674, filed 20 June 2022, the contents of which are herein incorporated by reference. [0002] The present invention broadly relates to methods for epigenetic modification of yeasts. The modified yeasts are useful in fermentation processes to alter flavour and aroma profiles of fermented products.

BACKGROUND TO THE INVENTION

[0003] Epigenetics is the study of phenotypic changes in organisms which predominantly result from alterations of nucleotides and histones instead of the deoxyribonucleic acid (DNA) sequence, and therefore epigenetic modifications are considered as a non-genetically modified organism (non-GMO) technique ^1,2 . DNA methylation and histone acetylation are the most common and well-studied epigenetic modifications. DNA methylation is a process by which methyl groups are added to the DNA molecule. Histone acetylation is a biological process by which an acetyl group is added to lysine residues in the histone N-terminal tail ³ . Histone modifications play an important role in epigenetic regulation of cellular events ⁴ . Bioactive compounds and phytochemicals which naturally occur in fruits and vegetables have been identified as carrying out epigenetic alterations in organisms ⁵ . In accordance with the common types of epigenetic modifications, these bioactive compounds and phytochemicals, termed epigenetic modifiers, can act on DNMT (DNA-methyl transferase) HD AC (histone deacetylase) ⁶ , in addition to HAT (Histone-acetyl transferase) ⁷ . Inhibition or activation of these enzymes can cause epigenetic modification.

[0004] Torres-Garcia et al ⁸ investigated the effect of caffeine on fission yeast Schizosaccharomyces pombe, and they concluded that the resistance which arose in fission yeast towards the stress caused by caffeine was a consequence of epigenetic heterochromatin alterations and heterochromatin-mediated gene silencing/less expression. Resistance was not due to changes in acetylation or methylation.

[0005] The production of fermented foods and beverages using yeasts has a very long history. Common fermented food products include wine, beer, cider, milk, yoghurt, kefir, kimchi, and sauerkraut amongst others. Changing consumer preferences and tastes means food and beverage manufacturers are constantly looking for ways of producing products with unique flavour and/or aroma profiles. For example, in the wine industry, there is a drive to produce wines with fruitier aromas, more floral characteristics, and/or a lower ethanol content.

[0006] Existing approaches to producing wine and beer with unique flavours and aromas have focused on breeding new grape and hop varieties with unique characteristics. However, the breeding process is slow and expensive. Accordingly, there is a need for a faster and more cost-effective processes for altering the characteristics of fermented food products. There is also a need for a non-GMO approach for efficiently altering characteristics of fermented food products.

[0007] It is an object of the present invention to go at least some way to meeting at least one of these needs; and/or to at least provide the public with a useful choice.

[0008] Other objects of the invention may become apparent from the following description which is given by way of example only.

[0009] Any discussion of documents, acts, materials, devices, articles, or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.

SUMMARY OF THE INVENTION

[00010] In a first aspect, the invention provides a process for epigenetic modification of a yeast starter culture, the process comprising a step of contacting the yeast starter culture with an effective amount of an epigenetic modifier which is selected from: an HD AC (histone deacetylase) inhibitor; a DNMT (DNA-methyl transferase) inhibitor; a HAT (histone acetyltransferase) inhibitor; an HD AC activator; a DNMT activator; and a HAT activator.

[00011] In a second aspect, the invention provides a process for epigenetic modification of a yeast starter culture in a growth medium, the process comprising a step of contacting the yeast starter culture with an effective amount of an epigenetic modifier which is exogenous to the growth medium, the epigenetic modifier being selected from: an HD AC (histone deacetylase) inhibitor; a DNMT (DNA-methyl transferase) inhibitor; a HAT (histone acetyltransferase) inhibitor; an HD AC activator; a DNMT activator; and a HAT activator. [00012] In various embodiments, contacting the yeast starter culture with an epigenetic modifier comprises growing the yeast starter culture in a growth medium comprising the epigenetic modifier.

[00013] In a third aspect, the invention provides a process for fermenting a food product, comprising steps of: contacting a yeast or a yeast starter culture with an effective amount of an epigenetic modifier which is selected from: an HD AC inhibitor; a DNMT inhibitor; a HAT (histone acetyltransferase) inhibitor; an HD AC activator; a DNMT activator; and a HAT activator; and fermenting said food product using said yeast or yeast starter culture.

[00014] In a fourth aspect, the invention provides a process for fermenting a food product, comprising steps of contacting a yeast or yeast starter culture with an effective amount of an epigenetic modifier which is exogenous to the food product, the epigenetic modifier being selected from: an HD AC inhibitor; a DNMT inhibitor; a HAT inhibitor; an HD AC activator; a DNMT activator; and a HAT activator; and fermenting said food product using said yeast or yeast starter culture.

[00015] In some embodiments, the yeast starter culture is contacted with the epigenetic modifier prior to the fermentation step.

[00016] In some embodiments, the process further comprises the step of isolating the modified yeast starter culture after the contacting step.

[00017] In some embodiments, the modified yeast starter culture is dried or freeze-dried, and the process further comprises reviving the modified starter culture before or during the fermentation step.

[00018] In some embodiments, the yeast or yeast starter culture is contacted with the epigenetic modifier during the fermentation step.

[00019] In some embodiments, the process comprises the steps of:

(a) adding a yeast or yeast starter culture to the food product:

(b) adding the epigenetic modifier to the food product; and

(c) fermenting the food product.

[00020] In some embodiments, step (b) is carried out prior to step (a). In other embodiments, step (a) is carried out prior to step (b). [00021] In some embodiments, the epigenetic modifier is an HD AC inhibitor.

[00022] In some embodiments, the epigenetic modifier is a dietary compound.

[00023] In some embodiments, the epigenetic modifier is a preservative for a food product.

[00024] In some embodiments, the epigenetic modifier is selected from: benzoic acid, 4- phenylbutyric acid, sodium butyrate, quercetin, genistein, anacardic acid, curcumin, and epigallocatechin gallate.

[00025] In some embodiments, the epigenetic modifier is selected from sodium butyrate, and epigallocatechin gallate.

[00026] In some embodiments, the epigenetic modifier is a lipophilic weak acid.

[00027] In some embodiments, the epigenetic modifier is benzoic acid.

[00028] In some embodiments, the yeast starter culture is contacted with benzoic acid at a concentration of from about 1 to about 30 mM, more preferably from about 5 mM to about

10 mM.

[00029] In some embodiments, the yeast starter culture is contacted with sodium butyrate at a concentration of from about 1 to about 30 mM, more preferably from about 5 mM to about 10 mM.

[00030] In some embodiments, the yeast is grown in the presence of the epigenetic modifier in a growth medium, for between 10 and 1000 hours, preferably for about 500 hours.

[00031] In some embodiments, the yeast is of the genus Saccharomyces. In some embodiments, the yeast is Saccharomyces cerevisiae.

[00032] In some embodiments of the third or fourth aspects, the food product is wine. In other embodiments of the third or fourth aspects, the food product is beer.

[00033] In a fifth aspect, the present invention provides a modified yeast when produced by the process of the first or second aspect.

[00034] In a sixth aspect, the present invention provides a composition comprising a modified yeast according to the fifth aspect.

[00035] In a seventh aspect, the present invention provides a food product which has been fermented by the process of the third or fourth aspect.

[00036] In an eighth aspect, the present invention provides a wine produced by the process of the third or fourth aspect.

[00037] In a ninth aspect, the present invention provides a beer produced by the process of the third or fourth aspect. [00038] In a tenth aspect, the present invention provides a wine produced by fermentation of a fruit juice using an epigenetically modified yeast, the wine having an increased content of one or more of: cis-3-hexen-l-ol; phenylethyl alcohol; ethyl lactate; ethyl decanoate; fructose; and glycerol; as compared to a wine prepared by fermentation of the fruit juice using an unmodified yeast.

[00039] In an eleventh aspect, the present invention provides a wine produced by fermentation of a fruit juice in the presence of an epigenetic modifier which is selected from: an HD AC inhibitor; a DNMT inhibitor; a HAT inhibitor; an HD AC activator; a DNMT activator; and a HAT activator;, the wine having one or more of: an increased content of a residual sugar such as glucose or fructose; a decreased content of glycerol; and a decreased content of ethanol; as compared to a wine prepared by fermentation of the fruit juice in the absence of the epigenetic modifier.

[00040] In some embodiments of the eleventh aspect, the wine has been produced by fermentation of a fruit juice using an epigenetically modified yeast.

[00041] In a twelfth aspect, the present invention provides a beer produced by fermentation of wort using an epigenetically modified yeast, the beer having an improved organoleptic attribute as compared to a beer prepared by fermentation of the wort using an unmodified yeast.

[00042] In some embodiments, the improved organoleptic attribute is one or more of: sweet, sour, dairy, smooth, and creamy. In some embodiments, the improved organoleptic attribute is measured using a penalty analysis method.

[00043] In some embodiments of the tenth or twelfth aspect, the yeast is epigenetically modified prior to the fermentation step.

[00044] In a thirteenth aspect, the present invention provides produced by fermentation of wort in the presence of an epigenetic modifier which is selected from: an HD AC inhibitor; a DNMT inhibitor; a HAT inhibitor; an HD AC activator; a DNMT activator; and a HAT activator.

[00045] In some embodiments of the thirteenth aspect, the beer has been produced by fermentation of wort using an epigenetically modified yeast.

[00046] In a fourteenth aspect, the present invention relates to the use of an epigenetic modifier which is selected from: an HD AC inhibitor; a DNMT inhibitor; a HAT inhibitor; an HD AC activator; a DNMT activator; and a HAT activator; to modify gene expression in a yeast for fermentation of a food. [00047] The following embodiments and preferences may relate alone or in any combination of any two or more to any of the above aspects.

[00048] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[00049] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[00050] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[00051] To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[00052] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples. BRIEF DESCRIPTION OF THE FIGURES

[00053] The present invention will be described with reference to the accompanying figures, in which:

Figure 1 is a bar graph showing results of a fluorometric HD AC assay, indicating HD AC inhibition capacity on HeLa nuclear extracts of candidate epigenetic modifiers benzoic acid, 4-PBA (4-phenylbutyric acid), sodium butyrate, quercetin, genistein, anacardic acid, curcumin, and EGCG (epigallocatechin gallate), was tested with reference to control compounds glucose, 5-aza-2’ -deoxycytidine and Trichostatin A (TSA) at different concentrations according to example 1;

Figure 2 is a bar graph showing the results of a colorimetric histone H3 gene modification multiplex assay, showing relative change in 21 histone H3 modification patterns using antibodies specific to respective histone H3 modifications: included were fifteen genes for methylation (me), four genes for acetylation (ac), and two genes for phosphorylation (p); in a strain of S. cerevisiae treated with 5 mM benzoic acid compared to untreated wild type S. cerevisiae strain, according to example 2A;

Figure 3 shows a heat map of gene expression levels resulting from an RNA expression assay which was carried out on 12 samples/24 genes, including 5 housekeeping genes as listed in Table 1, in nine strains of S. cerevisiae according to example 2B;

Figure 4 shows a schematic for a generation test of S. cerevisiae wild type strain and Epimutant 1. The strains were transferred from YPD broth onto YPD agar plates as follows: column 1, YPD agar containing 10 mM benzoic acid (first generation); column 2, YPD agar (first generation), YPD agar containing 10 mM benzoic acid (second generation); column 3, YPD agar (first generation), YPD agar (second generation), YPD agar containing 10 mM benzoic acid (third generation); column 4, YPD agar (first generation), YPD agar (second generation), YPD agar (third generation), YPD agar containing 10 mM benzoic acid (fourth generation); transfer to each YPD agar plate was performed after 36 hours.

Figure 5 shows the morphological differences in the two strains of S. cerevisiae, each plate was recorded at 18, 24, 26, and 48 hours after transfer onto the YPD agar plate to show generation changes according to example 3;

Figure 6 shows a schematic for a test of three strains of S. cerevisiae transferred from YPD broth onto YPD agar plates containing benzoic acid at concentrations of zero, 0.8 mM, 1.6 mM and 10 mM, and incubated at 25 °C, 30 °C, 32 °C, 35 °C, and 40 °C. Figure 7 shows the morphological differences in the three strains of S. cerevisiae, each recorded at 18, 24, 26, and 48 hours after transfer onto the YPD agar plates to show changes due to temperature variation according to example 3;

Figure 8 is a bar graph showing the content of eleven ester and higher alcohol compounds representing potential indication of wine aroma alterations in wine samples produced by fermentation using three strains of S. cerevisiae according to example 4, as measured by GC-MS;

Figure 9 shows principal component analysis (PCA) biplots and agglomerative hierarchical clustering (AHC) dendrograms for wine samples prepared in two groups of wine fermentation experiments according to example 4; three grape juice samples fermented using wild type, epimutant 1 and epimutant 2 (Group 1); six grape juice samples fermented using wild type and epimutant 1, each at a concentration of 0.8 mM and 1.6 mM respectively (Group 2). Figure 9(A) shows a PCA biplot of Group 1 wine samples and Figure 9(B) shows a PCA biplot of Group 2 wine samples.

Figure 10(A) shows Group 1 wine samples grouped using agglomerative hierarchical clustering (AHC) according to their dissimilarity levels. Figure 10(B) shows Group 2 wine samples grouped using AHC according to their dissimilarity levels;

Figure 11 is a bar graph showing chemical composition of wine samples fermented using three strains of S. cerevisiae in three grape juice samples fermented using wild type, epimutant 1 and epimutant 2 (Group 1 of example 4), as measured using an enzymatic test (glucose and fructose), glycerol assay (glycerol) and GC-MS (ethanol);

Figure 12 is a bar graph showing chemical composition of wine samples fermented using two strains of S. cerevisiae in three different fermentation conditions, in six grape juice samples fermented using wild type and epimutant 1, each at a concentration of 0.8 mM and 1.6 mM respectively (Group 2 of example 4), as measured using an enzymatic test (glucose and fructose), glycerol assay (glycerol) and GC-MS (ethanol);

Figure 13(A) shows a principal component analysis (PCA) biplot illustrating the relationship between beer samples fermented in five beer fermentation experiments according to example 5. Figure 13(B) shows beer samples grouped using agglomerative hierarchical clustering (HCA) according to their dissimilarity levels.

Figure 14 shows Just-About-Right (JAR) frequencies and penalty analysis results for attributes sweet, sour, dairy, smooth, and creamy of beer fermented using Wild Type 100 S. cerevisiae strain according to example 5; Figure 15 shows Just- About- Right (JAR) frequencies and penalty analysis results for attributes sweet, sour, dairy, smooth, and creamy of beer fermented using Wild Type 100 S. cerevisiae strain with 0.5 mM sodium butyrate addition during fermentation, according to example 5 ;

Figure 16 shows Just- About- Right (JAR) frequencies and penalty analysis results for attributes sweet, sour, dairy, smooth, and creamy of beer fermented using Epimutant 3 S. cerevisiae strain according to example 5;

Figure 17 shows Just-About-Right (JAR) frequencies and penalty analysis results for attributes sweet, sour, dairy, smooth, and creamy of beer fermented using Epimutant 3 S. cerevisiae strain with 0.5 mM sodium butyrate addition during fermentation according to example 5 ; and

Figure 18 shows Just-About-Right (JAR) frequencies and penalty analysis results for attributes sweet, sour, dairy, smooth, and creamy of beer fermented using Epimutant 4 S. cerevisiae strain according to example 5.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[00054] The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains.

[00055] The general chemical and biological terms used herein have their usual meanings.

[00056] Examples of definitions of common terms in microbiology, molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology, 3 ^rd Edition, C. A. Reddy, et al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nd ed., Joshua Lederburg, (ed.). Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of Microbiology and Molecular Biology, Singleton et al. (2d ed.) (1994); Biology of Microorganisms 11t h ed. Brock et al., Pearson Prentice Hall, (2006); Biodiversity of Fungi: Inventory and Monitoring Methods, Mueller et al. Academic Press, (2004); Genes IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994); and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995), Ausubel, Brent, Kingston, Moore, Seidman, Smith and Struhl. 1994. Current protocols in molecular biology, Current Protocols, Brooklyn, N.Y. Standard microbiological, molecular biology and biochemistry protocols and procedures that can be used to perform the present invention include the methods for measuring methylation of nucleic acids discussed in international patent publication WO 2021/032060, and the user guide for nCounter published at http://web.archive.org/web/20211105013514/https://nanostring .com/products/ncounter- analysis-svstem/ncounter-systems-overview/.

[00057] The term “comprising” as used in this specification and claims means “consisting at least in part of’. When interpreting each statement in this specification and claims that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise”, “comprised” and “comprises” are to be interpreted in the same manner.

[00058] As used herein the term “and/or” means “and” or “or”, or both.

[00059] As used herein “(s)” following a noun means the plural and/or singular forms of the noun.

[00060] As used herein, the term “dietary compound” includes any compound which is suitable for use in production of a food product for human or animal consumption.

[00061] As used herein, the term “food product” is used to refer to a broad range of foods and beverages, as well as to soft or liquid foods, which might alternatively be described as foods or beverages by a person skilled in the art.

[00062] The term an “effective amount” as used herein means an amount effective to cause epigenetic modification of a yeast in a starter culture or during a fermentation process, as measured by a histone modification assay or a gene expression assay.

[00063] In one embodiment the term “statistically significant” as used herein refers to the likelihood that a result or relationship is caused by something other than random chance. A result may be found to be statistically significant using statistical hypothesis testing as known and used in the art. Statistical hypothesis testing provides a "P-value" as known in the art, which represents the probability that the measured result is due to random chance alone. It is believed to be generally accepted in the art that levels of significance of 5% (0.05) or lower are considered to be statistically significant. [00064] The term “contacting” as used herein refers to both direct and indirect contact between the yeast starter culture and an epigenetic modifier. Indirect contact includes exposure of the yeast in its environment, including in a growth medium and a food product to be fermented, to an epigenetic modifier.

[00065] The term “exogenous” as used herein refers to a source of an epigenetic modifier that is outside the food product or growth medium. That is, the term exogenous refers to an epigenetic modifier that is introduced to the fermentation or starter culture from a source other than the food product or growth medium. The term is intended to exclude compounds that are otherwise endogenous to the food product, growth medium, or starter culture. The term is also intended to exclude compounds that may be produced in trace amounts by the yeast during fermentation. The present invention relates to alteration of the flavour and aroma profile of fermented food products.

[00066] A colony-forming unit (CFU) is a unit used to estimate the number of viable bacteria in a sample. Determining colony-forming units requires culturing the microbes and counts only viable cells, i.e., those able to multiply and form a visible colony.

Yeast

[00067] As used herein, the term “yeast” refers to any yeast useful in fermentation processes. In some embodiments the yeast is a yeast of the genus Saccharomyces. In some embodiments, the yeast is a Saccharomyces cerevisiae yeast.. In some embodiments the yeast is a non-cerevisiae Saccharomyces yeast which include, but are not limited to, S. uvarum, S. kudriavzevii, S. pastorianus, S. paradoxus, S. mikatae, S. bayanas, S. abulensis, and S. florentinus. In some embodiments the yeast is a fission yeast such as Schizosaccharomyces pombe.

[00068] As used herein, the term “yeast starter culture” or “wild type” yeast refers to a culture of yeast for use in fermentation of a food product which has not been contacted with an epigenetic modifier as defined herein. Yeast starter cultures may be commercially sourced yeasts such as Lalvin ECU 18™ (Lallemand Brewing, Canada). Lalvin ECU 18™ is a Saccharomyces cerevisae var cerevisiae (ex banyanus) yeast. Saccharomyces can be present in the starter culture at about IxlO ⁶ to about IxlO ¹⁴ , e.g., about IxlO ⁸ to about IxlO ¹² , about IxlO ¹⁰ to IxlO ¹² , or at least 1 x 10 ⁹ or at least IxlO ¹⁰ colony forming units (CFU)/g.

[00069] As used herein, the term “epimutant” refers to a yeast or a yeast starter culture which has been exposed to an epigenetic modifier as defined herein, under conditions which result in altered heritable gene expression by the yeast. The alteration results not from DNA sequence changes, but from an alteration (gain or loss) in DNA methylation or in histone acetylation. Similarly, the phrase “epimutation of the starter culture” refers to exposure of a yeast culture to an epigenetic modifier as defined herein, under conditions which result in altered gene expression, including upregulation or downregulation of certain genes. In some embodiments, the level of methylation or acetylation alteration in the yeast is statistically significant when compared to the level of methylation or acetylation in the yeast prior to alteration. The term “modified yeast” is used synonymously with “epimutant”, and the term “modified yeast starter culture” refers yeast starter culture comprising an epimutant/modified yeast.

Epigenetic modifier

[00070] The term “epigenetic modifier” as used herein refers to a compound which is an inhibitor or activator of HD AC (histone deacetylase), HAT (Histone acetyltransferase) and/or DNMT (DNA-methyl transferase). In some cases, an epigenetic modifier is a compound which will inhibit or activate both HD AC and DNMT, such as EGCG.

[00071] For fermentation purposes the epigenetic modifier should be acceptable for use in food processing, in other words it should be a dietary compound. In some embodiments, the epigenetic modifier is selected from a group of HD AC inhibitors including but not limited to benzoic acid, 4-phenylbutyric acid (4-PBA), sodium butyrate, quercetin, genistein, anacardic acid, curcumin, and epigallocatechin gallate (EGCG), diallyl disulfides, sulforaphane, and bis(4-hydroxybenzyl)sulfide. Other examples of HD AC inhibitors may be found in Akone et al, Front. Pharmacol., 13 August 2020 and in Zwergel et al., Curr Top Med Chem 2016;16(7):680-96. In some embodiments, the epigenetic modifier is a HD AC activator. Known HD AC activators include ITSA-1. In some embodiments, the epigenetic modifier is selected from a group of DNMT inhibitors including but not limited to EGCG, quercetin, resveratrol, sulforaphane, and Z-liguistilide. In some embodiments, the epigenetic modifier is a DNMT activator. As used herein, the term DNMT activator includes methyl donors, including but not limited to S-adenosyl methionine/S-adenynyl methionine, choline, folate, vitamin B12, methionine, and betaine. In some embodiments, the epigenetic modifier is a HAT inhibitor, for example anacardic acid. In some embodiments, the epigenetic modifier is a HAT activator, for example quercetin.

[00072] In some embodiments, the epigenetic modifier is a HD AC inhibitor which is a short-chain fatty acid such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, phenylacetic acid, phenylbutyric acid, valproic acid or a nutritionally acceptable salt thereof.

Epigenetic modification process

[00073] In one aspect, the invention relates to a process for epigenetic modification of yeast by contacting a yeast starter culture with an epigenetic modifier to produce modified yeast. In some embodiments, the contacting step comprises inoculating the yeast starter culture into, and growing the yeast starter culture in a growth medium comprising an epigenetic modifier. Inoculation may be carried out using known art techniques. Growth is typically under known art conditions, for example as discussed in Marin et al., J Vis Exp. 2018; (139): 58192.

[00074] In some embodiments of the invention, the growth medium is selected from any known art yeast growth mediums, including agars and broths, for example, as detailed in Marin et al. (above). In some embodiments, the growth medium is yeast peptone dextrose broth (YPD), yeast peptone dextrose agar, yeast nitrogen base with or without amino acids. In some embodiments the yeast growth medium is Difco™ YPD broth powder (Becton, Dickinson and Company (BD), USA) which comprises yeast extract, peptone, and dextrose in a weight ratio of 1:2:2, and can be made up by suspending in purified water at a ratio of 50g to IL. YPD agar contains a similar composition of yeast extract, peptone, dextrose and agar in a weight ratio of 1:2:2: 1.5, and can be prepared by suspending in purified water at a ratio of 65g to IL, heating with agitation, and boiling for 1 minute to completely dissolve the powder. The growth medium is typically autoclaved before use, for example at 121°C for 15 minutes. Typically, the growth medium will be sterilised prior to use.

[00075] In some embodiments growth is carried out in the growth medium in atmospheric conditions in a temperature range of from about 10 to about 40 °C, for example about 15 to about 40 °C, about 15 to about 38 °C, about 18 to about 38 °C, about 18 to about 35 °C, about 20 to about 35 °C, about 23 to about 35 °C, or about 25 to about 35 °C. In some embodiments growth is carried out at 30 to 33 °C, more preferably at 32 °C.

[00076] In some embodiments, an effective amount of the epigenetic modifier is present in the growth medium. An effective amount is commonly a concentration in the growth media of from about 10 pM to about 100 mM, for example from about 10 pM to about 50 mM, about 10 pM to about 40 mM, about 0.1 mM to about 9 mM, about 0.1 to about 8 mM, about 0.1 to about 7 mM, about 0.1 to about 6 mM, about 0.1 to about 5 mM, about 0.5 to about 18 mM, about 0.5 to about 16 mM, about 0.5 to about 15 mM, about 0.5 to about 14 mM, about 0.5 to about 12 mM, about 0.5 to about 10 mM, from about 1 to about 30 mM, about 1 to about 20 mM, about 1 to about 18 mM, about 1 to about 16 mM, about 1 to about 15 mM, about 1 to about 14 mM, about 1 to about 12 mM, or about 1 to about 10 mM. Reference herein to the unit |aM or mM in connection with a concentration means a concentration of pM.L ^-1 or mM.L ¹ respectively.

[00077] In some embodiments, an effective amount of the epigenetic modifier is present in the food product during fermentation, for example at a concentration in the food product of from about 10 pM to about 10 mM, for example from about 10 pM to about 8 mM, about 10 pM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 to about 5 mM, about 0.1 to about 4 mM, about 0.1 to about 3 mM, about 0.1 to about 2 mM, about 0.2 to about 2 mM, about 0.3 to about 1.8 mM, about 0.4 to about 1.8 mM, about 0.4 to about 1.6 mM, about 0.1 to about 1 mM, about 0.1 to about 1.5 mM, about 0.1 to about 1.3 mM, about 0.2 to about 1.3 mM, about 0.3 to about 1.3 mM, about 0.5 to about 1 mM, or about 0.5 to about 1.6 mM. [00078] In some embodiments, the epigenetic modifier is benzoic acid, and an effective amount of the epigenetic modifier is present in the growth medium at a concentration of about 10 pM to about 40 mM, for example from about 1 to about 30 mM, about 1 to about 20 mM, about 1 to about 18 mM, about 1 to about 16 mM, about 1 to about 15 mM, about 1 to about 14 mM, about 1 to about 12 m , about 1 to about 10 mM, 0.1 to about 9 mM, about 0.1 to about 8 mM, about 0.1 to about 7 mM, about 0.1 to about 6 mM, about 0.1 to about 5 mM, about 0.5 to about 18 mM, about 0.5 to about 16 mM, about 0.5 to about 15 mM, about 0.5 to about 14 mM, about 0.5 to about 12 mM, about 0.5 to about 10 mM. Benzoic acid is a lipophilic weak acid that occurs naturally in many fruits, vegetables, nuts, and even in cultured dairy products as a microbial metabolite ⁹ . It is commonly used as a preservative in many food products. As used herein, reference to benzoic acid includes benzoic acid salts. [00079] In some embodiments, the epigenetic modifier is sodium butyrate, and an effective amount of the epigenetic modifier is present in the growth medium at a concentration of about 10 pM to about 40 mM, for example from about 10 pM to about 20 mM, about 10 pM to about 10 mM, about 30 pM to about 40 mM, for example from about 30 pM to about 20 mM, about 30 pM to about 10 mM, about 50 pM to about 40 mM, for example from about 50 pM to about 20 mM, about 50 pM to about 10 mM, about 50 pM to about 5 mM, about 50 pM to about 3 mM, about 50 pM to about 1 mM, about 50 pM to about 0.5 mM. Sodium butyrate is the sodium salt of butyric acid and is present in foods such as butter. [00080] In some embodiments, the epigenetic modifier is quercetin, genistein, anacardic acid, curcumin, or EGCG, and an effective amount of the epigenetic modifier is present in the growth medium at a concentration from about 10 pM to about 40 mM, for example from about 0.1 to about 9 mM, about 0.1 to about 8 mM, about 0.1 to about 7 mM, about 0.1 to about 6 mM, about 0.1 to about 5 mM, about 0.5 to about 18 mM, about 0.5 to about 16 mM, about 0.5 to about 15 mM, about 0.5 to about 14 mM, about 0.5 to about 12 mM, about 0.5 to about 10 mM. EGCG is a phenolic antioxidant found in foods such as green and black tea. [00081] In some embodiments, yeast is maintained (grown) in the growth medium for a period sufficient for the epigenetic modifier to effect a change in the DNA methylation and/or histone acetylation pattern. Typically the growth period with the epigenetic modifier is from about 10 to 1000 hours, for example about 10 to 800 hours, about 10 to 600 hours, about 10 to 500 hours, about 10 to 400 hours, about 10 to 300 hours, about 10 to 200 hours, about 10 to 100 hours, about 50 to 1000 hours, about 50 to 800 hours, about 50 to 600 hours, about 50 to 500 hours, about 50 to 400 hours, about 50 to 300 hours, about 50 to 200 hours, about 50 to 100 hours, about 100 to 1000 hours, about 100 to 800 hours, about 100 to 600 hours, about 100 to 500 hours, about 100 to 400 hours, about 100 to 300 hours, about 100 to 200 hours. In some embodiments the yeast is maintained (grown) in the growth medium with the epigenetic modifier for a period of about 500 hours.

[00082] The modified yeast can be isolated from the growth media by clarification or centrifugation using known art techniques and further processed if required for example, by drying to enhance shelf-life or yeast viability. Further processing can for example include a step of drying, spray-drying, or freeze-drying the modified yeast. In drying processes, lyoprotectants and/or cryoprotectants may be used. The terms “lyoprotectant” and “cryoprotectant” refer to compositions that protect active ingredients, in this case, yeasts, including S. cerevisiae strains. Lyoprotectants protect during drying, while cryoprotectants protect during freezing. The same composition can have both functions, and unless otherwise specified, the terms are used interchangeably herein. Common cryoprotectants include dimethyl sulfoxide; polyhydroxy compounds such as glycerol, mannitol, sorbitol, inositol, thiol and polyethylene glycol; sugars such as sucrose; glucose, lactose and trehalose: amino acids such as proline and tryptophan, sodium glutamate, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine; polymers such as polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, and polyethyleneimine; salts such as calcium lactate, sodium sulfate, sodium glutamate, and potassium chloride: Tween 80 and other surfactants. Cryoprotectants can be used according to known art methods, such as those discussed in Cabrera et al., Genes 2020, 11(8), 835.

[00083] In some embodiments, the yeast may be further processed into forms suitable for storage or use in fermentation processes. In some embodiments the dried yeast is milled and/or pressed into powders, tablets, capsules or granules for use in fermentation. In some embodiments the dried yeast may be combined with additives including flowing agents, glidants, binding agents, bulking agents, anti-caking agents, lubricants, fillers, sweetners, excipients including pharmaceutically acceptable excipients, colourants, flavouring agents but not limited thereto.

[00084] Thus, in another aspect, the invention provides a composition comprising a modified yeast of the present invention, in other words, a composition comprising a yeast which has been modified by contact with an effective amount of an epigenetic modifier which is selected from: an HD AC (histone deacetylase) inhibitor; a DNMT (DNA-methyl transferase) inhibitor; a HAT (histone acetyltransferase) inhibitor; an HD AC activator; a DNMT activator; and a HAT activator. The composition can comprise, or be prepared from a modified yeast starter culture of the present invention. In some embodiments the yeast in the composition is in an inactive form, such as a dried or freeze-dried form. In some embodiments, the composition may further comprise a lyoprotectant or a cryoprotectant. In some embodiments the composition can comprise additives as set out above. In some embodiments, the composition is in a powder, tablet, capsule or granular form. In some embodiments, the yeast in the composition is in an active form, for example a live starter culture.

[00085] Where the modified yeast has been processed into an inactive form suitable for storage as discussed above, it can be revived or activated for fermentation by known art techniques, including hydrating and feeding the modified yeast.

Characterizing the gene expression due to histone modification

[00086] After contacting a yeast starter culture with an epigenetic modifier, the RNA and/or histone acetylation expression pattern of the yeast starter culture can be characterized. The characterization can be achieved by a number of methods known in the art, including those referred to herein. Briefly, isolation of RNA can be carried out by known methods including acid phenol extraction, glass fibre filter purification, and single-step reagents. Purity of the RNA can be measured by spectrophotometric techniques. RNA expression can be measured using a multiplex assay, NGS, PCR, qPCR or RNA sequencing. For example, RNA expression can be measured using the proprietary nCounter™ assay (NanoString Technologies, USA). Colorimetric multiplex assays can be used to determine histone H3 or H4 modification. Significant differences in RNA expression or histone H3 or H4 modification can be identified using the statistical methods defined herein, including use of P-values for statistical hypothesis testing using a confidence level of 95%, and use of analysis of variance (ANOVA), for example with a generalised linear model and post-hoc Tukey’s mean comparison test. Optionally, statistical software packages such as Minitab 20 (Minitab, LLC, U.S.A) or XLSTAT Statistical Software 2016 (Addinsoft, U.S.A.) nSolver 4.0 s can be used. Compared to the unmodified yeast, the modified yeast has a statistically significant different methylation pattern, acetylation pattern, or both.

Confirming the DNA sequence has not been modified

[00087] Optionally, it can be confirmed that the DNA sequence itself has not been modified by carrying out DNA sequencing of the epigenetically modified yeast. This can be carried out for example by chromatin immunoprecipitation sequencing (ChlP-Seq).

Changes in food products fermented by modified yeast

[00088] In some embodiments, a yeast is contacted with an epigenetic modifier in a starter culture, and epimutation of the starter culture increases the amount of one or more compounds in a food product fermented using the modified yeast, as compared to a food product fermented using a wild type yeast. In some embodiments the compounds are selected from: fructose; glycerol.

[00089] When the food product is a wine, the yeast can be contacted with the epigenetic modifier before and/or during fermentation of fruit juice. When the food product is a beer, the yeast can be contacted with the epigenetic modifier before and/or during fermentation of wort.

[00090] In some embodiments, a yeast is contacted with an epigenetic modifier during fermentation of a food product (e.g. wine or beer), and epimutation of the yeast during fermentation increases the amount of one or more compounds in the food product. In some embodiments, the compounds are residual sugars. For example, glucose or fructose.

[00091] In some embodiments, a yeast is contacted with an epigenetic modifier during fermentation of a food product (e.g. wine or beer), and epimutation of the yeast during fermentation decreases the amount of one or more compounds in the food product. In some embodiments, the compounds are selected from: glycerol; ethanol.

[00092] In some embodiments epigenetic modification of the yeast alters the expression of one or more genes responsible for production of flavours, aromas, and ethanol in the yeast. [00093] In some embodiments, genes involved in flavour/aroma production in yeast include ARO4 and TYR1, EEB1, and BATE

[00094] In some embodiments epigenetic modification of the yeast results in an increase in expression of the ARO4 and/or TYR1 genes, resulting in increased production of phenylethyl alcohol providing rosy aroma to a wine fermented by the yeast. In some embodiments epigenetic modification of the yeast results in a phenotype with increased production of czs-3-hexen-l-ol during fermentation, providing a kiwifruit and leaves aroma to a wine fermented by the yeast. In some embodiments epigenetic modification of the yeast results in a phenotype with increased production of ethyl decanoate during fermentation. In some embodiments epigenetic modification of the yeast results in a phenotype with increased production of ethyl lactate during fermentation, providing a strawberry aroma to a wine fermented by the yeast.

[00095] In some embodiments the epigenetic modification is one or more of: upregulation of a gene of a yeast such as Saccharomyces responsible for the over production of phenylethyl alcohol (ARO4 and TYR1); upregulation of a gene responsible for mediumchain fatty acid ethyl ester synthase/esterase important ester synthesis (EEB 1); upregulation of branched-chain-amino-acid transaminase (BAT1); downregulation of Histone deacetylase (RPD3) gene; upregulation of histone acetyltransferase (GCN5). In other embodiments the epigenetic modification upregulates or downregulates a different gene responsible for aroma. Kurat et al. ¹¹ has previously shown that upregulation of GCN5 gene led to histone acetylation and transcription activation.

[00096] In some embodiments, a yeast is contacted with an epigenetic modifier either in a starter culture, or during fermentation, or both, to produce wine. Epimutation of the starter culture or yeast during fermentation, or both, alters the amount of one or more aromatic compounds in the wine, selected from: ethyl acetate, ethyl isobutyrate, ethyl butanoate, ethyl isovalerate, isoamyl acetate, ethyl pentanoate, isoamyl alcohol, ethyl hexanoate, ethyl lactate, hexanol, Zrans-3-hexen-l-ol, cA-3-hexen-l-ol, ethyl octanoate, benzaldehyde, ethyl decanoate, phenylethyl alcohol, and p-tolualdehyde. In some embodiments, the wine has a statistically significant increase in the content of one or more of: phenylethyl alcohol, ethyl lactate, cA-3-hexen-l-ol and ethyl decanoate. [00097] In some embodiments, a yeast is contacted with an epigenetic modifier either in a starter culture, or during fermentation, or both, to produce beer. Epimutation of the starter culture or yeast during fermentation, or both, alters the amount of one or more aromatic compounds in the beer, selected from: isobutanol, phenylethyl alcohol, hexanol, isoamyl alcohol, 1-heptanol, benzaldehyde, and cis-3-hexenol. In some embodiments, the beer has an improved organoleptic attribute as compared to a beer prepared by fermentation of the wort using an unmodified yeast. The improved organoleptic attribute can be one or more of: sweet, sour, dairy, smooth, and creamy, and the improvement in the attribute can be identified using a penalty analysis method. For example, each organoleptic attribute for a beer can be rated by a panel of participants as either too little, too much, or just about right (JAR scale). Statistical penalty analysis methods can then be applied to the JAR results.

Fermentation process

[00098] Yeasts are useful in fermentation processes for producing a wide range of food products including beverages. Examples of fermented products include, but are not limited to, wine, beer, cider, milk, yoghurt, kefir, kimchi, tempeh sauerkraut, miso, and sourdough starter culture. Examples of products commonly fermented include grape juice, apple juice, tea, grains (e.g. barley, wheat, oats, malt, rice, corn), soya beans, and cabbage, but are not limited thereto.

[00099] In one aspect, the invention relates to a process for fermenting a food product, the process comprising fermenting the food product using an epigenetic ally modified yeast of the invention.

[000100] In one aspect, the invention relates to a process for fermenting a food product, the process comprising fermenting the food product using a starter yeast strain, and contacting the starter yeast strain with an epigenetic modifier prior to, during, or both prior to and during, the fermentation process.

[000101] In some embodiments of the invention, the yeast is contacted with the epigenetic modifier during a step of fermentation of a food product.

[000102] In various embodiments, the process comprises steps of

(a) adding the yeast to the food product:

(b) adding the epigenetic modifier to the food product; and

(c) fermenting the food product. [000103] In some embodiments of the invention, step (b) is carried out prior to step (a). In some embodiments step (a) is carried out prior to step (b).

[000104] In some embodiments, an effective amount of the epigenetic modifier is added to the food product at a concentration from about 0.1 to about 20 mM , for example from about 0.1 to about 18 mM, about 0.1 to about 16 mM, about 0.1 to about 15 mM, about 0.1 to about 14 mM, about 0.1 to about 12 m , about 0.1 to about 10 mM, about 0.1 to about 9 mM, about 0.1 to about 8 mM, about 0.1 to about 7 mM, about 0.1 to about 6 mM, about 0.1 to about 5 mM, about 0.1 to about 4 mM, about 0.1 to about 3 mM, about 0.1 to about 2 mM, about 0.1 to about 1 mM, about 0.5 to about 18 mM, about 0.5 to about 16 mM, about 0.5 to about 15 mM, about 0.5 to about 14 mM, about 0.5 to about 12 mM, about 0.5 to about 10 mM, about 0.5 to about 9 mM, about 0.5 to about 8 mM, about 0.5 to about 7 mM, about 0.5 to about 6 mM, about 0.5 to about 6 mM, about 0.5 to about 4 mM, about 0.5 to about 3 mM, about 0.5 to about 2 mM, about 0.5 to about 1 mM, about 0.8 to about 15 mM, about 0.8 to about 14 mM, about 0.8 to about 12 mM, about 0.8 to about 10 mM, about 0.8 to about 9 mM, about 0.8 to about 8 mM, about 0.8 to about 7 mM, about 0.8 to about 6 mM, about 0.8 to about 6 mM, about 0.8 to about 4 mM, about 0.8 to about 3 mM, about 0.8 to about 2 mM, about 0.8 to about 1.6 mM.

[000105] Yeast can be used for fermentation of various food products across a wide range of temperatures. The time period for fermentation can also vary widely and depends on the food product being fermented. Typically, the time period and temperature conditions for a fermentation in which a yeast is contacted with the epigenetic modifier either before or during a step of fermentation of a food product by a process of the invention will be understood by the person skilled in the art as being similar to the conditions for a fermentation of that food product by a prior art method.

Examples

Materials and methods

S. cerevisiae starter preparation

[000106] Epigenetic modification of yeast starter cultures was induced by growth of the starter culture in the presence of the epigenetic modifier. YPD broth was prepared either from a commercial YPD broth powder (Difco™, BD.com USA) or prepared to provide the same components of yeast extract, peptone, and dextrose in a weight ratio of 1:2:2, with the mixture then being suspended in purified water at a ratio of 50g to IL, then autoclaved before the strains were grown. Various strains of S. cerevisiae starters were prepared from the strain Lalvin ECU 18™ (Lallemand Brewing, Canada) as follows.

Wild type: A non-stressed/negative control strain, grown for 500 hours in regular yeast peptone dextrose (YPD) broth (BD, USA), 20 hrs/sub-culture;

Epimutant 1: A strain grown for 500 hours in YPD broth containing 10 mM benzoic acid; and

Epimutant 2: A strain grown for 1 Generation in YPD, followed by 500 hours growth in YPD broth containing 10 mM benzoic acid, and 20 hours growth (1 generation) in regular YPD broth, 20 hrs/sub-culture.

HDAC inhibition assay

[000107] The measurement of HDAC inhibition capacity of epigenetic modifiers was achieved by a fluorometric HDAC assay kit (Active Motif, USA), following manufacturer’s instructions. The kit provides HeLa nuclear extract as the HDAC source, with an input volume of 5 pL. The compounds, including the positive control TSA, were added at the volume of 10 pL. The volume of HDAC assay buffer was adjusted to reach a total volume of 50 pL in each well. Fluorescence was measured using FLUOstar Omega (BMG Labtech, Germany) microplate reader with excitation wavelength at 360 nm and emission wavelength at 460 nm.

RNA isolation

[000108] Total RNA from S. cerevisiae was isolated using RiboPure™ RNA Purification Kit (Invitrogen, USA). RNA purity was measured by DeNovix DS- 11 Spectrophotometer (DeNovix Inc., USA).

RNA expression analysis

[000109] The RNA expression was measured using nCounter technology (NanoString Technologies, USA). The nCounter assay was carried out on 12 samples/24 genes (including 5 housekeeping genes), the RNA input amount was 300 ng for each sample. Expression counts were normalised and analysed using the nSolver 4.0 software.

Histone H3 modification multiplex assay [000110] The 21 histone H3 modification patterns of 5 mM benzoic acid treated S. cerevisiae compared to untreated wild type strain were measured using EpiQuik™ Histone H3 modification multiplex assay kit (Colorimetric, EpiGentek, USA) following manufacturer’s instructions. The kit measures relative change in 21 histone H3 modification patterns using antibodies specific to respective histone H3 modifications: fifteen for methylation (me), four for acetylation (ac), and two for phosphorylation (p). Absorbance was measured using FLUOstar Omega microplate reader at 450 nm with a reference wavelength of 655 nm.

DAPI staining

[000111] S. cerevisiae was cultured overnight to an ODeoo = 0.8 - 2.0. Yeast culture was treated with 100% EtOH at a 1:2 ratio for 45 min at room temperature. Then the mixture was centrifuged at 2500 rpm for 1 min, 1 mL IxPBS was used to wash the cells, followed by centrifugation at 2500 rpm for 1 min. The pellet was resuspended in 200 pL of lxPBS/l:2000 dilution DAPI mixture, and was observed under fluorescence microscope (Nikon Eclipse 50i, USA) after 45 min.

Yeast morphology by generation and temperature changes

[000112] S. cerevisiae starter cultures were transferred from YPD broth onto YPD agar plates before starting the experiments, target isolates were serially diluted to ODeoo = 0.1, 5 pL strain solution was spotted onto YPD agar plates after an additional lOx dilution was applied, IxPBS was used for dilution. The growing temperature was at 32 °C for the generation test.

GC-MS analysis of wine and beer samples

[000113] The alcohol and ester aroma compounds analysis was conducted using HS- SPME and Shimadzu (Japan) QP-2010 GC-MS.

Chemical analysis of wine samples

[000114] Ethanol content was analysed by GC-FID which was carried out on a Shimadzu GC-2010 gas chromatograph-flame ionization detector equipped with an AOC-20i autoinjector and AOC-20s autosampler. The chromatography was performed using an 1909 IN- 133 HP-Innowax GC column, serial number US 151697611 (Polyethylene Glycol- Agilent Technologies, USA). Residual sugars including glucose and fructose were measured using Vintessential (Australia) enzymatic test kit, and glycerol content was measured using Megazyme (Ireland) glycerol assay kit.

Statistical analysis

[000115] Data were analysed using analysis of variance (ANOVA) with a generalised linear model, followed by post-hoc Tukey’s mean comparison test, by using Minitab 20 (Minitab, LLC, USA). Principal Component Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC) was used to analyse and categorize the relationship between samples fermented under different conditions. The dissimilarity of them was analysed based on the Euclidean distance and the Ward’s method. PCA and AHC were analysed using XLSTAT Statistical Software 2016 (Addinsoft, USA). A confidence level of 95% was applied to the statistical analysis, and data were presented as mean ± SD.

Wine fermentation

[000116] Three starters, namely wild type, epimutant 1 and epimutant 2 were inoculated at 10 ⁷ CFU/mL to 400 mL of Pinot Noir grape juice (Lincoln University Farm) contained in a 500 mL Schott bottle for fermentation at 32 °C, in triplicate. Brix values were monitored every two days as an indicator of the fermentation progress. Wine samples were collected and stored at 4 °C for downstream analysis upon completion of fermentation.

Beer fermentation

[000117] The wort used for beer fermentation was provided by Three Boys Brewery (Christchurch, New Zealand), which were filled into 2 L Schott bottles (total of 15 Schott bottles) and each was pitched with English style Ale yeast (Saccharomyces cerevisiae, Lallemand Brewing, Canada) at IxlO ⁶ CFU/mL. The fermentation process was monitored for 13 days and the vessels were kept at 20 °C in a water bath.

Organoleptic attributes of beer samples (Sensory Session)

[000118] Nine participants (n = 9) were recruited for the sensory session, during which the participants completed a questionnaire while tasting the beer samples. Participants were firstly asked to rate their overall liking of each beer sample based on a 9-point hedonic scale, which represents 9 hedonic responses using number 1 (dislike extremely) to 9 (like extremely) with a neutral response as 5 (neither like nor dislike). In the second part of the questionnaire, sweetness, sourness, dairy flavour, smoothness and creaminess were evaluated by just-about-right-scale (JAR) in terms of both intensity and acceptability (1 = too little, 2 = just about right, 3 = too much). Penalty analysis was applied to the JAR data in order to determine how much the overall liking and acceptance of beer samples were influenced by their attributes.

Example 1 - HDAC inhibition capacity of epigenetic modifiers

[000119] Compounds were investigated regarding their HDAC inhibition capacity. The candidate epigenetic modifiers benzoic acid, 4-phenylbutyric acid (4-PBA), sodium butyrate, quercetin, genistein, anacardic acid, curcumin, and epigallocatechin gallate (EGCG) were selected from literature for their known HDAC inhibition properties. Without wishing to be bound by theory, the hydrophobic properties of benzoic acid could perturb membrane dynamics in yeast and could therefore induce stress in the yeast at a relatively low concentration ¹⁰ .

[000120] Results are shown in Figure 1. Untreated HeLa nuclear extracts were used as the negative control. In addition, glucose and 5-aza-2’-deoxycytidine were also included as negative controls for the property of HDAC inhibition. The well-known HDAC inhibitor Trichostatin A (TSA) was used as the positive control. Relevant half-maximal inhibitory concentration (IC50) values were used as references for determining the testing concentrations of the compounds, except benzoic acid since it has not been previously applied as an inhibitor.

[000121] Following the results of the HDAC inhibition assay, benzoic acid was selected for further investigation due to its stress inducing capacity, solubility in aqueous system and cost-effectiveness. A 10 mM concentration was used since in addition to its HDAC inhibition properties, benzoic acid inhibits the growth of the yeast at higher concentrations.

Example 2 - epigenetic modifications

Example 2A - histone H3 modification patterns - 5mM benzoic acid

[000122] Variation in 21 histone H3 modifications was investigated by comparing modifications exhibited by a S. cerevisiae strain grown for 500 hours in YPD broth containing 5 mM benzoic acid, with those of untreated wild type strain.

[000123] Figure 2 shows the relative change in 21 histone H3 modification patterns in the strain grown in the presence of 5 mM benzoic acid for 500 hours compared to untreated wild type strain. Both stimulation and inhibition in histone marks were seen with exposure to 5 mM benzoic acid. H3K4me2, H3K9me3, H3K27me2, H3K9ac, H3K18ac and H3serl0p were stimulated to more than fourfold higher in the 5 mM benzoic acid strain compared to the wild type strain, whereas some methylation patterns in the 5 mM benzoic acid strain including H3K4me3, H3K9me2 and H3K27me3 were halved compared to the wild type strain, confirming that benzoic acid has impacts on several histone marks.

Example 2B - Gene expression in S. cerevisiae

[000124] Table 1 details the genes the expression of which was assayed in various yeast strains including wild type, epimutants 1, 2 and 3, and further strains as follows:

A strain grown overnight in YPD broth containing 0.9% NaCl; and

A strain grown for 500 hours in YPD broth containing 80 nM TSA; and

A strain grown overnight in YPD broth containing 80 nM TSA;

A strain grown overnight in YPD broth containing 500 pm EGCG; and A strain grown overnight in YPD broth containing 80 nm TSA.

[000125] Figure 3 shows the gene expression levels presented as a heat map graph after Z- score transformation, ranging from -3 to 3, blue (down-regulation) to orange (upregulation). Genes responsible for the over production of phenylethyl alcohol (ARO4 and TYR1), genes responsible for medium-chain fatty acid ethyl ester synthase/esterase important ester synthesis (EEB 1), branched-chain-amino-acid transaminase important for biosynthesis of higher alcohols that affects wine aroma and flavour (BAT1) were all upregulated. There was no significant change in expression levels of another ester synthase gene (EHT1) between samples. Several genes responsible for stress tolerance and cell cycle were also analysed. Histone deacetylase (RPD3) gene was downregulated and histone acetyltransferase (GCN5) was upregulated in both the 5 mM benzoic acid strain and epimutant 1, clearly suggesting the role of benzoic acid as an HD AC inhibitor.

Example 2C - DAPI staining

[000126] The protocol for DAPI staining is given in the Materials and Methods section above. Results are shown in Table 2, repeated as cultures A, B and C for each of the wild type and epimutant 1.

[000127] DAPI (4’, 6-diamidino-2-phenylindole) staining was applied to wild type and epimutant 1 (500 hrs growth in YPD broth containing 10 mM benzoic acid, 20 hrs/sub- culture) to visualise their nuclear DNA in terms of any size changes (expansion) which might have resulted from benzoic acid treatment. The corrected total cell fluorescence (CTCF) was calculated based on the integrated density in the nuclear region. The mean comparison results indicated that there is a significant difference between two samples (p < 0.05), which suggests an expansion to the nuclear DNA in S. cerevisiae when they were exposed to benzoic acid potentially due to histone modifications resulted in relaxed genome state. This confirms that cells treated with HDACi lead to relaxed genome state and more fluorescence.

Table 2: DAPI staining and corrected total cell fluorescence (CTCF) of 10 mM benzoic acid-directed S. cerevisiae compared to untreated wild type ^{a b} indicate significant difference based on Tukey pairwise mean comparison results, p < 0.05.

Example 3 - Yeast morphology in relation to epigenetic alteration and temperature changes

[000128] The morphology of S. cerevisiae strains including benzoic acid stressed S. cerevisiae was recorded at different growth points in order to depict any phenotypic differences compared to the untreated wild type strain. As shown in Figure 4, epimutant 1 was generally more robust when experiencing certain external stresses. Its robustness and adaptability faded soon after the stress was eliminated from the environment, demonstrating the transient nature of epigenetic changes. This data is line with the Nanostring assay of example 2B and Fig. 3, where the gene expression pattern of S. cerevisiae epimutant 1 showed very similar expression patterns to the 5 mM benzoic acid strain. However, epimutant 2 exhibited significantly different gene expression patterns when compared within the benzoic acid treatment group. Epimutant 2 gene expression patterns were more similar to wild type than other strains. This suggests that the alteration of gene expression caused by dietary epigenetic compounds is transient and tends to fade once the compound is eliminated from the environment.

[000129] Figures 4 and 5 show the schematic and results for a generation test for wild type and epimutant 1 (500 hours growth), as well as for wild type grown for 600 hours and a corresponding modified epimutant 1 (grown for 600 hours). Results are shown at 18, 24, 26, and 48 hours after transfer onto the YPD agar plate. As shown in Figure 5, the generation test suggested that S. cerevisiae stressed with 10 mM benzoic acid epimutant 1 was more robust compared to a wild type strain when facing the same stress on YPD agar. However, this robustness and better adaption faded out once the epimutant 1 was put back to regular YPD broth, regardless of the number of generations without stress.

[000130] Figures 6 and 7 show the schematic and morphological differences of the three S. cerevisiae starter strains wild type, epimutant 1 and epimutant 2 due to temperature changes. All three types of strains seem to grow better at 32 °C, and their colonies tend to become smaller when the benzoic acid concentration increased. In accordance with the generation test, the epimutant 2 lost its robustness and grew in the same pattern as the wild type strain, regardless of temperature changes. Interestingly, the robustness of the epimutant 1 was largely inhibited by temperature increase, and it became more obvious when benzoic acid concentration increased as well.

[000131] Overall, in the present invention the robustness of S. cerevisiae epimutants and their adaption to stressed environment were obviously improved by continuously treating the strain with the stress. However, the epigenetic changes were not heritable once the stress was removed from the environment.

Example 4 - Wine fermentation and changes in wine characteristics as a result of epigenetic alteration

[000132] Two groups of wine fermentation experiments were carried out. Pinot noir grape juice samples were fermented using different starter S. cerevisiae wild type, epimutant 1 and epimutant 2, and in some cases with different benzoic acid concentrations within the fermentation system, as follows. In Group 1, three grape juice samples were fermented using three types of starters: wild type, epimutant 1 and epimutant 2. These resulted in three wine categories which were distinct from each other in terms of their aromatic profiles (Figure 9(A) and 9(B)). In Group 2, six grape juice samples were fermented using wild type and epimutant 1. To test the effect of addition of benzoic acid, four of the fermentations contained benzoic acid at a concentration of 0.8 mM and 1.6 mM respectively.

[000133] The results of the GC-MS data analysis regarding alcohol and ester aroma compounds in the wine samples are shown in Table 3. Figure 8 shows the content of eleven ester and higher alcohol compounds representing potential indication of wine aroma alterations resulting from epimutation of the starter culture. The listed compounds confer pleasant aromatic profiles, including fruity aromas, as well as floral aroma.

Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of wine samples

[000134] Figure 9(A) shows a principal component analysis (PCA) biplot illustrating the relationship between Group 1 wine samples which had been fermented under different conditions and the variance on their alcohol and ester aroma compounds quantitatively generated by GC-MS analysis. Figure 10(A) shows Group 1 wine samples grouped using agglomerative hierarchical clustering (AHC) according to their dissimilarity levels.

[000135] The corresponding PCA and AHC analyses in respect of the wine samples of Group 2 are shown in Figures 9(B) and 10(B).

Aromatic compounds

[000136] The wines produced by wild type strain, epimutant 1 and epimutant 2 possessed distinct aromatic compounds p-tolualdehyde, cis-3-hexen-l-ol and ethyl decanoate respectively (p < 0.05). For wine samples fermented in Group 2 (Figure 9(B) and 10(B)), as expected, the PCA shows that all samples fermented with addition of benzoic acid were positively correlated with benzoic acid, and tended to negatively correlate with other aroma and ester compounds, and they were grouped within the same class. The two samples fermented using wild type and epimutant 1 were classified separately and their aromatic correlation was same as the pattern observed for the wine samples fermented in Group 1. Wine fermented by wild type strain was positively correlated with p-tolualdehyde, while wine fermented by epimutant 1 had significantly higher cA-3-hcxcn-l -ol content (p < 0.05). Overall, there is significant difference regarding the aromatic profiles between wine samples fermented by wild type strain and epimutant 1. Epimutant 1 significantly altered five compounds. Phenylethyl alcohol, known to give rosy like aromas and used in foods and perfumes, and ethyl lactate, strawberry aroma, was significantly higher. Furthermore, wines fermented by wild type strain, epimutant 1 and epimutant 2 possessed distinct aromatic compounds p-tolualdehyde, cA-3-hexen-l-ol and ethyl decanoate, respectively. These compounds were positively correlated with relevant wine samples (p < 0.05). p-Tolualdehyde in wild type wine sample would confer a cherry and fruity organoleptic perception, whereas cA-3-hexen-l-ol contained in epigenetically altered wine sample (epimutant 1) would have green kiwifruit and leaves aroma ¹² . Since cA-3-hexen-l-ol is an important aroma compound in many white wines, it can confer a complex aromatic profile on the altered wine produced by epimutant 1, by adding partial aromatic features of white wine. [000137] Figure 8 shows chemical composition changes due to epigenetic alterations in wine samples fermented in Group 1 and Group 2 under different conditions. Related data is tabulated in Table 3. Results were presented as mean and standard deviation (SD); Results with different superscripts within the same column indicate significant differences (Tukey’s HSD test, p < 0.05 ^{a d} different letters above the bars of each chemical composition indicate significant difference between treatments (p < 0.05).

Residual sugars, glycerol and ethanol

[000138] In addition to aromatic profiles of the wine samples, Figures 11 and 12 show the chemical composition changes in the wine due to epigenetic alterations. Generally, addition of benzoic acid during the wine fermentation process increased the residual sugar content, particularly at the higher concentration of 1.6 mM benzoic acid. In contrast, both glycerol (p

< 0.05) and ethanol (p > 0.05) content tended to decrease in Group 2 wine samples fermented with benzoic acid addition. With regard to wine samples fermented without benzoic acid treatment, the results were consistent for samples from both Group 1 and Group 2, where epimutant strains tended to increase their fructose (p < 0.05 for Group 1 samples) and glycerol (p < 0.05 for Group 2 samples) content, while not significantly changing the ethanol content (p > 0.05).

Example 5 - Beer fermentation and changes in beer characteristics as a result of epigenetic alteration

[000139] For this example, various strains of Saccharomyces cerevisiae starters were prepared from a commercial strain of English style Ale yeast (LalBrew London™, Lallemand Brewing, Canada) as follows

Wild type 100: A non-stressed/negative control strain, grown for 100 hours in regular yeast peptone dextrose (YPD) broth (BD, USA), 20 hrs/sub-culture;

Epimutant 3: A strain grown for 100 hours in YPD broth containing 5 mM sodium butyrate, 20 hrs/sub-culture; and

Epimutant 4: A strain grown for 100 hours in YPD broth containing 5 mM sodium butyrate, followed by 20 hours growth (1 generation) in regular YPD broth, 20 hrs/sub- culture.

[000140] Five wort samples were fermented using the three types of S. cerevisiae starters as outlined above. The wild type 100, epimutant 3 and epimutant 4 were used in the first three fermentations (WT100 - without SB, E3 - without SB, E4 - without SB, in Figure 13(A)). To test the effect of addition of sodium butyrate during the fermentation process, two additional fermentations used the wild type 100 and epimutant 3 starters, and sodium butyrate addition at a concentration of 500 pM (WT100 - 0.5 mM SB, E3 - 0.5 mM SB, in Figure 13(A)).

Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of beer samples

[000141] Figure 13(A) shows a principal component analysis (PCA) biplot illustrating the relationship between the beer samples which had been fermented under different conditions and the variance on their alcohol and ester aroma compounds quantitatively generated by GC-MS analysis. Figure 13(B) shows beer samples grouped using agglomerative hierarchical clustering (AHC) according to their dissimilarity levels.

[000142] As shown in Figure 13(A), most of the higher alcohol compounds were positively correlated with class 1, namely wild type S. cerevisiae fermented without sodium butyrate addition and Epimutant 3 fermented with addition of 0.5 mM sodium butyrate. The beer samples fermented by wild type S. cerevisiae with 0.5 mM sodium butyrate resulted in a separate class, which was negatively correlated with cA-3-hexenol. Class 3 which was composed of Epimutants 3 and 4, both fermented without sodium butyrate addition, was negatively correlated with the compound isobutanol. Generally, most of the higher alcohol compounds were positively correlated with class 1 samples with slightly different aromatic profiles respectively, indicating increased production in class 1 samples. Beer fermented using Epimutant 3 with sodium butyrate addition during fermentation showed a positive correlation with phenylethyl alcohol, in contrast to beer fermented using wild type S. cerevisiae without sodium butyrate addition, which was closely associated with benzaldehyde. As discussed above, phenylethyl alcohol provides a rosy aroma to foods.

[000143] As shown in Figures 14-18, beers fermented by Epimutants 3 and 4, both without sodium butyrate addition during the fermentation process (E3 - without SB and E4 - without SB; class 3) had the most selections for JAR (just about right) among all five beer samples, which resulted in good penalty analysis profiles by the sensory panellists. A good penalty analysis profile indicates no improvements are needed based on the five attributes (sweet, sour, dairy, smooth, and creamy). Beers fermented by wild type 100 with and without sodium butyrate addition (WT 100 - without SB and WT 100 - 0.5 mM SB), and the beer fermented by Epimutant 3 with sodium butyrate addition (E3 - 0.5 mM SB), were not sour enough to cater to the sensory panellists’ preference. While dairy flavour was generally just about right for the E3 - 0.5 mM SB beer sample, the other attributes sweetness, creaminess and smoothness need to be improved for this sample.

[000144] It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention as set out in the appended claims.

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