Method for producing low alcohol or reduced alcohol beverages

Abstract

The present invention relates to methods for producing a low alcohol or a reduced alcohol product from a base alcohol production biomass and products produced therefrom. The present invention also relates to enzyme compositions and strains, methods and uses thereof, and products produced therefrom.

Description

[0001] METHOD FOR PRODUCING LOW ALCOHOL OR REDUCED ALCOHOL BEVERAGES FIELD OF THE INVENTION

[0002] The present invention relates to methods for producing a low alcohol or a reduced alcohol product from a base alcohol production biomass and products produced therefrom. The present invention also relates to enzyme compositions and strains, methods and uses thereof, and products produced therefrom.

[0003] BACKGROUND OF THE INVENTION

[0004] Several methods have been explored over the last 30 years for manufacturing low and no alcohol wine with various degrees of success (Pickering, 2000). The main methods can be broadly categorised into pre-fermentation methods for reducing fermentable sugar prior to wine fermentation and post-fermentation methods for alcohol removal post wine fermentation (Liguori et al., 2018; Schmidtke et al., 2012). Some of the pre-fermentation approaches include using unripe grape with low sugar content, juice dilution with water where it is allowed (Schelezki et al., 2020), enzymatic reduction of glucose in the juice using glucose oxidase (Pickering et al., 1999a, 1999b, 1999c) or a combination of glucose oxidase with catalase (Roecker et al., 2016a), nanofiltration membrane to remove fermentable sugar from the juice (EP1459636; Salgado et al., 2015) and fermentation using low alcohol yeast such as Pichia stipitis or Candida tropicalis under aerobic condition to convert the fermentable sugar into yeast biomass resulting in a reduced alcohol product (DE3939064; Roecker et al., 2016b). Post-fermentation methods include evaporation, distillation, freeze-concentration, solvent extraction and membrane filtration techniques such as reverse osmosis and dialysis for alcohol removal post wine fermentation (Liguori et al., 2018; Pickering, 2001; Schmidtke et al., 2012). Currently, the main methods that are used commercially for manufacture of no and low alcohol wines are advanced vacuum distillation techniques such as spinning cone column (SCC) and reverse osmosis membrane technology (Pickering, 2000). These methods are effective for removal of alcohol from wine and other alcoholic beverages. However, volatile aroma and flavour compounds are lost during the alcohol removal process resulting in inferior quality compared to the full alcohol versions of the same product (Pickering, 2001). The processes are also costly.

[0005] Accordingly, there remains a need for improved methods for producing low alcohol and reduced alcohol products such as wine. SUMMARY OF THE INVENTION

[0006] The present inventors have developed methods, enzyme compositions and strains for producing a low alcohol or a reduced alcohol product from a base alcohol production biomass. In some embodiments, the product is wine.

[0007] In an aspect, the present invention provides a method of producing a low alcohol or a reduced alcohol product from a base alcohol production biomass. In an aspect, the present invention provides a method of producing a low alcohol or a reduced alcohol product from a base alcohol production biomass the method comprising: i) primary fermentation of the base alcohol production biomass with lactic acid bacteria to reduce fermentable sugar; ii) secondary alcohol fermentation of the material from i) with yeast forming a low alcohol or reduced alcohol product.

[0008] In some embodiments, the product is a low sugar or reduced sugar product. In some embodiments, the lactic acid bacteria are Leuconostoc me senter oides. In some embodiments, the Leuconostoc mesenteroides is selected from one or more of: i) Leuconostoc mesenteroides BF1 deposited under V17 / 021729 on 25 September 2017 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; ii) Leuconostoc mesenteroides BF2 deposited under V17 / 021730 on 25 September 2017 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; iii) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; iv) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; v) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; vi) Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof; and vii) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia of 1 / 153 Bertie St, Port Melbourne, VIC, Australia 3207 or a derivative thereof.

[0009] In an aspect, the present invention provides a lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product obtained, or obtainable, by the method as described herein. In an aspect, the present invention provides a lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product, wherein the composition comprises fermentation enzymes and exopolysaccharides.

[0010] In an aspect, the present invention provides a product comprising low alcohol or reduced alcohol product obtained, or obtainable, by the method as described herein.

[0011] In an aspect, the present invention provides a double fermented low alcohol or reduced alcohol product.

[0012] In an aspect, the present invention provides an isolated strain of Leuconostoc mesenteroides selected from: i) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; ii) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof; iii) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; iv) Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and v) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0013] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand examples of lactic acid bacteria outlined above for the methods of the invention equally apply to the enzyme compositions of the invention.

[0014] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0015] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0016] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures. BRIEF DESCRIPTION OF THE ACCOMPANING DRAWINGS

[0017] Figure 1. Comparison of the sugar profile of control shiraz grape juice and samples fermented by Leuconostoc mesenteroides cultures (Cl 2 and BF2) at culture dosage of 108CFU / mL for 72 hours at different temperatures under static condition.

[0018] Figure 2. Percentage sugar and alcohol reduction in shiraz wine samples prefermented for 72 hours by Leuconostoc mesenteroides cultures at 108CFU / mL dosage at different conditions.

[0019] Figure 3. Shows the sugar profile of Shiraz grape juice (with added sucrose (1%) and malolactic nutrient) prior to and after 48 and 72 hours of fermentation using BF2 culture at 108CFU / mL dosage.

[0020] Figure 4. Shows the A) percentage sugar reduction and B) percentage mannitol production in Pinot gris grape juice samples fermented by Leuconostoc mesenteroides strains for 72 hours at 30°C using a culture dosage of 108CFU / mL.

[0021] Figure 5. Shows the sugar profile of control Pinot gris grape juice and sample fermented using Leuconostoc mesenteroides C14 culture at 30°C for 72 hours at culture dosage of 108CFU / mL.

[0022] Figure 6. Shows the percentage sugar and alcohol reduction in Pinot gris samples pre-fermented using Leuconostoc mesenteroides C14 culture at 30°C for 72 hours at culture dosage of 108CFU / mL.

[0023] Figure 7. Shows the sugar reduction profile of pasteurised cloudy apple juice following 72 hours of fermentation at 30°C using C20 Leuconostoc mesenteroides culture at 108CFU / mL.

[0024] Figure 8. Shows the percentage sugar and alcohol reduction in Apple cider samples pre-fermented using Leuconostoc mesenteroides C20 culture at 30°C for 75 hours at culture dosage of 108CFU / mL.

[0025] Figure 9. Bioinformatics pipeline developed for the detection of the glycosyltransferases, levansucrase and dextransucrase related to different EPS biosynthesis pathways with raw whole genome sequences of novel L. mesenteroides strains.

[0026] Figure 10. Shows the number of genes encoding glycosyltransferase enzymes in Leuconostoc mesenteroides strains in CSIRO collection and the reference strain ATCC 8293. Left to right: levansucrase, dextransucrase, other non-1 evansucrase and glycoside hydrolase family 68 and 70 proteins collectively referred to as other glycoside transferases. Figure 11. Shows PC A score plots showing the impact of temperature and acid stress on glycosyltransferase production by two Leuconostoc mesenteroides strains C13 and C18.

[0027] Figure 12. Shows the microbial count of pre-stressed and control C13 and Cl 8 cultures after 24 hours of fermentation.

[0028] Figure 13. Shows the relative abundance and relative abundance per cell of levansucrases in C13 ferments prestressed at different conditions prior to fermentation.

[0029] Figure 14. Shows the relative abundance and relative abundance per cell of dextransucrase in C13 ferments prestressed at different conditions prior to fermentation.

[0030] Figure 15. Shows the relative abundance and relative abundance per cell of levansucrases in Cl 8 ferments prestressed at different conditions prior to fermentation.

[0031] Figure 16. Shows the relative abundance and relative abundance per cell of dextransucrases in C18 ferments prestressed at different conditions prior to fermentation.

[0032] Figure 17. Shows the evolution of pH during primary fermentation of Sauvignon Blanc samples at 30°C. Samples were inoculated with Leuconostoc mesenteroides C12 culture at 108CFU / mL.

[0033] Figure 18. Shows the evolution of pH during primary fermentation of Grenache grape juice at 30°C. Samples were inoculated with Leuconostoc mesenteroides C12 culture at 108CFU / mL.

[0034] Figure 19. Shows the evolution of pH during primary fermentation of Shiraz grape juice at 30°C. Samples were inoculated with Leuconostoc mesenteroides C12 culture at 108CFU / mL.

[0035] Figure 20. Shows fermentable sugar reduction (%) in LAB fermented grape juice samples after LAB fermentation at 30°C. LAB fermented samples were inoculated with Leuconostoc mesenteroides C12 culture at a dosage of 108CFU / mL. The fermentation time was 48 hours in the case of Sauvignon Blanc and Grenache samples and 91 hours in the case of Shiraz samples. Average of three replicates with the standard deviation as error bars are presented. Different letters within the same group (similar-coloured bars) represent significant difference (p<0.05).

[0036] Figure 21. Shows comparison of the average alcohol content of untreated control (samples on the left) wines with that of samples subjected to LAB primary fermentation (samples on the right). Average of three replicates with the standard deviation as error bars are presented. Different letters within the same group (similar-coloured bars) represent significant difference (p<0.05).

[0037] Figure 22. Shows a schematic representation of the method for producing alcohol reduced wine involving two fermentation steps together with resin adsorption or electro- membrane processing step to remove acetic acid after sugar reduction fermentation using lactic acid bacteria.

[0038] DETAILED DESCRIPTION

[0039] General techniques and definitions

[0040] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., enzyme, fermentation, inoculation).

[0041] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0042] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0043] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, of the designated value.

[0044] As used herein “alcohol” refers to an organic compound characterised by one or more hydroxyl ( — OH) groups attached to a carbon atom of an alkyl group (hydrocarbon chain) (e.g. ethanol). Alcohol is an intoxicant found in alcoholic beverages. It can be produced by fermentation. The volume of alcohol found in products, such as beverages are described for example in Wine Australia (2021).

[0045] As used herein “low alcohol” refers to less than about 1.15% alcohol by volume (Wine Australia, 2021). In an embodiment, a low alcohol product comprises about 0.1% to about 1.15% alcohol by volume. In an embodiment, a low alcohol product comprises about 0.5% to about 1.15% alcohol by volume. In an embodiment, a low alcohol product comprises about 0.5% to about 1% alcohol by volume. In an embodiment, a low alcohol product comprises about 0.5% to about 0.75% alcohol by volume. In an embodiment, a low alcohol product comprises about 1.15% alcohol by volume. In an embodiment, a low alcohol product comprises about 1% alcohol by volume. In an embodiment, a low alcohol product comprises about 0.75% alcohol by volume. In an embodiment, a low alcohol product comprises about 0.5% alcohol by volume.

[0046] As used herein “reduced alcohol” refers to product comprising a reduced amount of alcohol compared to a control product. In an embodiment, alcohol is reduced by at least about 5% compared to a control product. In an embodiment, alcohol is reduced by at least about 10% compared to a control product. In an embodiment, alcohol is reduced by at least about 15% compared to a control product. In an embodiment, alcohol is reduced by at least about 20% compared to a control product. In an embodiment, alcohol is reduced by at least about 30% compared to a control product. In an embodiment, alcohol is reduced by at least about 40% compared to a control product. In an embodiment, alcohol is reduced by at least about 50% compared to a control product. In an embodiment, alcohol is reduced by about 5% to about 50% compared to a control product. In an embodiment, alcohol is reduced by about 10% to about 50% compared to a control product. In an embodiment, alcohol is reduced by about 20% to about 50% compared to a control product. In an embodiment, alcohol is reduced by about 30% to about 50% compared to a control product. In an embodiment, alcohol is reduced by about 40% to about 50% compared to a control product. In an embodiment, alcohol is present at about 0.1% to about 15% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 14% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 13% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 12% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 11% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 10% alcohol by volume. In an embodiment, alcohol is present at about 0.1% to about 9% alcohol by volume. In an embodiment, alcohol is present at about 2% to about 9% alcohol by volume. In an embodiment, alcohol is present at about 4.5% to about 9% alcohol by volume.

[0047] As used herein “sugar” refers to a sweet soluble carbohydrate. In an embodiment, the sugar is a monosaccharide and / or a disaccharide.

[0048] As used herein “carbohydrate” refers to a class of molecules of the general formula Cx(H2O)y.

[0049] As used herein “fermentable sugar” refers to one or more of: fructose, glucose and sucrose. In an embodiment, the fermentable sugar is fructose. In an embodiment, the fermentable sugar is glucose. In an embodiment, the fermentable sugar is sucrose. As used herein “total sugar” refers to the combination of sucrose, glucose and fructose. In an embodiment, total sugar further comprises one or more of xylose, arabinose, mannose and lactose.

[0050] As used herein “low sugar” refers to a sugar concentration of about 0 g / L to about 30 g / L. In an embodiment, low sugar refers to low fermentable sugar. In an embodiment, low sugar refers to low total sugar.

[0051] As used herein “reduced sugar” refers to where fermentable sugar in the product is reduced compared to a control product. In an embodiment, the sugar in the product is reduced at least about 30% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 40% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 50% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 60% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 70% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 80% compared to a control product. In an embodiment, the sugar in the product is reduced at least about 90% compared to a control product. In an embodiment, the sugar in the product is reduced at least 100% compared to a control product. In an embodiment, the sugar in the product is reduced about 30% to 100% compared to a control product. In an embodiment, the sugar in the product is reduced about 40% to about 95% compared to a control product. In an embodiment, the sugar in the product is reduced about 40% to about 90% compared to a control product. In an embodiment, the sugar in the product is reduced about 50% to about 80% compared to a control product. In an embodiment, fermentable sugar is reduced in the product. In an embodiment, total sugar is reduced in the product.

[0052] As used herein a “control product” is a product produced using a method described herein but lacking the primary fermentation step. In an embodiment, the control product does not comprise mannitol. In an embodiment, the control product comprises about 6 g / L to about 220 g / L sugar. In an embodiment, the control product comprises about 21 g / L to about 220 g / L sugar. In an embodiment, the control product comprises about 71 g / L to about 220 g / L sugar. In an embodiment, the sugar is fermentable sugar. In an embodiment, the sugar is total sugar.

[0053] As used herein "derivative" or “derivatives” of a given strain refers to mutants and homologues thereof.

[0054] As use herein “fermentation” occurs when an organism converts any carbohydrate into an acid or alcohol. In an embodiment, the organism is a lactic acid bacteria. In an embodiment, the organism is a yeast. Fermentation is a metabolic process that produces chemical changes in organic substances through the action of enzymes.

[0055] As used herein “food grade” refers to components / materials permitted to come into direct contact with food meant for human consumption.

[0056] As used herein “wine” refers to an alcohol containing beverage produced by the fermentation of grape must or grape juice, including but not limited to red wine, white wine, sparkling wine etc. The alcohol contents are expressed by the volume of alcohol in relation to the total volume.

[0057] As used herein “cider” refers to an alcohol containing beverage produced by the fermentation of fruit must or fruit juice, including, but not limited to from apple, pear, peach etc. The alcohol contents are expressed by the volume of alcohol in relation to the total volume.

[0058] As used herein a “de-alcoholised” product as described herein is a product produced using a secondary fermentation step as described herein, after secondary fermentation, the alcohol is removed / extracted from the product e.g. by one or more of e.g. vacuum distillation, evaporation, distillation, freeze-concentration, solvent extraction and membrane filtration techniques such as reverse osmosis and dialysis for alcohol removal (Liguori et al., 2018; Pickering, 2001; Schmidtke et al., 2012). A person skilled in the art would appreciate that the de-alcoholising process additionally removes favorable flavour and / or odour components from the product.

[0059] As used herein “no alcohol” refers to a product comprising no quantifiable amount of alcohol.

[0060] As used herein “component” refers to a part or element of a larger whole.

[0061] As used herein “increase” or “increased” means that the level of an indicated component (e.g. mannitol) is higher than that present in a material before a step in the method commenced or is higher in a product described herein compared to a control product as described herein. In an embodiment, the step is the primary fermentation step. In an embodiment, the level of the indicated component is increased from about 5% to about 100%. In an embodiment, the level of the indicated component is increased from about 5% to about 90%. In an embodiment, the level of the indicated component is increased from about 5% to about 80%. In an embodiment, the level of the indicated component is increased from about 5% to about 70%. In an embodiment, the level of the indicated component is increased by at least about 5%. In an embodiment, the level of the indicated component is increased by at least about 10%. In an embodiment, the level of the indicated component is increased by at least about 15%. In an embodiment, the level of the indicated component is increased by at least about 20%. In an embodiment, the level of the indicated component is increased by at least about 30%. In an embodiment, the level of the indicated component is increased by at least about 40%. In an embodiment, the level of the indicated component is increased by at least about 50%. In an embodiment, the level of the indicated component is increased by at least about 60%. In an embodiment, the level of the indicated component is increased by at least about 70%. In an embodiment, the level of the indicated component is increased by at least about 80%. In an embodiment, the level of the indicated component is increased by at least about 90%. In an embodiment, the level of the indicated component is increased by at least about 100%. As used herein “reduced” means that the level of an indicated component (e.g. fermentable sugar or total sugar) in lower in a product described herein compared to a control product as described herein. In an embodiment, the level of the indicated component is reduced by about 5% to about 100%. In an embodiment, the level of the indicated component is reduced by about 5% to about 90%. In an embodiment, the level of the indicated component is reduced by about 5% to about 80%. In an embodiment, the level of the indicated component is reduced by about 5% to about 70%. In an embodiment, the level of the indicated component is reduced by about 5% to about 60%. In an embodiment, the level of the indicated component is reduced by about 20% to about 60%. In an embodiment, the level of the indicated component is reduced by about 30% to about 60%. In an embodiment, the level of the indicated component is reduced by about 40% to about 60%. In an embodiment, the level of the indicated component is reduced by about 5% to about 50%. In an embodiment, the level of the indicated component is reduced by about 5% to about 40%. In an embodiment, the level of the indicated component is reduced by about 5% to about 30%. In an embodiment, the level of the indicated component is reduced by about 5% to about 20%. In an embodiment, the level of the indicated component is reduced at least by about 5%. In an embodiment, the level of the indicated component is reduced at least by about 10%. In an embodiment, the level of the indicated component is reduced at least by about 15%. In an embodiment, the level of the indicated component is reduced at least by about 20%. In an embodiment, the level of the indicated component is reduced at least by about 30%. In an embodiment, the level of the indicated component is reduced at least by about 40%. In an embodiment, the level of the indicated component is reduced at least by about 50%. In an embodiment, the level of the indicated component is reduced at least by about 60%. In an embodiment, the level of the indicated component is reduced at least by about 70%. In an embodiment, the level of the indicated component is reduced at least by about 80%. In an embodiment, the level of the indicated component is reduced at least by about 90%. In an embodiment, the level of the indicated component is reduced by 100%.

[0062] As used herein “glycosyltransferase” refers to an enzyme that catalyzes the formation of glycosidic bonds. Such enzymes transfer the saccharide from a sugar nucleotide donor to an acceptor and are responsible for the biosynthesis of polymers. In an embodiment, the polymer is an oligosaccharide. In an embodiment, the polymer is a polysaccharide. In an embodiment, the glycosyltransferases hydrolyse the sugar nucleotide sucrose. In an embodiment, the glycosyltransferases hydrolyse the sugar nucleotide raffinose. In an embodiment, the glycosyltransferase is levansucrase. In an embodiment, the glycosyltransferase is dextransucrase. In an embodiment, the glycosyltransferase is alternansucrase.

[0063] As used herein “levansucrase” refers to an enzyme that catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain.

[0064] As used herein “dextransucrase” refers to an enzyme that catalyses the biosynthesis of dextran from sucrose.

[0065] As used herein “pasteurised” refers to a heat treatment to destroy or inactivate spoilage microorganisms and enzymes.

[0066] Method of producing a low alcohol or a reduced alcohol product

[0067] The present invention relates to methods for producing a low alcohol or a reduced alcohol product from a base alcohol production biomass.

[0068] In an aspect, the present invention provides a method of producing a low alcohol or a reduced alcohol product from a base alcohol production biomass the method comprising: i) primary fermentation of the base alcohol production biomass with lactic acid bacteria to reduce fermentable sugar; ii) secondary alcohol fermentation of the material from i) with yeast forming a low alcohol or reduced alcohol product.

[0069] In an embodiment, the base alcohol production biomass is juice obtained from crushed fruit wherein the skin is separated from the juice (juice separated from the skin).

[0070] In some embodiments, the method further comprises producing a low sugar or reduced sugar product. In some embodiments, sugar is fermentable sugar. In some embodiments, the sugar is total sugar.

[0071] In some embodiments, lactic acid bacteria are removed from the fermented material produced by step i) and the fermented material, an extract thereof, or dried powder produced from the fermented material or an extract thereof, is incubated with unfermented base alcohol production biomass before step ii).

[0072] In some embodiments, lactic acid bacteria are deactivated from the fermented material produced by step i) and the fermented material, an extract thereof, or dried powder produced from the fermented material or an extract thereof, is incubated with unfermented base alcohol production biomass before step ii).

[0073] In some embodiments, the ratio of fermented material to unfermented base alcohol production biomass is about 1:100 to about 1:5.

[0074] In some embodiments, the ratio of fermented material to unfermented base alcohol production biomass is about 1:5. In some embodiments, incubation is for about 2 to about 50 hours at about 25°C to about 50°C. In some embodiments, incubation is for about 5 to about 50 hours at about 25°C to about 50°C. In some embodiments, incubation is for about 10 to about 50 hours at about 25°C to about 50°C. In some embodiments, incubation is for about 20 to about 50 hours at about 25°C to about 50°C. In some embodiments, incubation is for about 24 to about 48 hours at about 30°C.

[0075] In some embodiments, sucrose is added before step i), during step i) or before step ii). In some embodiments, sucrose is added before step i). In some embodiments, sucrose is added during step i). In some embodiments, sucrose is added before step ii).

[0076] In some embodiments, yeast extract, ammonium sulphate or other nitrogen source is added before step i).

[0077] In some embodiments, the base alcohol production biomass may be diluted with water before step i).

[0078] In some embodiments, calcium chloride is added before step i) or before step ii). In some embodiments, the base alcohol production biomass is adjusted to a pH of about 5 to about 7 before step i). In some embodiments, the base alcohol production biomass is adjusted to a pH of about 5.5 to about 6.5 before step i).

[0079] In some embodiments, the pH is adjusted with the addition of a food grade salt. In some embodiments, the food grade salt is selected from one or more of: CaCCh, Ca(OH)2, KHCCh or K2HPO4. In some embodiments, the food grade salt is CaCCh. In some embodiments, the food grade salt is Ca(OH)2. In some embodiments, the food grade salt is KHCO3. In some embodiments, the food grade salt is K2HPO4.

[0080] In some embodiments, before step i) the base alcohol production biomass is pretreated to reduce the microbial load. As used herein “microbial load” refers to the number and type of microorganisms present in / on the base alcohol production biomass.

[0081] In some embodiments, pre-treatment comprises one or more of: addition of a sulphite, addition of dimethyl decarbonate, microfiltration or heat treatment. In some embodiments, pre-treatment comprises the addition of sulphite. In some embodiments, pre-treatment comprises addition of dimethyl decarbonate. In some embodiments, pretreatment comprises microfiltration. In some embodiments, pre-treatment comprises heat treatment. In some embodiments, the heat treatment is flash detent.

[0082] In some embodiments, the sulphite is selected from one or both of: sulphur dioxide or potassium metabisulphite. In some embodiments, the sulphite is sulphur dioxide. In some embodiments, the sulphite is potassium metabisulphite.

[0083] In some embodiments, after step i) the pH is adjusted to a pH of about 3.4 to about 3.7. In some embodiments, the pH is adjusted with the addition of tartaric acid. In some embodiments, before step ii) the level of acetic is in the ferment from i) is reduced.

[0084] In some embodiment, acetic acid is removed by one or both of a weak anionic resin and activated carbon. In some embodiments, the weak anionic resin is selected from one or more of: Amberlite IRA-67, Amberlyst A21, Dowex optipore L-493, Indion 850, Tuision A-8X MP, Indion 810, Amberlite IRA-96, Purolite A1335, Sepra NH2, Sepra SAX, Sepra ZT-AX, Dowex 1x8-100 (CI), Dowex 21K XLT, Dowex marathon A2 (CI), Amberlite IRA-400 (CI), Amberlite IRA-900 (CI), Daion HP-20 or polyvinyl pyrrolidone (PVP) resins such as reillex 425. In an embodiment, the weak anionic resin is Amberlite IRA-67.

[0085] In some embodiments, acetic acid is removed by electro-membrane processing. In some embodiments, acetic acid is removed with electro-membrane processing. In some embodiments, the reduction of acetic acid is followed by reacidification prior to step ii). In some embodiments, the pH is adjusted to a pH of about 3.4 to about 3.7. In some embodiments, the reacidification comprises addition of an acid. In some embodiments, the acid is tartaric acid.

[0086] In some embodiments, the methods described herein produce a low alcohol or reduced alcohol product that has increase flavour and / or aroma compounds compared to a control product as described herein.

[0087] A person skilled in the art would appreciate that the methods as described herein can be performed using existing fermentation infrastructure, for beverage production e.g. wine, thus reducing the production cost in comparison to current methods.

[0088] Base alcohol production biomass

[0089] As used herein "base alcohol production biomass” refers to a biomass suitable for producing an alcohol product via fermentation.

[0090] In an embodiment, the base alcohol production biomass is selected from one or more of: must, juice, juice concentrate, puree, wort, reconstituted fruit or vegetable powder, rehydrated dried fruit pieces and sugary fraction of fruit and vegetable processing. In an embodiment, the base alcohol production biomass comprises must. In an embodiment, the base alcohol production biomass comprises juice. In an embodiment, the base alcohol production biomass comprises juice concentrate. In an embodiment, the base alcohol production biomass comprises puree. In an embodiment, the base alcohol production biomass comprises wort. In an embodiment, the base alcohol production biomass comprises reconstituted fruit or vegetable powder. In an embodiment, the base alcohol production biomass comprises rehydrated dried fruit pieces and sugary fraction of fruit and vegetable processing.

[0091] In an embodiment, the base alcohol production biomass is produced by crushing fruit and separating juice from the skin.

[0092] In an embodiment, the base alcohol production biomass is produced by macerating the fruit.

[0093] In an embodiment, the base alcohol production biomass is pasteurised.

[0094] In an embodiment, the base alcohol production biomass is sulphite treated.

[0095] As used herein “must” refers to the material obtained by pressing fruit and contains the juice, skins, seeds and stems of the fruit. In an embodiment, the must is mixed fruit must. In an embodiment, the must is grape must. In an embodiment, the must is apple must. In an embodiment, must is used for wine production. In an embodiment, must is used for cider production.

[0096] As used herein “juice” refers to the liquid extracted from processed fruit or vegetable material. In an embodiment, the base alcohol production biomass is juice. In an embodiment, the juice is fruit juice. In an embodiment, the juice is mixed fruit juice. In an embodiment, the juice is grape juice. In an embodiment, the juice is apple juice. In an embodiment, the juice is fresh juice, juice treated with a preservative, pasteurised juice, sterilised juice or frozen juice (after thawing). In an embodiment, the juice is fresh juice. In an embodiment, the juice is treated with a preservative. In an embodiment, the juice is pasteurised juice. In an embodiment, the juice is sterilised juice. In an embodiment, the juice is frozen juice. In an embodiment, the juice is treated with SO2, dimethyl dicarbonate (Velcorin®) or any other preservative or a combination thereof.

[0097] In an embodiment, the base alcohol production biomass is juice obtained from crushed fruit wherein the juice is separated from the skin (the separated skin may be stored and added after the primary fermentation step).

[0098] In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 80% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 85% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 90% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 95% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 98% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 99% of the skin is separated from the juice.

[0099] In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 80% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 85% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 90% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 95% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 98% of the skin is separated from the juice. In an embodiment, the base alcohol production biomass is grape juice separated from the skin of the grape, wherein at least 99% of the skin is separated from the juice. In an embodiment, of the methods described herein the juice is present in step i) and the skin is added before step ii). In an embodiment, of the methods described herein the juice is present in step i) and the skin stored separately during step i) and is added before step ii).

[0100] In an embodiment, the base alcohol production biomass is juice concentrate. In an embodiment, the juice concentrate is fruit juice concentrate. In an embodiment, the juice concentrate is grape juice concentrate. In an embodiment, the base alcohol production biomass is puree. In an embodiment, the base alcohol production biomass is wort. In an embodiment, the base alcohol production biomass is reconstituted fruit or vegetable powder. In an embodiment, the base alcohol production biomass is rehydrated dried fruit pieces. In an embodiment, the base alcohol production biomass is the sugary fraction of fruit and vegetable processing.

[0101] As used herein “wort”, refers to the liquid extracted from the process of mashing cereal grains.

[0102] In an embodiment, the fruit is selected from one or more of: a simple, aggregate and multiple fruit. In an embodiment, the fruit is from one or more family / families selected from: Arecaceae, Myrtaceae, Rosaceae, Musaceae, Ericaceae, Saxifragaceae, Cucurbitaceae, Nightshade, Capparaceae, Adoxaceae, Vitaceae, Rutaceae, Actinidiaceae, Sapindaceae, Anacardiaceae, Moraceae, Oleaceae, Cactaceae, Passifloraceae, Bromeliaceae, Cactaceae, Lythraceae, Polygonaceae, Oxalidaceae and Caesalpinioideae .

[0103] In an embodiment, the family is Rosaceae, preferably an apple. In an embodiment, the fruit is selected from one or more or all of: apple, apricot, avocado, banana, bilberry, blackberry, blackcurrant, blueberry, coconut, currant, cherry, cherimoya, clementine, cloudberry, damson, durian, elderberry, fig, feijoa, gooseberry, grape, grapefruit, orange, guava, huckleberry, jackfruit, jambul, jujube, kiwifruit, kumquat, lemon, lime, loquat, lychee, mandarin, mango, melon, cantaloupe, honeydew, watermelon, nectarine, orange, passionfruit, paw paw, peach, pear, plum, plumcot, pineapple, pomegranate, pomelo, purple mangosteen, raspberry, rambutan, redcurrant, satsuma, star fruit, strawberry, tangerine, tomato, and ugli fruit.

[0104] In an embodiment, the fruit is apple. In an embodiment, the variety of apple is selected from one or more of: royal gala, golden delicious, red delicious, fuji, cripps pink (pink lady), granny smith, Jonathan, jonagold, jazz, sundowner, and braebum.

[0105] In an embodiment, the fruit is grape from Vitaceae. In an embodiment, the fruit is grape from a Vitis.

[0106] In an embodiment, the fruit is orange. In an embodiment, the variety of orange is selected from one or more or all of: amold blood, bali, belladonna, bergamont, berna, biondo comune, biondo riccio, byeonggyul, cadanera, cara cara, carvalhal, castellana, cherry orange, clanor, clementine, dom Joao, fukuhara, gardner, hamlin, homosassa, jaffa orange, joppa, khettmali, kona, lima (acidless orange), lue gim gong, macetera, malta, maltaise blonde, maltaise ovale, marrs, medan, midsweet, moro tarocco, navel, navelina, newhall, parson brown, pera, pera coroa, pera natal, pera rio, pineapple, pontianak, premier, rhode red, roble, queen, salustiana, sanguine (blood orange), sathgudi, seleta, Seville (bitter orange), shamouti masry, sunstar, tomango, Valencia, vema, vicieda, Washington, and westin. In an embodiment, the variety of orange is selected from one or more of: navel, Valencia, clementine, hamlin, salustiana, sanguine (blood orange), bergamont, and cara cara.

[0107] In an embodiment, the must or juice is derived from berries of a Vitaceae. In an embodiment, the must or juice is derived from berries of a Vitis. In an embodiment, the Vitis is a wine grape, table grape or dried grape varieties. In an embodiment, the berries of the Vitis is suitable for producing wine. In an embodiment, the wine is a red wine. In an embodiment, the wine is a white wine. In an embodiment, the Vitis is a member of the sub-genera Muscadinia. In an embodiment, the Vitis is a member of the sub-genera Euvitis. In an embodiment, the Vitis is Vinifera.

[0108] In an embodiment, the must or grape juice is derived from grapes selected from: Shiraz, Chardonnay, Sauvingnon Blanc, Grenache, Pinot Gris, Kyoho, Cabernet Sauvignon, Sultanina, Merlot, Tempranillo, Airen, Syrah, Pinot Noir / Blauer Burgunder, Trebbiano Toscana / Ugni Blanc, Semilion, Riesling and Muscat of Alexandria. In an embodiment, the must or grape juice is derived from Shiraz grapes. In an embodiment, the must or grape juice is derived from Shiraz grapes. In an embodiment, the must or grape juice is derived from Chardonnay grapes. In an embodiment, the must or grape juice is derived from Pinot Gris grapes. In an embodiment, the must or grape juice is derived from Kyoho grapes. In an embodiment, the must or grape juice is derived from Cabernet Sauvignon grapes. In an embodiment, the must or grape juice is derived from Sultanina grapes. In an embodiment, the must or grape juice is derived from Merlot grapes. In an embodiment, the must or grape juice is derived from Tempranillo grapes. In an embodiment, the must or grape juice is derived from Airen grapes. In an embodiment, the must or grape juice is derived from Syrah grapes. In an embodiment, the Grenache is derived from Garnacha Tinta / Grenache Noir grapes. In an embodiment, the must or grape juice is derived from Sauvingnon Blanc grapes. In an embodiment, the must or grape juice is derived from Pinot Noir / Blauer Burgunder grapes. In an embodiment, the must or grape juice is derived from Trebbiano Toscana / Ugni Blanc grapes. In an embodiment, the must or grape juice is derived from Semilion grapes. In an embodiment, the must or grape juice is derived from Riesling grapes. In an embodiment, the must or grape juice is derived from Muscat of Alexandria grapes.

[0109] In an embodiment, the vegetable is a root vegetable. In an embodiment, the vegetable is selected from one or more of: potato, sweet potato, cassava, carrot or other starchy vegetable.

[0110] Primary fermentation

[0111] As used herein “primary fermentation” refers to fermentation of the base alcohol production biomass with lactic acid bacteria to reduce fermentable sugar.

[0112] In some embodiments, primary fermentation further comprises malolactic fermentation (conversion of malic acid to lactic acid).

[0113] In some embodiments, exopolysaccharides are produced during the primary fermentation. In an embodiment, the exopolysaccharides are predominantly homopolysaccharides (HoPS). As used herein, a homopolysaccharide refers to polysaccharide composed of repeating units of a single type of monosaccharide.

[0114] As used herein, “lactic bacteria” or “lactic acid bacteria” are bacteria that produce lactic acid as an end product of carbohydrate fermentation, and can include, but are not limited to including bacteria from the genera Leuconostoc, Oenococcus, Lactobacillus, Weissela, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, Fuctobacillus, Bacillus and Zymomonas or a combination thereof. In an embodiment, the lactic acid bacteria are from the genera Leuconostoc. In an embodiment, the lactic acid bacteria are from the genera Oenococcus. In an embodiment, the lactic acid bacteria are from the genera Lactobacillus. In an embodiment, the lactic acid bacteria are from the genera Pediococcus. In an embodiment, the lactic acid bacteria are from the genera Lactococcus. In an embodiment, the lactic acid bacteria are from the genera Streptococcus. In an embodiment, the lactic acid bacteria are from the genera Aerococcus. In an embodiment, the lactic acid bacteria are from the genera Carnobacterium. In an embodiment, the lactic acid bacteria are from the genera Enterococcus. In an embodiment, the lactic acid bacteria are from the genera Sporolactobacillus. In an embodiment, the lactic acid bacteria are from the genera Tetragenococcus. In an embodiment, the lactic acid bacteria are from the genera Vagococcus. In an embodiment, the lactic acid bacteria are from the genera Weissella. In an embodiment, the lactic acid bacteria are from the genera Bacillus. In an embodiment, the lactic acid bacteria are from the genera Zymomonas.

[0115] In an embodiment, the lactic acid bacteria are selected from one or more of: Leuconostoc mesenteroides, Oenococcus Oeni, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactococus lactis, Pediococcus pentosaceus, Lactobacillus rhamnosus, Pedicoccus acidilacti, Bacillus subtilis, Bacillus subtilis natto, Bacillus circulans and Zymomonas mobilis or a combination thereof. In an embodiment, the lactic acid bacteria are Leuconostoc mesenteroides. In an embodiment, the lactic acid bacteria are Oenococcus Oeni. In an embodiment, the lactic acid bacteria are Lactobacillus gasseri. In an embodiment, the lactic acid bacteria are Lactobacillus reuteri. In an embodiment, the lactic acid bacteria are Lactobacillus plantarum. In an embodiment, the lactic acid bacteria are Lactobacillus pentosus. In an embodiment, the lactic acid bacteria are Lactobacillus brevis. In an embodiment, the lactic acid bacteria are Lactococus lactis. In an embodiment, the lactic acid bacteria are Pediococcus pentosaceus. In an embodiment, the lactic acid bacteria are Lactobacillus rhamnosus. In an embodiment, the lactic acid bacteria are Pedicoccus acidilacti. In an embodiment, the lactic acid bacteria are Bacillus subtilis. In an embodiment, the lactic acid bacteria are Bacillus subtilis natto. In an embodiment, the lactic acid bacteria are Bacillus circulans. In an embodiment, the lactic acid bacteria are Zymomonas mobilis.

[0116] In an embodiment, the lactic acid bacteria produce an enzyme encoded by a gene selected from one or more of: gtfA, LCIT_00500, LEUM_1409, LEUMJ747, DIS10 08860, LEUM_1410, LEUM 411, LEMES_01041, H2060_03810, LPJSA22_01242, LPJSA22_01063, levS_2, NHN79_00835, KE630_00215, levS_l, C1T23_O1282, sacB, sacB_2, sacB_3, levS_3, EUZ72J0755, NQL29_06645, EFP04_04965, LAP8962_01701, ASU25_00535, levS_4, S101174_00409, SAC12_0369, DQM12_01810, ADS73 1930, Nizol839_2806, LJA01_10220, SF2A35B_1062, JK161_01400, CHH57 18525, C2I06J6290, HLK66_22900, C2I17_14445, C2H98 18060, levS, S100434_00436, GB995_04720, G7B65J2340, S100434_00483, GB995_04960, S102022_02672, LAP8962_01024, ADS73 1700, IAR49_00640, SRCM101060_02609, S 101258 01184, N876_05150, BMS99_08665, S 101520_02514, D2U09_07590, FXE14_08880, CJP43_14315, DB342_0690, S101258_03346, S102022_01132, H6X79_02665, J6K68_08370, J3330_05935, LPJSA22_02538, EZV74_01260, A0F18_09060, CD198_06875, CD198_06870, gtfC_6, gtfC_2, HFP47 05615, EQK02_06830, NX809_06765, MI1_O7585, ARA01_07835, H2060_03815, EQZ98_07210, C7M31_00572, C7M32_02485, C7M33_00571,Nizo2262_1509, C1940J3880, Nizo3894_1514, CFN49J3740, mtlD, Lpl6_0215, H073J3211, L103_03153, N574J5285, mdh, SH83_00985, ALX04_13535, N876_0202495, FXE15_01185, J8138 12190, mleS, I526_0921, Nizo2726_2007, Nizo2830_2922, LEUM_1005, HFP47_03725, maeA, DQM12_10300, Nizo2891_2382, Nizo2535_2209, KGI16_04450, RI536_12465, sfcA, N876_0213650, N876_04935, A9F05_07390, HFP47_07275, dsrF, LEUM_1752 or LEUM 0857. In an embodiment, the gene is gtfA. In an embodiment, the gene is LCIT 00500. In an embodiment, the gene is LEUM 1409. In an embodiment, the gene is LEUM 1747. In an embodiment, the gene is DIS10 08860. In an embodiment, the gene is LEUM 1410. In an embodiment, the gene is LEUM 1411. In an embodiment, the gene is LEMES 01041. In an embodiment, the gene is H2060 03810. In an embodiment, the gene is LPJSA22 01242. In an embodiment, the gene is LPJSA22 01063. In an embodiment, the gene is levS_2. In an embodiment, the gene is NHN79 00835. In an embodiment, the gene is KE630 00215. In an embodiment, the gene is levS l. In an embodiment, the gene is C1T23 01282. In an embodiment, the gene is sacB. In an embodiment, the gene is sacB_2. In an embodiment, the gene is sacB_3. In an embodiment, the gene is levS_3. In an embodiment, the gene is EUZ72 10755. In an embodiment, the gene is NQL29 06645. In an embodiment, the gene is EFP04 04965. In an embodiment, the gene is LAP8962 01701. In an embodiment, the gene is ASU25 00535. In an embodiment, the gene is levS_4. In an embodiment, the gene is S101174_00409. In an embodiment, the gene is SAC12_0369. In an embodiment, the gene is DQM12 01810. In an embodiment, the gene is ADS73 11930. In an embodiment, the gene is Nizol839_2806. In an embodiment, the gene is LJA01 10220. In an embodiment, the gene is SF2A35B 1062. In an embodiment, the gene is JK161 01400. In an embodiment, the gene is CHH57 18525. In an embodiment, the gene is C2I06 16290. In an embodiment, the gene is HLK66 22900. In an embodiment, the gene is C2I17 14445. In an embodiment, the gene is C2H98 18060. In an embodiment, the gene is levS. In an embodiment, the gene is S100434 00436. In an embodiment, the gene is GB995 04720. In an embodiment, the gene is G7B65_12340. In an embodiment, the gene is S100434_00483. In an embodiment, the gene is GB995_04960. In an embodiment, the gene is S102022_02672. In an embodiment, the gene is LAP8962 01024. In an embodiment, the gene is ADS73 11700. In an embodiment, the gene is IAR49 00640. In an embodiment, the gene is SRCM101060 02609. In an embodiment, the gene is S101258 01184. In an embodiment, the gene isN876_05150. In an embodiment, the gene is BMS99 08665. In an embodiment, the gene is S101520 02514. In an embodiment, the gene is D2U09 07590. In an embodiment, the gene is FXE14 08880. In an embodiment, the gene is CJP43 14315. In an embodiment, the gene is DB342 0690. In an embodiment, the gene is S101258_03346. In an embodiment, the gene is S102022_01132. In an embodiment, the gene is H6X79 02665. In an embodiment, the gene is J6K68 08370. In an embodiment, the gene is J3330 05935. In an embodiment, the gene is LPJSA22 02538. In an embodiment, the gene is EZV74 01260. In an embodiment, the gene is A0F18 09060. In an embodiment, the gene is CD198 06875. In an embodiment, the gene is CD198 06870. In an embodiment, the gene is gtfC_6. In an embodiment, the gene is gtfC_2. In an embodiment, the gene is HFP47 05615. In an embodiment, the gene is EQK02 06830. In an embodiment, the gene is NX809 06765. In an embodiment, the gene is MI1 07585. In an embodiment, the gene is ARA01 07835. In an embodiment, the gene is H2060 03815. In an embodiment, the gene is EQZ98 07210. In an embodiment, the gene is C7M31 00572. In an embodiment, the gene is C7M32_02485. In an embodiment, the gene is C7M33_00571. In an embodiment, the gene is Nizo2262_1509. In an embodiment, the gene is C1940_13880. In an embodiment, the gene isNizo3894_1514. In an embodiment, the gene is CFN49_13740. In an embodiment, the gene is mtlD. In an embodiment, the gene is Lpl6_0215. In an embodiment, the gene is H073 13211. In an embodiment, the gene is L103 03153. In an embodiment, the gene is N574 15285. In an embodiment, the gene is mdh. In an embodiment, the gene is SH83 00985. In an embodiment, the gene is ALX04 13535. In an embodiment, the gene is N876_0202495. In an embodiment, the gene is FXE15 01185. In an embodiment, the gene is J8138 12190. In an embodiment, the gene is mleS. In an embodiment, the gene is 1526 0921. In an embodiment, the gene is Nizo2726_2007. In an embodiment, the gene is Nizo2830_2922. In an embodiment, the gene is LEUM 1005. In an embodiment, the gene is HFP47 03725. In an embodiment, the gene is maeA. In an embodiment, the gene is DQM12 10300. In an embodiment, the gene is Nizo2891_2382. In an embodiment, the gene is Nizo2535_2209. In an embodiment, the gene is KGI16 04450. In an embodiment, the gene is RI536 12465. In an embodiment, the gene is sfcA. In an embodiment, the gene is N876 0213650. In an embodiment, the gene is N876_04935. In an embodiment, the gene is A9F05_07390. In an embodiment, the gene is HFP47 07275. In an embodiment, the gene is dsrF. In an embodiment, the gene is LEUM 1752. In an embodiment, the gene is LEUM 0857.

[0117] In an embodiment, the lactic acid bacteria produce one or more glycosyltransferase / s.

[0118] In an embodiment, the lactic acid bacteria produce an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme, oxalacetate dehydrogenase, and glycosyltransferase. In an embodiment, the lactic acid bacteria produce malic enzyme. In an embodiment, the lactic acid bacteria produce L-lactate dehydrogenase. In an embodiment, the lactic acid bacteria produce malate dehydrogenase. In an embodiment, the lactic acid bacteria produce mannitol dehydrogenase. In an embodiment, the lactic acid bacteria produce malolactic enzyme oxalacetate dehydrogenase. In an embodiment, the lactic acid bacteria produce glycosyltransferase. In an embodiment, the glycosyltransferase is dextransucrase. In an embodiment, the glycosyltransferase is levansucrase. In an embodiment, the glycosyltransferase is alternansucrase. In an embodiment, the glycosyltransferase is a glycoside hydrolase family 68 protein. In an embodiment, the glycosyltransferase is a glycoside hydrolase family 70 protein.

[0119] In an embodiment, the lactic acid bacteria produce an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme oxalacetate dehydrogenase, dextransucrase, levansucrase, alternansucrase, glycoside hydrolase family 68 protein and glycoside hydrolase family 70 protein.

[0120] In an embodiment, the lactic acid bacteria produce an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, alternansucrase, malolactic enzyme oxalacetate dehydrogenase, dextransucrase and levansucrase.

[0121] In an embodiment, the lactic acid bacteria produce dextransucrase encoded by a gene selected from one or more of: gtfA, LCIT_00500, LEUM_1747, LEMES_01041, LPJSA22_01242, LPJSA22_01063, S101174_00409, SAC12_0369, S102022_02672, SRCM101060_02609, S 101258_01184, N876_05150, S 101520_02514, D2U09_07590, FXE14_08880, CJP43_14315, DB342_0690, S102022_01132, HFP47_07275, dsrF, LEUM 1752 or LEUM 0857. In an embodiment, the gene is gtfA. In an embodiment, the gene is LCIT 00500. In an embodiment, the gene is LEUM 1747. In an embodiment, the gene is LEMES 01041. In an embodiment, the gene is LPJSA22 01242. In an embodiment, the gene is LPJSA22 01063. In an embodiment, the gene is S101174 00409. In an embodiment, the gene is SAC12 0369. In an embodiment, the gene is S102022_02672. In an embodiment, the gene is SRCM101060_02609. In an embodiment, the gene is S101258 01184. In an embodiment, the gene is N876_05150. In an embodiment, the gene is S101520 02514. In an embodiment, the gene is D2U09 07590. In an embodiment, the gene is FXE14 08880. In an embodiment, the gene is CJP43 14315. In an embodiment, the gene is DB342 0690. In an embodiment, the gene is S102022_01132. In an embodiment, the gene is HFP47_07275. In an embodiment, the gene is dsrF. In an embodiment, the gene is LEUM 1752. In an embodiment, the gene is LEUM 0857.

[0122] In an embodiment, the lactic acid bacteria produce levansucrase encoded by a gene selected from one or more of: LEUM_1409, LEUM_1410, LEUM_1411, levS_2, NHN79_00835, KE630_00215, levS_l, C1T23_O1282, sacB, sacB_2, sacB_3, levS_3, EUZ72_10755, NQL29_06645, EFP04_04965, LAP8962_01701, ASU25_00535, levS_4, ADS73_11930, Nizol839_2806, LJA01_10220, SF2A35BJ062, CHH57 18525, C2I06J6290, HLK66_22900, C2I17J4445, C2H98 18060, levS, S100434_00436, GB995_04720, G7B65J2340, S100434_00483, GB995_04960, LAP8962_01024, ADS73_11700, IAR49_00640, S101258_03346, H6X79_02665, LPJSA22_02538, C7M31_00572, C7M32_02485, C7M33_00571, Nizo2262_1509, C1940_13880, Nizo3894_1514 or CFN49_13740. In an embodiment, the gene is LEUM 1409. In an embodiment, the gene is LEUM 1410. In an embodiment, the gene is LEUM 1411. In an embodiment, the gene is levS_2. In an embodiment, the gene is NHN79 00835. In an embodiment, the gene is KE630 00215. In an embodiment, the gene is C1T23 01282. In an embodiment, the gene is sacB. In an embodiment, the gene is sacB_2. In an embodiment, the gene is sacB_3. In an embodiment, the gene is levS_3. In an embodiment, the gene is EUZ72 10755. In an embodiment, the gene is NQL29 06645. In an embodiment, the gene is EFP04 04965. In an embodiment, the gene is LAP8962_01701. In an embodiment, the gene is ASU25_00535. In an embodiment, the gene is levS_4. In an embodiment, the gene is ADS73 11930. In an embodiment, the gene is Nizol839_2806. In an embodiment, the gene is LJA01 10220. In an embodiment, the gene is SF2A35B 1062. In an embodiment, the gene is CHH57 18525. In an embodiment, the gene is C2I06 16290. In an embodiment, the gene is HLK66 22900. In an embodiment, the gene is C2I17 14445. In an embodiment, the gene is C2H98 18060. In an embodiment, the gene is levS. In an embodiment, the gene is S100434_00436. In an embodiment, the gene is GB995_04720. In an embodiment, the gene is G7B65_12340. In an embodiment, the gene is S100434_00483. In an embodiment, the gene is GB995 04960. In an embodiment, the gene is LAP8962 01024. In an embodiment, the gene is ADS73 11700. In an embodiment, the gene is IAR49 00640. In an embodiment, the gene is S101258 03346. In an embodiment, the gene is H6X79 02665. In an embodiment, the gene is LPJSA22 02538. In an embodiment, the gene is C7M31 00572. In an embodiment, the gene is C7M32_02485. In an embodiment, the gene is C7M33_00571. In an embodiment, the gene is Nizo2262_1509. In an embodiment, the gene is C1940_13880. In an embodiment, the gene isNizo3894_1514. In an embodiment, the gene is CFN49_13740.

[0123] In an embodiment, the lactic acid bacteria produce a glycosyltransferase encoded by a gene selected from one or more of: H2060 03810, DQM12 01810, JK161 01400, BMS99 08665, J6K68_08370, J3330_05935, EZV74_01260, A0F18_09060, CD198_06875, CD198_06870, gtfC_6, gtfC_2, HFP47_05615, EQK02_06830, NX809_06765, MIl_07585, ARA01_07835, H2060_03815, EQZ98_07210, sfcA, N876_0213650, N876_04935 or A9F05_07390. In an embodiment, the gene is H2060 03810. In an embodiment, the gene is DQM12 01810. In an embodiment, the gene is JK161 01400. In an embodiment, the gene is BMS99 08665. In an embodiment, the gene is J6K68 08370. In an embodiment, the gene is J3330 05935. In an embodiment, the gene is EZV74 01260. In an embodiment, the gene is A0F18 09060. In an embodiment, the gene is CD198 06875. In an embodiment, the gene is CD198 06870. In an embodiment, the gene is gtfC_6. In an embodiment, the gene is gtfC_2. In an embodiment, the gene is HFP47 05615. In an embodiment, the gene is EQK02 06830. In an embodiment, the gene is NX809 06765. In an embodiment, the gene is MI1 07585. In an embodiment, the gene is ARA01 07835. In an embodiment, the gene is H2060 03815. In an embodiment, the gene is EQZ98 07210. In an embodiment, the gene is sfcA. In an embodiment, the gene is N876 0213650. In an embodiment, the gene is N876_04935. In an embodiment, the gene is A9F05_07390.

[0124] In an embodiment, the mannitol dehydrogenase is mannitol- 1 -phosphate 5-dehydrogenase.

[0125] In an embodiment, the lactic acid bacteria produce mannitol dehydrogenase encoded by a gene selected from one or more of: mtlD, Lpl6_0215, H073 13211, L103_03153, N574_15285, mdh, SH83_00985, ALX04_13535, N876_0202495 or FXE15 01185. In an embodiment, the gene is mtlD. In an embodiment, the gene is Lpl6_0215. In an embodiment, the gene is H073 13211. In an embodiment, the gene is L103 03153. In an embodiment, the gene is N574_15285. In an embodiment, the gene is mdh. In an embodiment, the gene is SH83 00985. In an embodiment, the gene is ALX04_13535. In an embodiment, the gene is N876_0202495. In an embodiment, the gene is FXE15_01185.

[0126] In an embodiment, the lactic acid bacteria produce a malolactic enzyme encoded by a gene selected from one or more of: J8138 12190, mleS, 1526 0921, Nizo2726_2007, Nizo2830_2922, LEUM_1005, HFP47_03725, maeA, DQM12_10300, Nizo2891_2382, Nizo2535_2209, KGI16_04450 or RI536J2465. In an embodiment, the gene is J8138 12190. In an embodiment, the gene is mleS. In an embodiment, the gene is I526_0921. In an embodiment, the gene is Nizo2726_2007. In an embodiment, the gene is Nizo2830_2922. In an embodiment, the gene is LEUM 1005. In an embodiment, the gene is HFP47 03725. In an embodiment, the gene is maeA. In an embodiment, the gene is DQM12 10300. In an embodiment, the gene is Nizo2891_2382. In an embodiment, the gene is Nizo2535_2209. In an embodiment, the gene is KGI16 04450. In an embodiment, the gene is RI536 12465.

[0127] In an embodiment, the lactic acid bacteria produce greater than 1 mg / mL of exopolysaccharides. In an embodiment, the lactic acid bacteria produce greater than 1 mg / mL of exopolysaccharides when cultivated at 37°C for 20 hours with 10% sucrose supplementation. In an embodiment, the lactic acid bacteria produce about 1 mg / mL of exopolysaccharides to about 15 mg / mL when cultivated at 37°C for 20 hours with 10% sucrose supplementation. In an embodiment, exopolysaccharides are predominantly HOPS.

[0128] In an embodiment, the lactic acid bacteria are Leuconostoc mesenteroides. In an embodiment, the Leuconostoc mesenteroides xdwa isolated from Umbelliferae . In an embodiment, the Leuconostoc mesenteroides produces malolactic enzyme.

[0129] In an embodiment, the Leuconostoc mesenteroides is selected from one or more of: i) Leuconostoc mesenteroides BF1 deposited under V17 / 021729 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof; ii) Leuconostoc mesenteroides BF2 deposited under V17 / 021730 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof; iii) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; iv) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof; v) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; vi) Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and vii) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0130] In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides BF1 deposited under VI 7 / 021729 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides BF2 deposited under V17 / 021730 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof. In an embodiment, the Leuconostoc mesenteroides is Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0131] In an embodiment, the primary fermentation is for about 10 to about 24 hours. In an embodiment, the primary fermentation is for about 10 hours to about 20 days. In an embodiment, the primary fermentation is for about 10 hours to about 17 days. In an embodiment, the primary fermentation is for about 10 hours to about 15 days. In an embodiment, fermentation is for about 10 days. In an embodiment, fermentation is for about 13 days. In an embodiment, fermentation is for about 15 days. In an embodiment, fermentation is for about 18 days. In an embodiment, fermentation is for about 20 days. In an embodiment, fermentation is for about 24 days.

[0132] In an embodiment, the primary fermentation occurs till the pH is reduced to about 4.1. In an embodiment, the primary fermentation occurs till the pH is reduced to about 4. In an embodiment, the primary fermentation occurs till the pH is reduced to about 3.9. In an embodiment, the primary fermentation is at about 22°C to about 34°C. In an embodiment, the primary fermentation is at about 26°C to about 34°C. In an embodiment, the primary fermentation is at about 28°C to about 34°C. In an embodiment, the primary fermentation is at about 28°C to about 32°C. In an embodiment, the primary fermentation is at about 30°C.

[0133] In an embodiment, the lactic acid bacteria are deactivated after step i). In an embodiment, the lactic acid bacteria are deactivated by the addition of a bactericidal agent.

[0134] In an embodiment, the lactic acid bacteria are removed after step i). In an embodiment, the lactic acid bacteria are removed by microfiltration.

[0135] In an embodiment, the bactericidal agent is selected from sulphur dioxide, potassium metabisulphite or dimethyl decarbonate. In an embodiment, the bactericidal agent is sulphur dioxide. In an embodiment, the bactericidal agent is potassium metabisulphite. In an embodiment, the bactericidal agent is dimethyl decarbonate.

[0136] In an embodiment, after step i) the pH is adjusted to a pH of about 3.4 to about 3.7. In an embodiment, after step i) the pH is adjusted to a pH of about 3.5 to about 3.6. In an embodiment, the pH is adjusted with the addition of tartaric acid.

[0137] In an embodiment, the fermentation culture is stirred. In an embodiment, stirring is intermittent. In an embodiment, stirring is continuous.

[0138] In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass by about 10% to about 70%. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass by about 20% to about 50%. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass by about 20% to about 40%. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass by about 25% to about 40%. In an embodiment, the primary fermentation reduces the total fermentable sugar in the base alcohol production biomass by at least 25%. In an embodiment, the primary fermentation reduces the total fermentable sugar in the base alcohol production biomass by at least 33%. In an embodiment, the primary fermentation reduces the total fermentable sugar in the base alcohol production biomass by at least 44%.

[0139] In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by about 30% to about 50%. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by about 20% to about 50%. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by about 20% to about 40%. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by at least 30%. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by at least 35%. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass by at least 45%.

[0140] In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by about 30% to about 50%. In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by about 20% to about 50%. In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by about 20% to about 40%. In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by at least 30%. In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by at least 35%. In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass by at least 45%.

[0141] In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass to about 150 g / L to about lOOg / L. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass to about 120 g / L to about 140 g / L. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass to at least about 100 g / L. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass to at least about 120 g / L. In an embodiment, the primary fermentation reduces the total sugar in the base alcohol production biomass to at least about 130 g / L.

[0142] In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass to about 40 g / L to about 60g / L. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass to at least about 40 g / L. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass to at least about 50 g / L.

[0143] In an embodiment, the primary fermentation reduces the glucose in the base alcohol production biomass to about 60 g / L to about 85g / L. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass to at least about 80 g / L. In an embodiment, the primary fermentation reduces the fructose in the base alcohol production biomass to at least about 70 g / L.

[0144] In an embodiment, the primary fermentation increases the mannitol in the base alcohol production biomass. In an embodiment, the primary fermentation increases the mannitol in the base alcohol production biomass to about 10 g / L to about 55 g / L. In an embodiment, the primary fermentation increases the mannitol in the base alcohol production biomass to about 47 g / L. In an embodiment, the primary fermentation reduced the growth of yeast in the base alcohol production biomass.

[0145] In an embodiment, the primary fermentation reduces the alcohol concentration in the final product by about 24% to about 70% compared to a final product prepared using the same method lacking the primary fermentation step. In an embodiment, the primary fermentation reduces the alcohol concentration in the final product by about 24% to about 45% compared to a final product prepared using the same method lacking the primary fermentation step. In an embodiment, the primary fermentation reduces the alcohol concentration in the final product by about 24% to about 42% compared to a final product prepared using the same method lacking the primary fermentation step. In an embodiment, the primary fermentation reduces the alcohol concentration in the final product by at least about 24% compared to a final product prepared using the same method lacking the primary fermentation step. In an embodiment, the primary fermentation reduces the alcohol concentration in the final product by at least about 41% compared to a final product prepared using the same method lacking the primary fermentation step. In an embodiment, the final product is a wine. In an embodiment, the primary fermentation reduces the alcohol concentration in Sauvignon Blanc by at least about 42%. In an embodiment, the primary fermentation reduces the alcohol concentration in Grenache by at least about 41%. In an embodiment, the primary fermentation reduces the alcohol concentration in Shiraz by at least about 24%.

[0146] Secondary alcohol fermentation

[0147] As used herein “secondary alcohol fermentation” refers to fermentation of the material from i) with yeast to product alcohol. A person skilled in the art will appreciate that there are a number of different yeasts, including various wine fermentation yeasts, suitable for use in the methods described herein. These include, for example, those described in Chen et al., 2024, Romano et al., 2003, Oliveira et al., 2005 and Hranilovic et al., 2020).

[0148] In some embodiments, the skin of the biomass is added before secondary fermentation.

[0149] In an embodiment, the yeast is suitable for producing wine. In an embodiment, the yeast is suitable for producing cider.

[0150] In an embodiment, the yeast is a Saccharomyces. In an embodiment, the yeast is a non-Saccharomyces cerevisiae (for example as described in Chen et al., 2024, Oliveira et al., 2005, Romano et al., 2003). In an embodiment, the non-Saccharomyces cerevisiae yeast is selected from the genera: Candida, Pichia, Candida stellata, Zigosaccharomyces fermentati, Hanseniaspora, Schizosaccharomyces, Dekkera, Metschnikowia, and Zygosaccharomyces.

[0151] In an embodiment, the non-Saccharomyces cerevisiae yeast is Candida. In an embodiment, the non-Saccharomyces cerevisiae yeast is Pichia. In an embodiment, the non-Saccharomyces cerevisiae yeast is Candida stellata. In an embodiment, the non-Saccharomyces cerevisiae yeast is Hanseniaspora. In an embodiment, the non-Saccharomyces cerevisiae yeast is Schizosaccharomyces. In an embodiment, the non-Saccharomyces cerevisiae yeast is Dekkera. In an embodiment, the non-Saccharomyces cerevisiae yeast is Metschnikowia. In an embodiment, the non-Saccharomyces cerevisiae yeast is Zygosaccharomyces .

[0152] In an embodiment, the yeast is selected from one or more of: Saccharomyces cerevisiae, Pichia stipitis and Candida tropicalis. In an embodiment, the yeast is Saccharomyces cerevisiae. In an embodiment, the yeast is Pichia stipites. In an embodiment, yeast is Candida tropicalis.

[0153] In an embodiment, the Saccharomyces cerevisiae is selected from: LALVIN EC 1118 and VitiFerm™ Rubion Extra Bio. In an embodiment, the Saccharomyces cerevisiae is LALVIN EC 1118. In an embodiment, the Saccharomyces cerevisiae is VitiFerm™ Rubion Extra Bio.

[0154] In an embodiment, the yeast is a low alcohol yeast (for example as described in Hranilovic et al., 2020).

[0155] In an embodiment, the secondary alcohol fermentation comprises malolactic fermentation.

[0156] In an embodiment, secondary fermentation is for about 5 days to about 40 days. In an embodiment, secondary fermentation is for about 10 days to about 40 days. In an embodiment, secondary fermentation is for about 10 days to about 38 days. In an embodiment, secondary fermentation is for about 15 days to about 35 days. In an embodiment, secondary fermentation is for about 20 days to about 33 days. In an embodiment, secondary fermentation is for about 25 days to about 30 days. In embodiment, secondary fermentation is for about 10 days. In embodiment, secondary fermentation is for about 15 days. In embodiment, secondary fermentation is for about 20 days. In embodiment, secondary fermentation is for about 25 days. In embodiment, secondary fermentation is for about 30 days. In embodiment, secondary fermentation is for about 35 days. In embodiment, secondary fermentation is for about 40 days. In an embodiment, secondary fermentation is at about 15°C to about 34°C. In an embodiment, secondary fermentation is at about 15°C to about 32°C. In an embodiment, secondary fermentation is at about 15°C to about 30°C. In an embodiment, secondary fermentation is at about 18°C to about 30°C. In an embodiment, secondary fermentation is at about 20°C to about 30°C. In an embodiment, secondary fermentation is at about 22°C to about 30°C. In an embodiment, secondary fermentation is at about 24°C to about 30°C. In an embodiment, secondary fermentation is at about 26°C to about 30°C. In an embodiment, secondary fermentation is at about 28°C to about 30°C.

[0157] In an embodiment, the secondary fermentation is anaerobic.

[0158] Post-treatment

[0159] In some embodiments, the method as described herein comprises post-treating the product described herein.

[0160] In an embodiment, the method as described herein comprises post-treating after step ii) of the method as described herein.

[0161] In some embodiments, post-treating comprises microfiltration.

[0162] In some embodiments, post-treating comprises fining. In an embodiment, the finning agent is selected from one or more of: gelatine, isinglass, egg white (egg albumen), casein, skim milk, bentonite, carbon and polyvinylpolypyrrolidone (PVPP).

[0163] In some embodiments, post-treating comprises the addition of preservatives. In an embodiment, the preservative is selected from one or more of: sulphur dioxide and potassium metabisulphite, dimethyl dicarbonate (Velcorin®), potassium sorbate, calcium propionate, sodium benzoate, benzoic acid, propionic acid and sorbic acid.

[0164] In some embodiments, post-treatment comprises addition of an antioxidant. In an embodiment the antioxidant is ascorbic acid.

[0165] In some embodiments, post-treating comprises addition of further biomass. In some embodiments, the biomass is a base alcohol production biomass as described herein. In some embodiments, post-treating comprises reverse osmosis. In an embodiment, the further biomass is juice. In an embodiment, the further biomass is must.

[0166] In some embodiment, post-treating comprises a de-alcoholising treatment selected from one or more of: vacuum distillation, evaporation, distillation, freezeconcentration, solvent extraction and membrane filtration techniques such as reverse osmosis and dialysis for alcohol removal (Liguori et al., 2018; Pickering, 2001; Schmidtke et al., 2012).

[0167] In some embodiments, post-treating comprises inactivating microbes. As used herein “microbes” refers to bacterial, viral, fungal or eukaryotic activity that can result in degradation or spoilage of the isothiocyanate containing product. As used herein “inactivate” or “inactivation” of microbes refers to reducing the viable microbes by about 1 to about 7 logs. In an embodiment, the viable microbes are reduced by about 1 to 6 logs. In an embodiment, the viable microbes are reduced by about 2 to 6 logs. In an embodiment, the viable microbes are reduced by about 3 to 6 logs. In an embodiment, the microbe is a fermentation bacteria or yeast used in step i) or ii).

[0168] In some embodiments, post-treating comprises malolactic fermentation (a separate malolactic fermentation).

[0169] In some embodiments, post treating comprises reducing the level of acetic acid.

[0170] Reduction of acetic acid level

[0171] In some embodiments, the method as described herein comprises reducing the level of acetic acid before step ii) and / or after step ii). In some embodiments, the level of acetic acid is reduced before step ii). In some embodiment, the level of acetic is reduced after step ii).

[0172] In some embodiments, acetic acid is removed via absorption onto a weak anionic resin or activated carbon. In some embodiments, the weak anionic resin is selected from one or more of: Amberlite IRA-67, Amberlyst A21, Dowex optipore L-493, Indion 850, Tuision A-8X MP, Indion 810, Amberlite IRA-96, Purolite A1335, Sepra NH2, Sepra SAX, Sepra ZT-AX, Dowex 1x8-100 (CI), Dowex 21K XLT, Dowex marathon A2 (CI), Amberlite IRA-400 (CI), Amberlite IRA-900 (CI),Daion HP-20 or polyvinyl pyrrolidone (PVP) resins such as reillex 425. In some embodiments, the weak anionic resin is Amberlite IRA-67. In some embodiments, the weak anionic resin is Amberlyst A21. In some embodiments, the weak anionic resin is Dowex optipore L-493. In some embodiments, the weak anionic resin is Indion 850. In some embodiments, the weak anionic resin is Tuision A-8X MP. In some embodiments, the weak anionic resin is Indion 810. In some embodiments, the weak anionic resin is Amberlite IRA-96. In some embodiments, the weak anionic resin is Purolite A1335. In some embodiments, the weak anionic resin is Sepra NH2. In some embodiments, the weak anionic resin is Sepra SAX. In some embodiments, the weak anionic resin is Sepra ZT-AX. In some embodiments, the weak anionic resin is Dowex 1x8-100 (CI). In some embodiments, the weak anionic resin is Dowex 2 IK XLT. In some embodiments, the weak anionic resin is Dowex marathon A2 (CI). In some embodiments, the weak anionic resin is Amberlite IRA-400 (CI). In some embodiments, the weak anionic resin is Amberlite IRA-900 (CI). In some embodiments, the weak anionic resin is Daion HP -20. In some embodiments, the resin is a reillex 425. In some embodiments, acetic acid is removed via absorption onto activated carbon.

[0173] In some embodiments, acetic acid is removed with electro-membrane processing. In an embodiment, the level of acetic acid is reduced by about 40% to about 99%. In an embodiment, the level of acetic acid is reduced by about 40% to about 95%. In an embodiment, the level of acetic acid is reduced by about 40% to about 90%. In an embodiment, the level of acetic acid is reduced by about 40% to about 85%. In an embodiment, the level of acetic acid is reduced by about 40% to about 80%. In an embodiment, the level of acetic acid is reduced by about 40% to about 70%. In an embodiment, the level of acetic acid is reduced by about 40% to about 60%. In an embodiment, the level of acetic acid is reduced by at least about 60%. In an embodiment, the level of acetic acid is reduced by at least about 40%. In an embodiment, the level of acetic acid is reduced by at least about 40%.

[0174] In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 1.5 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 1.4 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 1.2 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 1 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 0.8 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L to about 0.5 g / L acetic acid.

[0175] In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 1.5 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 1.4 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 1.2 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 1 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 0.8 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L to about 0.5 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.5 g / L to about 1.5 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.5 g / L to about 1.4 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.5 g / L to about 1.2 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.5 g / L to about 1 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.5 g / L to about 0.8 g / L acetic acid.

[0176] In an embodiment, the level of acetic acid is reduced to about 1.5 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 1.4 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 1.2 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 1 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.8 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.6 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.4 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.2 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.1 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.05 g / L acetic acid. In an embodiment, the level of acetic acid is reduced to about 0.01 g / L acetic acid.

[0177] Method of producing fermentation enzymes

[0178] In an aspect, the present invention provides a method of producing a lactic acid bacteria enzyme composition comprising primary fermentation of a base alcohol production biomass with lactic acid bacteria to produce a fermented product comprising fermentation enzymes.

[0179] In some embodiments, the lactic acid bacteria are removed after fermentation. In some embodiments, the lactic acid bacteria are deactivated after fermentation.

[0180] In some embodiments, the fermented material, an extract thereof, or dried powder produced from the fermented material or an extract thereof, is incubated with unfermented base alcohol production biomass.

[0181] In some embodiments, the ratio of fermented material to unfermented base alcohol production biomass is about 1:100 to about 1:5.

[0182] In some embodiments, the ratio of fermented material to unfermented base alcohol production biomass is about 1:5.

[0183] In some embodiments, the incubation is for about 2 to about 50 hours at about 25°C to about 50°C. In some embodiments, the incubation is for about 5 to about 50 hours at about 25°C to about 50°C. In some embodiments, the incubation is for about 10 to about 50 hours at about 25°C to about 50°C. In some embodiments, the incubation is for about 15 to about 50 hours at about 25°C to about 50°C. In some embodiments, the incubation is for about 20 to about 50 hours at about 25°C to about 50°C. In some embodiments, the incubation is for about 20 to about 50 hours at about 30°C to about 45°C. In some embodiments, the incubation is for about 20 to about 50 hours at about 35°C to about 40°C.

[0184] In some embodiments, the incubation is for about 24 to about 48 hours at about 25°C to about 35°C.

[0185] In some embodiments, the incubation is for about 24 to about 48 hours at about 30°C. In some embodiments, the incubated product is stored at about 1°C to about 14°C. In some embodiments, the incubated product is stored at about 1°C to about 4°C. In some embodiments, the incubated product is frozen.

[0186] In some embodiments, the incubated product is dried.

[0187] Fermentation enzymes

[0188] In an aspect, the present invention provides a lactic acid bacteria enzyme composition.

[0189] In an aspect, the present invention provides a lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product obtained, or obtainable, by the method as described herein.

[0190] In an aspect, the present invention provides a lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product, wherein the composition comprises fermentation enzymes and exopolysaccharides.

[0191] In an embodiment, the composition comprises exopolysaccharides. In an embodiment, the exopolysaccharides are homopolysaccharides (HoPS). In an embodiment, the homopolysaccharides comprise dextran. In an embodiment, the homopolysaccharides comprise levan.

[0192] In an embodiment, the composition comprises an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme oxalacetate dehydrogenase, and a glycosyltransferase. In an embodiment, the composition comprises malic enzyme. In an embodiment, the composition comprises L-lactate dehydrogenase. In an embodiment, the composition comprises malate dehydrogenase. In an embodiment, the composition comprises mannitol dehydrogenase. In an embodiment, the composition comprises malolactic enzyme. In an embodiment, the composition comprises oxalacetate dehydrogenase. In an embodiment, the composition comprises a glycosyltransferase. In an embodiment, the glycosyltransferase is dextransucrase. In an embodiment, the glycosyltransferase is levansucrase. In an embodiment, the glycosyltransferase is alternansucrase. In an embodiment, the glycosyltransferase is a glycoside hydrolase family 68 protein. In an embodiment, the glycosyltransferase is a glycoside hydrolase family 70 protein. In an embodiment, the composition comprises dextransucrase. In an embodiment, the composition comprises levansucrase. In an embodiment, the glycosyltransferase is alternansucrase.

[0193] In an embodiment, the composition does not comprise inulosucrase and / or reuteransucrase. In an embodiment, the composition comprises an enzyme encoded by a gene selected from one or more of: gtfA, LCIT_00500, LEUM_1409, LEUM_1747, DIS10 08860, LEUM_1410, LEUM_1411, LEMES_01041, H2060_03810, LPJSA22_01242, LPJSA22_01063, levS_2, NHN79_00835, KE630_00215, levS_l, C1T23_O1282, sacB, sacB_2, sacB_3, levS_3, EUZ72_10755, NQL29_06645, EFP04_04965, LAP8962_01701, ASU25_00535, levS_4, S101174_00409, SAC12_0369, DQM12_01810, ADS73_11930, Nizol839_2806, LJA01_10220, SF2A35B_1062, JK161_01400, CHH57_18525, C2I06_16290, HLK66_22900, C2I17_14445, C2H98 18060, levS, S100434_00436, GB995_04720, G7B65_12340, S100434_00483, GB995_04960, S102022_02672, LAP8962_01024, ADS73_11700, IAR49_00640, SRCM101060_02609, S101258 01184, N876_05150, BMS99_08665, S 101520_02514, D2U09_07590, FXE14_08880, CJP43J4315, DB342_0690, S101258_03346, S102022_01132, H6X79_02665, J6K68_08370, J3330_05935, LPJSA22_02538, EZV74_01260, A0F18_09060, CD198_06875, CD198_06870, gtfC_6, gtfC_2, HFP47 05615, EQK02_06830, NX809_06765, MIl_07585, ARA01_07835, H2060_03815, EQZ98_07210, C7M31_00572, C7M32_02485, C7M33_00571,Nizo2262_1509, C1940J3880, Nizo3894_1514, CFN49J3740, mtlD, Lpl6_0215, H073J3211, L103_03153, N574J5285, mdh, SH83_00985, ALX04_13535, N876_0202495, FXE15_01185, J8138 12190, mleS, I526_0921, Nizo2726_2007, Nizo2830_2922, LEUM_1005, HFP47_03725, maeA, DQM12_10300, Nizo2891_2382, Nizo2535_2209, KGI16_04450, RI536_12465, sfcA, N876_0213650, N876_04935, A9F05_07390, HFP47_07275, dsrF, LEUM_1752 and LEUM 0857. In an embodiment, the gene is gtfA. In an embodiment, the gene is LCIT 00500. In an embodiment, the gene is LEUM 1409. In an embodiment, the gene is LEUM 1747. In an embodiment, the gene is DIS10 08860. In an embodiment, the gene is LEUM 1410. In an embodiment, the gene is LEUM 1411. In an embodiment, the gene is LEMES 01041. In an embodiment, the gene is H2060 03810. In an embodiment, the gene is LPJSA22 01242. In an embodiment, the gene is LPJSA22 01063. In an embodiment, the gene is levS_2. In an embodiment, the gene is NHN79 00835. In an embodiment, the gene is KE630 00215. In an embodiment, the gene is levS l. In an embodiment, the gene is C1T23 01282. In an embodiment, the gene is sacB. In an embodiment, the gene is sacB_2. In an embodiment, the gene is sacB_3. In an embodiment, the gene is levS_3. In an embodiment, the gene is EUZ72 10755. In an embodiment, the gene is NQL29 06645. In an embodiment, the gene is EFP04 04965. In an embodiment, the gene is LAP8962 01701. In an embodiment, the gene is ASU25 00535. In an embodiment, the gene is levS_4. In an embodiment, the gene is S101174_00409. In an embodiment, the gene is SAC12_0369. In an embodiment, the gene is DQM12 01810. In an embodiment, the gene is ADS73 11930. In an embodiment, the gene is Nizol839_2806. In an embodiment, the gene is LJA01 10220. In an embodiment, the gene is SF2A35B 1062. In an embodiment, the gene is JK161 01400. In an embodiment, the gene is CHH57 18525. In an embodiment, the gene is C2I06 16290. In an embodiment, the gene is HLK66 22900. In an embodiment, the gene is C2I17 14445. In an embodiment, the gene is C2H98 18060. In an embodiment, the gene is levS. In an embodiment, the gene is S100434 00436. In an embodiment, the gene is GB995 04720. In an embodiment, the gene is G7B65_12340. In an embodiment, the gene is S100434_00483. In an embodiment, the gene is GB995_04960. In an embodiment, the gene is S102022_02672. In an embodiment, the gene is LAP8962 01024. In an embodiment, the gene is ADS73 11700. In an embodiment, the gene is IAR49 00640. In an embodiment, the gene is SRCM101060 02609. In an embodiment, the gene is S101258 01184. In an embodiment, the gene isN876_05150. In an embodiment, the gene is BMS99 08665. In an embodiment, the gene is S101520 02514. In an embodiment, the gene is D2U09 07590. In an embodiment, the gene is FXE14 08880. In an embodiment, the gene is CJP43 14315. In an embodiment, the gene is DB342 0690. In an embodiment, the gene is S101258_03346. In an embodiment, the gene is S102022_01132. In an embodiment, the gene is H6X79 02665. In an embodiment, the gene is J6K68 08370. In an embodiment, the gene is J3330 05935. In an embodiment, the gene is LPJSA22 02538. In an embodiment, the gene is EZV74 01260. In an embodiment, the gene is A0F18 09060. In an embodiment, the gene is CD198 06875. In an embodiment, the gene is CD198 06870. In an embodiment, the gene is gtfC_6. In an embodiment, the gene is gtfC_2. In an embodiment, the gene is HFP47 05615. In an embodiment, the gene is EQK02 06830. In an embodiment, the gene is NX809 06765. In an embodiment, the gene is MI1 07585. In an embodiment, the gene is ARA01 07835. In an embodiment, the gene is H2060 03815. In an embodiment, the gene is EQZ98 07210. In an embodiment, the gene is C7M31 00572. In an embodiment, the gene is C7M32_02485. In an embodiment, the gene is C7M33_00571. In an embodiment, the gene is Nizo2262_1509. In an embodiment, the gene is C1940_13880. In an embodiment, the gene isNizo3894_1514. In an embodiment, the gene is CFN49_13740. In an embodiment, the gene is mtlD. In an embodiment, the gene is Lpl6_0215. In an embodiment, the gene is H073 13211. In an embodiment, the gene is L103 03153. In an embodiment, the gene is N574 15285. In an embodiment, the gene is mdh. In an embodiment, the gene is SH83 00985. In an embodiment, the gene is ALX04 13535. In an embodiment, the gene is N876_0202495. In an embodiment, the gene is FXE15 01185. In an embodiment, the gene is J8138 12190. In an embodiment, the gene is mleS. In an embodiment, the gene is 1526 0921. In an embodiment, the gene is Nizo2726_2007. In an embodiment, the gene is Nizo2830_2922. In an embodiment, the gene is LEUM 1005. In an embodiment, the gene is HFP47 03725. In an embodiment, the gene is maeA. In an embodiment, the gene is DQM12 10300. In an embodiment, the gene is Nizo2891_2382. In an embodiment, the gene is Nizo2535_2209. In an embodiment, the gene is KGI16 04450. In an embodiment, the gene is RI536 12465. In an embodiment, the gene is sfcA. In an embodiment, the gene is N876 0213650. In an embodiment, the gene is N876_04935. In an embodiment, the gene is A9F05_07390. In an embodiment, the gene is HFP47 07275. In an embodiment, the gene is dsrF. In an embodiment, the gene is LEUM 1752. In an embodiment, the gene is LEUM 0857.

[0194] In an embodiment, the composition comprises dextransucrase encoded by a gene selected from one or more of: gtfA, LCIT_00500, LEUM_1747, LEMES_01041, LPJSA22_01242, LPJSA22_01063, S101174_00409, SAC12_0369, S102022_02672, SRCM101060_02609, S 101258_01184, N876_05150, S 101520_02514, D2U09_07590, FXE14_08880, CJP43_14315, DB342_0690, S102022_01132, HFP47_07275, dsrF, LEUM 1752 and LEUM 0857. In an embodiment, the gene is gtfA. In an embodiment, the gene is LCIT 00500. In an embodiment, the gene is LEUM 1747. In an embodiment, the gene is LEMES 01041. In an embodiment, the gene is LPJSA22 01242. In an embodiment, the gene is LPJSA22 01063. In an embodiment, the gene is S101174 00409. In an embodiment, the gene is SAC12 0369. In an embodiment, the gene is S102022_02672. In an embodiment, the gene is SRCM101060_02609. In an embodiment, the gene is S101258 01184. In an embodiment, the gene is N876_05150. In an embodiment, the gene is S101520 02514. In an embodiment, the gene is D2U09 07590. In an embodiment, the gene is FXE14 08880. In an embodiment, the gene is CJP43 14315. In an embodiment, the gene is DB342 0690. In an embodiment, the gene is S102022_01132. In an embodiment, the gene is HFP47_07275. In an embodiment, the gene is dsrF. In an embodiment, the gene is LEUM 1752. In an embodiment, the gene is LEUM 0857.

[0195] In an embodiment, the composition comprises levansucrase encoded by a gene selected from one or more of: LEUM_1409, LEUM_1410, LEUM_1411, levS_2, NHN79_00835, KE630_00215, levS_l, C1T23_O1282, sacB, sacB_2, sacB_3, levS_3, EUZ72J0755, NQL29_06645, EFP04_04965, LAP8962_01701, ASU25_00535, levS_4, ADS73_11930, Nizol839_2806, LJA01_10220, SF2A35B_1062, CHH57 18525, C2I06_16290, HLK66_22900, C2I17_14445, C2H98_18060, levS, S100434_00436, GB995_04720, G7B65J2340, S100434_00483, GB995_04960, LAP8962_01024, ADS73 1700, IAR49_00640, S101258_03346, H6X79_02665, LPJSA22_02538, C7M31_00572, C7M32_02485, C7M33_00571, Nizo2262_1509, C1940_13880, Nizo3894_1514 and CFN49_13740. In an embodiment, the gene is LEUM 1409. In an embodiment, the gene is LEUM 1410. In an embodiment, the gene is LEUM 1411. In an embodiment, the gene is levS_2. In an embodiment, the gene is NHN79 00835. In an embodiment, the gene is KE630 00215. In an embodiment, the gene is C1T23 01282. In an embodiment, the gene is sacB. In an embodiment, the gene is sacB_2. In an embodiment, the gene is sacB_3. In an embodiment, the gene is levS_3. In an embodiment, the gene is EUZ72 10755. In an embodiment, the gene is NQL29 06645. In an embodiment, the gene is EFP04 04965. In an embodiment, the gene is LAP8962_01701. In an embodiment, the gene is ASU25_00535. In an embodiment, the gene is levS_4. In an embodiment, the gene is ADS73 11930. In an embodiment, the gene is Nizol839_2806. In an embodiment, the gene is LJA01 10220. In an embodiment, the gene is SF2A35B 1062. In an embodiment, the gene is CHH57 18525. In an embodiment, the gene is C2I06 16290. In an embodiment, the gene is HLK66 22900. In an embodiment, the gene is C2I17 14445. In an embodiment, the gene is C2H98 18060. In an embodiment, the gene is levS. In an embodiment, the gene is S100434_00436. In an embodiment, the gene is GB995_04720. In an embodiment, the gene is G7B65_12340. In an embodiment, the gene is S100434_00483. In an embodiment, the gene is GB995 04960. In an embodiment, the gene is LAP8962 01024. In an embodiment, the gene is ADS73 11700. In an embodiment, the gene is IAR49 00640. In an embodiment, the gene is S101258 03346. In an embodiment, the gene is H6X79 02665. In an embodiment, the gene is LPJSA22 02538. In an embodiment, the gene is C7M31 00572. In an embodiment, the gene is C7M32_02485. In an embodiment, the gene is C7M33_00571. In an embodiment, the gene is Nizo2262_1509. In an embodiment, the gene is C1940_13880. In an embodiment, the gene isNizo3894_1514. In an embodiment, the gene is CFN49_13740.

[0196] In an embodiment, the composition comprises a glycosyltransferase encoded by a gene selected from one or more of: H2060 03810, DQM12 01810, JK161 01400, BMS99 08665, J6K68_08370, J3330_05935, EZV74_01260, A0F18_09060, CD198_06875, CD198_06870, gtfC_6, gtfC_2, HFP47_05615, EQK02_06830, NX809_06765, MIl_07585, ARA01_07835, H2060_03815, EQZ98_07210, sfcA, N876_0213650, N876_04935 and A9F05_07390. In an embodiment, the gene is H2060 03810. In an embodiment, the gene is DQM12 01810. In an embodiment, the gene is JK161 01400. In an embodiment, the gene is BMS99 08665. In an embodiment, the gene is J6K68 08370. In an embodiment, the gene is J3330 05935. In an embodiment, the gene is EZV74 01260. In an embodiment, the gene is A0F18 09060. In an embodiment, the gene is CD198 06875. In an embodiment, the gene is CD198 06870. In an embodiment, the gene is gtfC_6. In an embodiment, the gene is gtfC_2. In an embodiment, the gene is HFP47 05615. In an embodiment, the gene is EQK02 06830. In an embodiment, the gene is NX809 06765. In an embodiment, the gene is MI1 07585. In an embodiment, the gene is ARA01 07835. In an embodiment, the gene is H2060 03815. In an embodiment, the gene is EQZ98 07210. In an embodiment, the gene is sfcA. In an embodiment, the gene is N876 0213650. In an embodiment, the gene is N876_04935. In an embodiment, the gene is A9F05_07390.

[0197] In an embodiment, the mannitol dehydrogenase is mannitol- 1 -phosphate 5-dehydrogenase.

[0198] In an embodiment, the composition comprises mannitol dehydrogenase encoded by a gene selected from one or more of: mtlD, Lpl6_0215, H073 13211, L103 03153, N574_15285, mdh, SH83_00985, ALX04_13535, N876_0202495 and FXE15_01185. In an embodiment, the gene is mtlD. In an embodiment, the gene is Lpl6_0215. In an embodiment, the gene is H073 13211. In an embodiment, the gene is L103 03153. In an embodiment, the gene is N574 15285. In an embodiment, the gene is mdh. In an embodiment, the gene is SH83 00985. In an embodiment, the gene is ALX04 13535. In an embodiment, the gene is N876_0202495. In an embodiment, the gene is FXE15_01185.

[0199] In an embodiment, the composition comprises a malolactic enzyme encoded by a gene selected from one or more of: J8138 12190, mleS, I526_0921, Nizo2726_2007, Nizo2830_2922, LEUM_1005, HFP47_03725, maeA, DQM12_10300, Nizo2891_2382, Nizo2535_2209, KGI16_04450 and RI536J2465. In an embodiment, the gene is J8138 12190. In an embodiment, the gene is mleS. In an embodiment, the gene is I526_0921. In an embodiment, the gene is Nizo2726_2007. In an embodiment, the gene is Nizo2830_2922. In an embodiment, the gene is LEUM_1005. In an embodiment, the gene is HFP47 03725. In an embodiment, the gene is maeA. In an embodiment, the gene isDQM12_10300. In an embodiment, the gene isNizo2891_2382. In an embodiment, the gene is Nizo2535_2209. In an embodiment, the gene is KGI16 04450. In an embodiment, the gene is RI536 12465.

[0200] In an embodiment, the composition comprises coenzyme nicotinamide adenine dinucleotide phosphate (NADPH).

[0201] Products In an aspect, the present invention provides a product comprising low alcohol or reduced alcohol product obtained, or obtainable, by the method as described herein.

[0202] In an aspect, the present invention provides a double fermented low alcohol product.

[0203] In an aspect, the present invention provides a double fermented reduced alcohol product. In an embodiment, the product is a beverage. In an embodiment the product is wine. In an embodiment, the product is a cider.

[0204] As used herein, the “double fermented” product refers to product that is treated with a primary fermentation and a secondary fermentation as described herein (a lactic acid fermentation and yeast fermentation).

[0205] In an embodiment, the product is a low alcohol product. In an embodiment, the low alcohol product comprises about 0.1% to about 1.15% alcohol by volume.

[0206] In an embodiment, the product is a reduced alcohol product. In an embodiment the reduced alcohol product comprises about 0.1% to about 15% alcohol by volume. In an embodiment the reduced alcohol product comprises about 0.1% to about 13% alcohol by volume. In an embodiment the reduced alcohol product comprises about 0.1% to about 11% alcohol by volume. In an embodiment the reduced alcohol product comprises about 0.1% to about 9% alcohol by volume. In an embodiment the reduced alcohol product comprises about 0.5% to about 9% alcohol by volume. In an embodiment the reduced alcohol product comprises about 0.5% to about 8.5% alcohol by volume. In an embodiment the reduced alcohol product comprises about 1% to about 8% alcohol by volume. In an embodiment the reduced alcohol product comprises about 2% to about 7% alcohol by volume. In an embodiment the reduced alcohol product comprises about 3% to about 6% alcohol by volume. In an embodiment the reduced alcohol product comprises about 3.5% to about 5.5% alcohol by volume. In an embodiment the reduced alcohol product comprises about 4% to about 5% alcohol by volume. In an embodiment the reduced alcohol product comprises about 4.5% alcohol by volume. In an embodiment the reduced alcohol product comprises about 5% alcohol by volume.

[0207] In an embodiment, the product is a low alcohol wine. In an embodiment, the low alcohol wine comprises about 0.1% to about 1.15% alcohol by volume.

[0208] In an embodiment, the product is a reduced alcohol wine. In an embodiment the reduced alcohol wine comprises about 0.1% to about 15% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 14% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 13% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 12% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 11% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 10% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.1% to about 9% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.5% to about 9% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 0.5% to about 8.5% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 1% to about 8% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 2% to about 7% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 3% to about 6% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 3.5% to about 5.5% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 4% to about 5% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 5% alcohol by volume. In an embodiment the reduced alcohol wine comprises about 4.5% alcohol by volume.

[0209] In an embodiment, the product is low alcohol cider. In an embodiment, the product is reduced alcohol cider.

[0210] In an embodiment, product is a low sugar or reduced sugar product. In an embodiment, the product is a low sugar product. In an embodiment, the product is a reduced sugar product. In an embodiment, the composition comprises about 0 g / L to about 30 g / L total sugar. In an embodiment, the composition comprises about 1 g / L to about 28 g / L total sugar. In an embodiment, the composition comprises about 5 g / L to about 25 g / L total sugar. In an embodiment, the composition comprises about 10 g / L to about 20 g / L total sugar. In an embodiment, the composition comprises about 0 g / L to about 30 g / L fermentable sugar. In an embodiment, the composition comprises about 1 g / L to about 28 g / L fermentable sugar. In an embodiment, the composition comprises about 5 g / L to about 25 g / L fermentable sugar. In an embodiment, the composition comprises about 10 g / L to about 20 g / L fermentable sugar.

[0211] In an embodiment, the composition comprises about 0 g / L to about 30 g / L sucrose. In an embodiment, the composition comprises about 1 g / L to about 28 g / L sucrose. In an embodiment, the composition comprises about 5 g / L to about 25 g / L sucrose. In an embodiment, the composition comprises about 5 g / L to about 20 g / L sucrose. In an embodiment, the composition comprises about 5 g / L to about 15 g / L sucrose. In an embodiment, the composition comprises about 5 g / L to about 10 g / L sucrose.

[0212] In an embodiment, the composition comprises about 0 g / L to about 30

[0213]

[0214] glucose. In an embodiment, the composition comprises about 1 g / L to about 28 g / L glucose. In an embodiment, the composition comprises about 5 g / L to about 25 g / L glucose. In an embodiment, the composition comprises about 5 g / L to about 20 g / L glucose. In an embodiment, the composition comprises about 5 g / L to about 15 g / L glucose. In an embodiment, the composition comprises about 5 g / L to about 10 g / L glucose.

[0215] In an embodiment, the composition comprises about 0 g / L to about 30 g / L fructose. In an embodiment, the composition comprises about 1 g / L to about 28 g / L fructose. In an embodiment, the composition comprises about 5 g / L to about 25 g / L fructose. In an embodiment, the composition comprises about 5 g / L to about 20 g / L fructose. In an embodiment, the composition comprises about 5 g / L to about 15 g / L fructose. In an embodiment, the composition comprises about 5 g / L to about 10 g / L fructose.

[0216] In an embodiment, the product comprises one or more of: mannitol, fructan and glucan.

[0217] In an embodiment, the product comprises mannitol. In an embodiment, the product comprises about 5 g / L to about 100 g / L mannitol. In an embodiment, the comprises about 5 g / L to about 80 g / L mannitol. In an embodiment, the comprises about 5 g / L to about 70 g / L mannitol. In an embodiment, the comprises about 5 g / L to about 60 g / L mannitol. In an embodiment, the comprises about 5 g / L to about 50 g / L mannitol. In an embodiment, the comparative control product comprises 0 g / L of mannitol.

[0218] In an embodiment, the product comprises fructan. In an embodiment, the comprises about 0 g / L to about 175 g / L fructan. In an embodiment, the comprises about 0 g / L to about 125 g / L fructan. In an embodiment, the comprises about 0 g / L to about 75 g / L fructan.

[0219] In an embodiment, the product comprises glucan. In an embodiment, the comprises about 0 g / L to about 175 g / L glucan. In an embodiment, the comprises about 0 g / L to about 125 g / L glucan. In an embodiment, the comprises about 0 g / L to about 75 g / L glucan.

[0220] In an embodiment, the product comprises about 0.01 g / L to about 1.5 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L to about 1.4 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L to about 1.2 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L to about 1 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L to about 0.8 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L to about 0.5 g / L acetic acid.

[0221] In an embodiment, the product comprises about 0.05 g / L to about 1.5 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L to about 1.4 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L to about 1.2 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L to about 1 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L to about 0.8 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L to about 0.5 g / L acetic acid. In an embodiment, the product comprises about 0.5 g / L to about 1.5 g / L acetic acid. In an embodiment, the product comprises about 0.5 g / L to about 1.4 g / L acetic acid. In an embodiment the product comprises about 0.5 g / L to about 1.2 g / L acetic acid. In an embodiment, the product comprises about 0.5 g / L to about 1 g / L acetic acid. In an embodiment, the product comprises about 0.5 g / L to about 0.8 g / L acetic acid.

[0222] In an embodiment, the product comprises about 1.5 g / L acetic acid. In an embodiment, the product comprises about 1.4 g / L acetic acid. In an embodiment, the product comprises about 1.2 g / L acetic acid. In an embodiment, the product comprises about 1 g / L acetic acid. In an embodiment, the product comprises about 0.8 g / L acetic acid. In an embodiment, the product comprises about 0.6 g / L acetic acid. In an embodiment, the product comprises about 0.4 g / L acetic acid. In an embodiment, the product comprises about 0.2 g / L acetic acid. In an embodiment, the product comprises about 0.1 g / L acetic acid. In an embodiment, the product comprises about 0.05 g / L acetic acid. In an embodiment, the product comprises about 0.01 g / L acetic acid.

[0223] In an embodiment, the fructan is selected from one or more of: levan, inulin and a fructooligosaccharide. In an embodiment, the fructan is levan.

[0224] In an embodiment, the fructan is inulin. In an embodiment, the fructan is fructooligosaccharide.

[0225] In an embodiment, the glucan is selected from one or more of: dextran, mutan, reuteran, alternan and a glucooligosacchaide. In an embodiment, the glucan is dextran. In an embodiment, the glucan is mutan. In an embodiment, the glucan is reuteran. In an embodiment, the glucan is alternan. In an embodiment, the glucan is a glucooligosacchaide.

[0226] In an embodiment, the product comprises exopolysaccharides. In an embodiment, the exopolysaccharides are predominantly homopolysaccharides (HoPS). In an embodiment, the homopolysaccharides comprise dextran. In an embodiment, the homopolysaccharides comprise levan.

[0227] In an embodiment, the product comprises no detectable level of malic acid. In an embodiment, the product comprises a low malic acid level (0 to about 10 g / L or 0 to about 6 g / L).

[0228] In an embodiment, the product comprises a favorable odour activity value. In an embodiment, the odour activity value is more favourable (closer to wine) than de-alcoholised wine. In an embodiment, the product is wine.

[0229] In an embodiment, the wine is a white wine. In an embodiment, the wine is a red wine. In an embodiment, the wine is a bone-dry wine. In an embodiment, the wine is a dry wine. In an embodiment, the wine is an off-dry wine. In an embodiment, the wine is a sweet wine. In an embodiment, the wine is a very sweet wine.

[0230] In an embodiment, the wine is selected from one or more of: Shiraz, Chardonnay, Sauvingnon Blanc, Grenache, Pinot Gris, Kyoho, Cabernet Sauvignon, Sultanina, Merlot, Tempranillo, Airen, Syrah, Pinot Noir / Blauer Burgunder, Trebbiano Toscana / Ugni Blanc, Semilion, Riesling and Muscat of Alexandria. In an embodiment, the wine is Shiraz. In an embodiment, the wine is Chardonnay. In an embodiment, the wine is Pinot Gris. In an embodiment, the wine is Kyoho. In an embodiment, the wine is Cabernet Sauvignon. In an embodiment, the wine is Sultanina. In an embodiment, the wine is Merlot. In an embodiment, the wine is Tempranillo. In an embodiment, the wine is Airen. In an embodiment, the wine is Syrah. In an embodiment, the Grenache is Garnacha Tinta / Grenache Noir. In an embodiment, the wine is Sauvingnon Blanc. In an embodiment, the wine is Pinot Noir / Blauer Burgunder. In an embodiment, the wine is Trebbiano Toscana / Ugni Blanc. In an embodiment, the wine is Semilion. In an embodiment, the wine is Riesling. In an embodiment, the wine is Muscat of Alexandria.

[0231] In an embodiment, the product is not a de-alcoholised product.

[0232] In an embodiment, the product is not a no alcohol product.

[0233] Isolated strain

[0234] In an aspect, the present invention provides isolated strains of Leuconostoc mesenteroides suitable for use in the methods and products as described herein.

[0235] In an embodiment, the Leuconostoc mesenteroides comprises a glycosyltransferase.

[0236] In an embodiment, the Leuconostoc mesenteroides comprises one or more enzymes selected from of: dextransucrase, levansucrase, alternansucrase, malolactic enzyme, and mannitol dehydrogenase.

[0237] In an embodiment, the Leuconostoc mesenteroides comprises dextransucrase. In an embodiment, the Leuconostoc mesenteroides comprises levansucrase. In an embodiment, the Leuconostoc mesenteroides comprises malolactic enzyme. In an embodiment, the Leuconostoc mesenteroides comprises mannitol dehydrogenase.

[0238] In an aspect, the present invention provides an isolated strain of Leuconostoc mesenteroides selected from: i) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;

[0239] ii) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof;

[0240] iii) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;

[0241] iv) Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and v) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0242] In an embodiment, the isolated strain is Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0243] In an embodiment, the isolated strain is Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof.

[0244] In an embodiment, the isolated strain is Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0245] In an embodiment, the isolated strain is Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and

[0246] In an embodiment, the isolated strain is Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0247] In an embodiment, the present invention provides a starter culture for producing a low alcohol or reduced alcohol product from a base alcohol production biomass comprising one or more of the isolated strains as described herein. As used herein a “starter culture” is a culture of live microorganisms for primary fermentation as described herein. In an embodiment, the present invention provides a starter culture for producing a low alcohol or reduced alcohol product from a base alcohol production biomass selected from one or more of:

[0248] i) Leuconostoc mesenteroides BF 1 deposited under VI 7 / 021729 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof;

[0249] ii) Leuconostoc mesenteroides BF2 deposited under V17 / 021730 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof; iii) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;

[0250] iv) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof;

[0251] v) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;

[0252] vi) Leuconostoc mesenteroides C18 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and vii) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

[0253] Strain BF1, C12, C14, C18 and C20 as described herein comprise malolactic enzyme. Strain BF2 as described herein does not comprise malolactic enzyme and cannot undertake malolactic fermentation.

[0254] In an embodiment, the base alcohol production biomass is inoculated with at least about 105CFU / mL of a starter culture as described herein. In an embodiment, the base alcohol production biomass is inoculated with at least 106about CFU / mL of a starter culture as described herein. In an embodiment, the base alcohol production biomass is inoculated with at least about 107CFU / mL of a starter culture as described herein. In an embodiment, the base alcohol production biomass is inoculated with at least about 108CFU / mL of a starter culture as described herein. In an embodiment, the base alcohol production biomass is inoculated with at least about 1010CFU / mL of a starter culture as described herein. In an embodiment, the base alcohol production biomass is inoculated with about 105CFU / mL to about 1010CFU / mL of a starter culture as described herein.

[0255] EXAMPLES

[0256] Example 1 - Materials and methods

[0257] Grape juice samples

[0258] Unpasteurised frozen shiraz juice (2021 Langhorne Creek Shiraz Juice), 1747 unpasteurised frozen 2022 Riverland, Chardonnay Juice and 2022 Adelaide Hills Pinot Gris Juice were purchased from Australian Wine Research Institute (AWRI). Nude cloudy apple juice was purchased from a local supermarket.

[0259] Chemical and biochemical reagents

[0260] Food grade calcium carbonate and potassium bicarbonate were sourced from Food depo (Melbourne, Australia) whereas potassium metabisulphite was sourced from Home Make It (Melbourne, Australia). Malolactic nutrient (MaloControl BIO) was purchased from Kauri Australia. Reagents and strips for SENTIA™ Analyser were sourced from AIM Scientific (Prospect, South Australia). All other chemical reagents were sourced from Sigma Aldrich Australia (VIC, Australia).

[0261] Lactic acid bacteria cultures

[0262] The lactic acid bacteria (LAB) cultures for this study were selected based on a short term study conducted on pasteurised Shiraz and Sauvignon Blanc grape juices. Leuconostoc mesenteroides culture strains (BF1, BF2, Cl 2, C13, Cl 4, C20, RG1, and RG3) were used in the experimental trials. All the LAB master cultures were kept at -80°C, in De Man Rogosa Sharpe (MRS) with a cryoprotectant (15% glycerol). The strains were first activated and propagated in an MRS broth CN0359 (Oxoid Ltd., Basingstoke, Hampshire, England) at 30°C for 48-72 hours. The colony forming unit (CFU) of cells and purity was confirmed by optical density (OD) and by plating on MRS agar CM0929 (Oxoid Ltd., Basingstoke, Hampshire, England) at 30°C under anaerobic condition for 48-72 hours.

[0263] Yeast cultures

[0264] The commercial yeast strains LALVIN EC 1118 (Saccharomyces cerevisiae) and VitiFerm™ Rubion Extra Bio (Saccharomyces cerevisiae used for elegant & fruity red wines), were purchased from a local brewing company. The yeast cultures were activated following the instruction of the manufacturers and were used together with nutrients supplied by the suppliers.

[0265] Microbial analysis

[0266] The microbial quality of the samples were assessed prior to and following LAB fermentation in accordance with methods described in Cai et al. (2019). The enumeration of yeast and mould, Enterobacteriaceae and LAB in samples (raw, pretreated, before or after LAB fermentation and after yeast fermentation) were done by Malt Extract Agar (MEA) (Oxoid Ltd., Cat. No. CM0059), Violet Red Bile Fructose Agar (VRBGA) (Oxoid Ltd., Cat. No. CM0485), and De Man Rogosa Sharpe (MRS) (Oxoid Ltd., Cat. No. CM0361).

[0267] Chemical analysis: pH measurement, titratable acidity, acetic acid and malic acid contents

[0268] The pH of both the unfermented and LAB preferment samples were assessed using the Dual pH, Temperature kit WP-80D model: WP80DZ Rev 2.1. The SENTIA™ Analyser Model A0295 from Universal Biosensors PTY LTD, Australia was used for measuring free-SCh, malic acid, titratable acidity, acetic acid, fructose and fructose for rapid measurement during and at the end of LAB and wine fermentation trials. Wine samples were sent to an external laboratory (Wine Check) for alcohol, acetic acid, ethyl acetate, and acetaldehyde analyses.

[0269] High performance liquid chromatography (HPLC) for sugar analysis

[0270] The sugar content of the samples was analysed prior to and after fermentation as described in (Xu et al., 2020; Ye et al., 2019) . In short, samples were mixed with three volumes of alcohol. This was followed by centrifugation to remove the alcohol insoluble residues. The supernatant was filtered and loaded to the HPLC for sugar analysis. HPLC analysis of soluble sugars in grape juice was performed by using an Arc HPLC system coupled with a 2414 refractive index detector (Waters Corp., Wilford, MA, USA) at 35°C. A Shodex Asahipak NH2P-504E (4.6 x 250 mm) and NH2P-50G column (4.6 x 50 mm) (Showa Denko K.K, Japan) was used to separate the sugars at temperature of 30°C with 68% acetonitrile as mobile phase. During operation, the samples and the standard mixture were kept at 5°C. Chromatograms were recorded and compared with 5mg / mL sugar standard mixture of erythritol PHR1479 (Sigma), fructose F0127 (Sigma), mannitol 291484B (BDH Reagents & Chemicals), fructose G8270 (Sigma) and sucrose VWRC27480.294 (AnalaRNORMAPUR®). Sugar content was calculated based on the sugar standard curve. The result of sugar reduction was reported as percentage compared to initial sugar present in non-fermented grape juice.

[0271] Gas chromatography - mass spectrometry (GC-MS) for volatile analysis

[0272] Volatile compounds in wine samples were analysed using head space solid-phase microextraction and gas chromatography - mass spectrometry (HS-SPME-GC-MS). One mL of sample was added to a 20 mL headspace vial containing 5uL internal standard (2-octanol, 100.3ug / L; dl2-hexanal, 136.8ug / L; dl3-hexanol, 273.1ug / L) and 0.5 g NaCl. The vial was tightly capped and heated at 40°C for 10 min prior to extraction using a solid-phase fiber (DVB / C-WR / PDMS, Agilent) at 40°C for 30 min. Subsequently, the fiber was desorbed in the GC injector for 2 min at 250°C.

[0273] Gas chromatography conditions: The QP2010 GC-MS system (Shimadzu Corporation) coupled with a DB-Wax column (30 m x 0.25 mm x 0.25 pm) was used to analyze volatile compounds. The carrier gas was ultrapure helium (purity >99.999%) in the splitless mode at 1.56 mL / min flow rate. The oven temperature program was 40°C for 5 min, ramping to 250°C at 7°C / min, and maintaining for 15 min. Total run time was 50 min.

[0274] Mass spectrometer conditions: Electron ionization (El) mass spectrometric data from m / z 35 to 350 were scanned at 0.3 s intervals. The ion source and the injector temperature were 230°C and 250°C, respectively.

[0275] Data analysis

[0276] Data analysis was conducted using OriginPro 2022b. Analysis of variance and Tukey means comparison test were used to evaluate significant differences between treatments.

[0277] Example 2 - Trial on red wine

[0278] Benchtop trials

[0279] LAB fermentation for sugar -reduction

[0280] The frozen shiraz grape juice was thawed overnight in a cold room at 4°C. The initial pH was ~3.5 and was adjusted to ~5.0 by adding CaCCh. For the benchtop low-alcohol wine fermentation (500 mL duplicate samples), LAB fermentation was first performed to reduce fermentable sugar content in the juice. For this process, two Leuconostoc mesenteroides cultures C12 and BF2 were used at a dosage of 108CFU / mL. The LAB fermentation was conducted under static condition for 72 hours, with some samples directly incubated at 25°C without prior incubation and others incubated at 30°C. The two culture strains and the fermentation conditions were selected based on preliminary screening experiments.

[0281] The sugar profiles of the LAB pre-fermented juices after 72 hours fermentation is presented in Figure 1. The average sugar reduction of the samples was 31.5% for BF2 fermentation at 30°C, 37.7% for C12 fermentation at 25°C and 32.8% for C12 fermentation at 30°C. The sugar content of the LAB fermented samples was significantly different from that of the control (p<0.05). However, there was no statistically significant difference between the samples fermented under different conditions in terms of sugar or mannitol content. The microbial quality of the fermented samples was also assessed following fermentation (Table 1). There was no outgrowth of Enterobacteriaceae . Both the lactic acid bacteria and total plate count ranged from 105to 107CFU / mL indicating that the main microorganisms active in the samples are the inoculated Leuconostoc mesenteroides cultures. Table 1: Microbial quality of samples fermented by Leuconostoc mesenteroides cultures (C12 and BF2) at culture dosage of 108CFU / mL for 72 hours at different temperatures under static condition.

[0282]

[0283] Yeast fermentation for production of reduced alcohol red wine

[0284] Following LAB fermentation, pH levels were measured, and samples were analysed for titratable acidity, malic acid, and fermentable sugars. Then samples were inoculated with EC-1118 yeast at 106CFU / mL in accordance with the protocol of the manufacturer, a yeast nutrient supplied with the culture was added to the samples and fermentation was conducted at 22°C until the fermentable sugar level dropped to <5 g / L. A control wine yeast fermentation without prior LAB fermentation was conducted for comparison. The sugar content and other parameters of the ferments during wine fermentation was continuously monitored using SENTIA™ Analyser. In cases where the fermentation was stuck for a prolonged period, samples were re-inoculated with yeast culture. Wine samples were sent to an external laboratory (Winechek, Melbourne) for alcohol and other chemical analyses.

[0285] The yeast fermentation took 20 to 33 days. Control non-fermented samples were also fermented using the same yeast strain under the same condition. The yeast fermentation took 11, 20 and 24 days for control, BF2 and C12 30°C (a) samples respectively whereas the other samples required 33 days of fermentation. Additional benchtop wine fermentation experiments were conducted after pre-fermenting grape juice (with added nitrogen source) using C12 culture at 30°C for six hours (preincubation) followed by 66 hours fermentation at 25°C. The total sugar reduction was about 11% in this case, whereas the average mannitol content of the samples was 26.7 g / L. Selected chemical attributes of the benchtop wine experiment samples after LAB and yeast fermentation are presented in Table 2. The percentage alcohol reduction (based on the average alcohol content of the control wine) in the pre-fermented samples together with percentage sugar reduction are presented in Figure 2. The alcohol reduction ranged from 10.8% to 39.5%. It correlated well with the level of sugar reduction except in the case of sample C12 25°C (a) (Figure 2), which may be due to contamination with yeast (outgrowth of indigenous yeast initially present in the juice) and most of the sugar reduction in that sample appears to be due to prior conversion to alcohol. A statistical analysis of the data showed a significant reduction (p<0.05) in alcohol content of the LAB pre-fermented samples compared to control. There was substantial level of mannitol specially in wines pre-fermented by C12 cultures indicating that at least 2% lower or 16% alcohol reduction is expected from conversion of fructose to the non-fermentable mannitol alone. Table 2: Chemical profile of benchtop shiraz wine pre-fermented with Leuconostoc mesenteroides cultures (C12, BF2) for 72 hours at different conditions for reducing fermentable sugar and alcohol content following yeast fermentation.

[0286] > < > > < > <

[0287]

[0288] Small pilot scale trials

[0289] LAB fermentation for sugar -reduction

[0290] To assess the scalability of the benchtop process and improve the quality of the trial wines, further trials using 25L fermentation tanks were conducted. Frozen Shiraz juice (15L) was used in the trial after thawing the juice overnight under refrigerated condition (4°C). The pH of the juice was adjusted to pH ~ 5.0 using CaCCh as in the benchtop trials. Sucrose (1%) and 0.6g malolactic nutrient (MaloControl BIO, which is essentially a yeast extract) were added to the juice before inoculation with BF2 (Culture dose of 108CFU / mL) with the goal of improving the level of sugar reduction. The fermentation was conducted for 72 hours, which consisted of 6 hours pre-incubation at 30°C and 66 hours incubation at 25°C. The sugar profile of the LAB-fermented sample after 72 hours of fermentation is shown in Figure 3.

[0291] The fructose, fructose and mannitol contents of the sample after 72 hours LAB fermentation was 98 g / L, 82 g / L and 23.6 g / L respectively (Figure 3), with total sugar reduction of 27.6% relative to control. The microbial quality of the sample was analysed prior and after fermentation. The sample was overall cleaner than other samples from the same batch. Enter obacteriaceae, yeast, mould and lactic acid bacteria were not detected in the sample prior to fermentation. The lactic acid bacteria and total plate count were 6.23xl07and 2.54xl07respectively after fermentation, whereas Enterobacteriaceae, yeast and mould were not detected after fermentation. However, 50 CFU / mL acetic acid bacteria was detected in the sample after wine fermentation.

[0292] The level of pH and sugar reduction after 72 hrs of fermentation was in a similar range with that of the benchtop samples fermented using the same culture indicating that the process is scalable. The added sucrose or nutrient did not have measurable effect on acidification rate or sugar reduction level.

[0293] Yeast fermentation trial

[0294] Following the completion of the LAB fermentation, dry ice was added to the LAB-fermented juice and the juice was kept chilled at 4°C for 4 hours before yeast inoculation to reduce oxidation (and production of acetic acid) by creating anaerobic condition. A different yeast culture (VitiFerm™ Rubion Extra Bio) was used in this case at 106CFU / mL dosage following the instruction of the manufacturer together with a yeast nutrient supplied with the culture. The yeast fermentation was conducted at 22°C until the total sugar reached 3.3 g / L after three weeks of fermentation. The alcohol content and other chemical parameters of the wine are given in Table 2. The alcohol content of the samples was 30% less than the average alcohol content without pre-fermentation. That correlated well with the observed sugar reduction (Table 2). The study showed that the production of reduced alcohol red wine is feasible through a two-step fermentation process involving 1) Fermentation using Leuconostoc mesenteroides cultures for reducing fermentable sugar followed by 2) conventional yeast fermentation for wine production.

[0295] Example 3 - Trial on white wine

[0296] The objective of this study was to evaluate sugar reduction fermentation using Leuconostoc mesenteroides cultures prior to alcoholic fermentation for production of reduced alcohol white wine and determine the level of alcohol reduction achievable using this approach and the quality of the final product. The experiment was conducted using Pinot gris grape juice as substrate.

[0297] LAB fermentation for sugar -reduction

[0298] LAB fermentation trials were conducted to evaluate the performance of various Leuconstoc mesenteroides strains (C13, C14, BF1, BF2, RG3) for reducing fermentable sugar in Pinot gris grape juices with and without added extra nutrients to identify strains most suited for reducing the fermentable sugar content of the juice and enable the production of reduced alcohol wine. All the experiments were conducted at 30°C. The pH of the juice was adjusted from the initial -3.49 to at - 5 by using CaCCh in the initial experiments and KHCCh in later experiments. The inoculation dosage was 108CFU / mL to minimize the outgrowth of indigenous microflora in the juice. The effects of adding malolactic nutrient (MaloControl BIO) and sucrose to the juices on the level of sugar reduction were also evaluated. All experiments were conducted using 100 mL glass containers.

[0299] The rate of fermentation was similar for all cultures except BF1, with pH reduced to 4.0 or below after 72 hours of fermentation. In the case of BF1, the pH of the samples was around 4.2 after 72 hours of fermentation. The total sugar reduction ranged from 23% to 30% for samples with added malolactic nutrient (Figure 4A).

[0300] Sugar reduction was accompanied by substantial mannitol production (Figure 4B) indicating that the starter cultures were the predominant cultures active in the juice. The lowest sugar reduction of -23% was observed with RG3 fermented samples. The sugar reduction level with all the other cultures was around 30%. The highest mannitol production was observed with C14 fermented sample. Thus, C14 was selected for yeast fermentation trial. Yeast fermentation

[0301] Following preliminary benchtop trials, a small pilot trial was conducted using a 25 L fermentation tank to evaluate the scalability of the sugar reduction fermentation and evaluate the level of alcohol reduction achievable following yeast fermentation. Frozen pinot gris juice (20 L) was thawed overnight under refrigerated condition (4°C). Its pH was adjusted to pH ~5.0 using KHCCh and 0.92 g malolactic nutrient was added into the sample. Then it was inoculated with C14 culture, which was selected based on the results of the LAB screening trials at 108CFU / mL dosage. Nitrogen gas was pumped into the juice for about 4 minutes at flow rate of 4 L / min to create anaerobic condition during LAB fermentation and reduce acetic acid production. The LAB fermentation was conducted at 30°C for 72 hours. Following the completion of the LAB fermentation, dry ice and sulphiting agent (Pure E224 Potassium Metabisulfite) were added to the sample to inactivate the LAB starter culture and stop fermentation. The sample was kept overnight in a cold room (4°C). The following day, it was inoculated with yeast (Rubion Extra Bio) at 106CFU / mL and was incubated at 17°C until the sugar content of the juice was below 5 g / L. Yeast fermentation was also conducted on control non-pre-fermented juice under the same condition using the same yeast under the same condition. Sulphiting agent (Pure E224 Potassium Metabisulfite) was also added to the control juice to increase the free SO2 level from 3.0 ppm to 50 ppm. In both the pre-fermented and control samples, yeast nutrient that was supplied with the yeast culture was added to the ferments in accordance with the protocol of the yeast manufacturer. The sugar content and other parameters of the ferments during wine fermentation were continuously monitored using SENTIA™ Analyser.

[0302] The LAB fermentation at the small pilot scale using C14 culture proceeded in a similar way as the benchtop fermentation. The pH dropped to 3.85 after 72 hours of fermentation. There was also a significant change in the sugar profile of the sample following fermentation. Production of mannitol was also observed indicating that the starter culture was active during fermentation. However, the percentage total sugar reduction was much lower (16.9% compared to 29.9%) compared to the benchtop trial (Figure 4A and Figure 5). The mannitol formed was also lower (21.4 g / L compared to 27.6 g / L) (Figure 4B and Figure 5). The sugar profile of the juice prior to and after LAB fermentation is presented in Figure 5. The percentage sugar and alcohol reduction are presented in Figure 6. The alcohol content and other chemical attributes of the control and pre-fermented samples are presented in Table 3. The percentage alcohol reduction (19.2%) was within the range of the percentage sugar reduction (Table 3). The acetic acid content of the alcohol reduced wine was 2 g / L, which is much lower than the red wine trial samples (Table 2). However, it is still higher than the maximum 1.2 g / L tolerated in white wine ((AWRI), 2024). The acetaldehyde and ethyl acetate levels are within acceptable range ((AWRI), 2024).

[0303] The study showed that a two-step fermentation process using Leuconostoc mesenteroides mediated fermentation for fermentable sugar reduction followed by conventional yeast fermentation enables the production of reduced alcohol white wine.

[0304] Table 3: Selected chemical attributes of pre-fermented and control Pinot gris wines produced at small pilot scale.

[0305]

[0306] Example 4 - Trial on apple cider

[0307] This study was aimed to evaluate producing reduced alcohol cider through a two-step process involving fermentation with Leuconostoc mesenteroides culture for sugar reduction followed by conventional yeast fermentation.

[0308] LAB fermentation for sugar -reduction

[0309] Lactic acid bacteria fermentation was conducted using C20 Leuconostoc mesenteroides culture from CSIRO collection. The pH of the mild pasteurised cloudy apple juice was adjusted from 3.28 to ~5.0 using potassium bicarbonate. The fermentation was conducted at 30°C for 75 hours until the pH dropped to ~4.0. The culture dosage was 108CFU / mL. Experiments were conducted in duplicates using 500 mL glass containers. The sugar reduction profile of the cloudy apple juice following LAB fermentation is presented in Figure 7. Substantial sugar reduction was observed following fermentation with over 92% reduction in sucrose and 44.6% reduction in total fermentable sugar.

[0310] Yeast fermentation

[0311] Following the completion of the LAB fermentation, potassium metabisulfite (Pure E224 Potassium Metabisulfite) was added to the sample to raise the free SO2 level to 50 ppm and inactivate the Leuconostoc mesenteroides culture. The sample was stored at 4°C overnight. Next day, it was inoculated with yeast culture (LALVIN EC 1118) at 106CFU / mL and yeast nutrient was added into the sample in accordance with the protocol provided by the supplier. The yeast fermentation of the sample was conducted at 17°C until the sugar content of the sample dropped to less than 5 g / L. Control yeast fermentation was also conducted on non-pre-fermented cloudy apple juice after adding potassium metabisulfite to a free SO2 level of 50 ppm. The fermentation of the control sample was conducted using the same yeast culture under the same condition as the prefermented sample. The percentage sugar and alcohol reductions are presented in Figure 8. As can be seen, prior sugar reduction through LAB fermentation enabled about 41% reduction in alcohol content of apple cider, which was close to what is expected based on the total sugar reduction. Data on the chemical attributes of the pre-fermented and control apple ciders are given in Table 4. Only one data is presented for the prefermented cider since the duplicate was lost (the bottle broke) during yeast fermentation. The acetic level in the pre-fermented reduced alcohol cider is quite high and could be reduced with further optimisation.

[0312] Table 4: Selected chemical attributes of pre-fermented and control Apple cider samples produced at laboratory scale.

[0313] < <

[0314] <

[0315]

[0316] Example 5 - Genomic insights into exopolysaccharide biosynthesis pathways in novel Leuconostoc mesenteroides strains

[0317] Some lactic acid bacteria synthesize slime-like substances called exopolysaccharides (EPS) as a protective mechanism against harsh environmental conditions, such as extreme temperatures, pH, and osmotic stress. These bacterial EPS have recently received attention in the food industry due to their potential applications. The unique structure imparted by bacterial EPS contributes to distinct rheological properties, texture, stability, emulsification ability, and gelling capacity in food products (Silva et al., 2019; Zeidan et al., 2017). Furthermore, some EPS can have prebiotic benefits, and some exhibit bioactive properties such as antioxidant and anti-tumour activities (Du et al., 2020; Silva et al., 2019).

[0318] Among the LAB species, Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) and Leuconostoc mesenteroides have gained prominence in the food industry. They are considered Generally Recognized as Safe (GRAS), and some strains of these species produce EPS (Deo et al., 2019; Patel et al., 2012). The discovery of new LAB strains capable of producing EPS at high yield is useful for improving the commercial feasibility of using these organisms for fermentative EPS production and expanding their application in the food and other industries.

[0319] The exopolysaccharides (EPS) production potential of novel strains of Leuconostoc mesenteroides, along with experimental evaluation of their actual EPS production capabilities was assessed. The genomic analysis revealed that strains L. mesenteroides C13, C12, C18, and C20 possess the highest genetic potential for EPS production among the studied strains, which was confirmed by experimental EPS production quantification. These three strains produced 1.67 mg / mL, 1.57 mg / mL, 1.34 mg / L and 0.82 mg / mL of EPS, respectively, during cultivation at 37°C for 20 hours in MRS medium with 10 % sucrose supplementation. The strains predominantly utilise the extracellular EPS biosynthesis pathway, suggesting that they primarily produce homopolysaccharides (HoPS), viz. dextran and levan. Hence, these strains can be potentially used for production of EPS for food and other biotechnological applications.

[0320] Materials and methods

[0321] Bacterial strain source

[0322] Bacterial cultures, previously isolated for separate studies at CSIRO Agriculture and Food in Werribee, Australia were used for the study. These cultures were isolated from broccoli, apple, and carrot. The strains were selected for the study because they were novel and had not been assessed for their potential to produce EPS before. DNA extraction and whole genome sequencing

[0323] DNA extraction and whole genome sequencing of the bacterial strains were conducted in a separate study at CSIRO Agriculture and Food in Werribee described above. DNA extraction was carried out following the method given in Gautam et al. (2019). The quality and quantity of DNA were assessed using the Nanodrop (absorbance at 600 nm), DNA tape station, and Qubit. Extracted DNA samples were sent for Nextera XT DNA Whole Genome Sequencing at the Ramaciotti Centre for Genomics, University of New South Wales, Sydney.

[0324] Whole genome sequence (WGS) data analysis

[0325] A bioinformatics pipeline was developed using multiple bioinformatics tools (Figure 9) which supports raw WGS data generated by means of Illumina technology.

[0326] Quality control of the sequences (FastQC)

[0327] For quality control, Babraham bioinformatics FastQC (version 01-03-23) was utilised to detect potential issues in high-throughput raw data sequences at the onset of the pipeline. FastQC was performed with the default settings, which include an average Q score of >15, a minimum length of <40, and the requirement that the number of N bases is >15 (Atxaerandio-Landa et al., 2022). Problematic areas were discernible through the summary output generated using various parameters (Brown et al., 2017), which can be visualised on a web server dashboard for subsequent downstream analysis. These parameters include per-base sequence quality, per-sequence quality scores, perbase sequence content, and the requisite criteria that sequences must meet to proceed.

[0328] Trim adapters (TrimGalore)

[0329] Read trimming with TrimGalore was employed under defined parameters to remove adapter sequences and low-quality bases. This process utilised a tool known as Cutadapt (Liao & Shi, 2020). This approach enhances alignment accuracy, eliminates adapter contamination, and reduces the occurrence of false positives (Yang et al., 2023).

[0330] Assembly of genes (MEGAHIT)

[0331] The assembly of short sequencing reads was carried out using MEGAHIT (version 1.2.9 using the flags -t 2 -m 1.0 — k-list 29,39,59,79,99,119) , a next-generation de novo assembler utilised for efficiently assembling a large volume of complex metagenomic data (Li et al., 2015). Quality assessment of gene assembly (BUSCO)

[0332] The quality of the genome assemblies and the completeness of annotation were assessed using BUSCO (version 5.5.2 and Lactobacillales odblO), which evaluates both the completeness of gene sets and transcriptomes by comparing them with the gene content of reference sequences (Seppey et al., 2019; Simao et al., 2015).

[0333] Genomic annotation (Prokka)

[0334] For gene annotation, Prokka (rapid prokaryotic genome annotation; version 1.14.5) was incorporated to identify predicted features such as coding sequences (CDS), rRNA and tRNA regions and non-coding RNA (Seemann, 2014).

[0335] Database (NCBI)

[0336] All available amino acid sequences related to various EPS biosynthesis pathways in L. mesenteroides were downloaded from the NCBI database in FASTA format and used as the EPS gene database.

[0337] BLAST BLAST+ (version 2.14.0) tool identifies regions of similarity between a query sequence and the known EPS database. It compares nucleotide or protein sequences and calculates statistical significance. BLAST results were filtered based on the criteria of e-value (le'5°), bit score (>50), and percentage identity (>40 %) (Deo et al., 2019; Du et al., 2020). For the visualisation of the identified gene sequences Geneious Prime software (version 2022.2.2) was used.

[0338] Evaluation of actual EPS production bacterial storage and growth conditions

[0339] The Leuconostoc mesenteroides strains (CSIRO library collection) were used throughout this study. De Man Rogosa and Sharpe media (MRS broth and agar, Oxoid, Australia) was used as a non-selective medium for bacterial growth. To obtain the primary inoculum, bacteria from a -80°C stock were first isolated on an MRS agar and incubated anaerobically at 30°C for 48 hours. A single bacterial colony from the plates was inoculated into 10 mL MRS broth and then incubated anaerobically at 37°C for 20 hours until the cultures reached an optical density of 1 at 600 nm, which was measured using a spectrophotometer (Novaspec, Amersham Biosciences, UK).

[0340] These cells were harvested by centrifugation at 4000 rpm at 8,000 g for 10 minutes and washed twice with phosphate-buffered saline (PBS) to remove traces of MRS broth media. The bacterial pellet was then resuspended in a glycerol and PBS solution (final glycerol concentration 15 % v / v) and adjusted to a turbidity equivalent to 109CFU / mL. The actual colony-forming units were confirmed by bacterial plating. The cell stocks prepared were stored at -80°C in glycerol and phosphate-buff ered saline (PBS) solutions as single-use cultures.

[0341] For EPS quantification, in each fermentation trial, bacterial cells from -80°C stock were collected, washed twice, and resuspended in 10 mL of MRS broth supplemented with 10 % sucrose. The initial CFU / mL at the beginning of fermentation was adjusted to 107CFU / mL for all the cultures. The cultures were grown at 37°C for 20 hours. Following incubation, samples were subjected to EPS isolation and quantification as describe in upcoming section.

[0342] EPS isolation and quantification

[0343] Based on the results of the whole genome sequence data analysis, three bacterial strains were selected due to their high number of wzx / wzy pathway-related genes and glycosyltransferase genes. These strains were L. mesenteroides strains C13, C18, C12 and C20.

[0344] For EPS isolation, the incubated cultures were centrifuged at 8,000 *g for 20 minutes at 4°C, to separate the bacterial cells. The resulting supernatant was carefully removed and subjected to 14 % trichloroacetic acid treatment to induce protein denaturation and precipitation. Next, the culture was subjected to additional homogenization in a shaker operating at 90 rpm for a duration of 30 to 40 minutes. Following this, another round of centrifugation was conducted at 8,000 *g for 20 minutes at 4°C and the pellet was discarded. The supernatant was combined with cold absolute ethanol, maintaining a proportion of two-fold volume in comparison to the supernatant, and left to incubate at 4°C for a period of 24 hours. A subsequent centrifugation step at 8,000 *g and 4°C for 20 minutes facilitated the precipitation of EPS (Bajpai et al., 2016). Finally, the obtained precipitate was dried using a speed vacuum (Savant SC250EXP SpeedVac concentrator and Savant RVT4104 refrigerated vapor trap) until get a stable weight (Bajpai et al., 2016).

[0345] Statistical analysis

[0346] The data for the EPS quantification was analysed using one-way ANOVA with Minitab software (version 21.1) and Tukey pairwise comparison was done at 95 % level of confidence. Results

[0347] General nucleotide properties

[0348] The detailed information on the nucleotide statistics for the whole genome sequence data analysis using the bioinformatics pipeline is presented in Table 5, along with the origins of the respective strains.

[0349] Table 5: Summary of general nucleotide properties as investigated through bioinformatics analysis.

[0350]

[0351] L. mesenteroides strains displayed variations in GC content within the dataset, with values ranging from 37.6 % to 44.6 %.

[0352] The genome lengths of L. mesenteroides sequences exhibited a broad range, spanning from approximately 1,088 to 4,049 kilobases (kb). This variation can be attributed to higher genomic diversity among the tested L. mesenteroides strains.

[0353] Open Reading Frames (ORFs) are critical elements in whole genome analysis, representing regions of DNA with the potential to encode proteins. ORFs are pivotal as they serve as the starting point for gene prediction and functional annotation. In terms of general properties of the strains investigated L. mesenteroides consisted of 1,710 to 4,551 ORFs, which may harbor the EPS-producing genes.

[0354] Identification of Leuconostoc mesenteroides strains with genes encoding levansucrase and dextransucrase

[0355] A customised bioinformatics pipeline was used to identify lactic acid bacteria strains with the highest genetic potential for production of dextransucrase and levansucrase and enable fermentable sugar reduction in grape juice and other fruit juices. The pipeline uses raw whole genome sequence data as an input and several data analysis algorithms including quality check (FastQC), removal of adapter sequence (TrimGalore), assembly of genes (MEGAHIT), quality assessment of gene assembly (BUSCO), genomic annotation (Prokka) and blasting to databases selected based on the target proteins. BLAST+ (version 2.14.0) tool was used to identify regions of similarity between a query sequence and the known glycosyltransferase databases. BLAST results were filtered based on the criteria of e-value (<lxe'5°), bit score (>50), and percentage identity (>40 %) (Deo et al., 2019; Du et al., 2020). The total number of genes encoding dextransucrases, levansucrases and other glycoside hydrolases in 17 C RQ Leuconostoc mesenteroides strains were compared with that of the reference Leuconostoc mesenteroides strain ATCC 8293. Data are presented in Figure 10. Details of the glycosyltransferase enzyme genes in each strain are presented in Table 6.

[0356] The result showed that C18 has the highest genetic potential for production of glycosyltransferase enzymes with 29 glycosyltransferase genes followed by C13 (26), C12 (22) and C14 and C20 (19), which were all higher than that of the reference strain ATCC8293 (16 genes). In these strains, the number of levansucrase genes are higher than that of dextransucrase. For instance, Cl 8 possess 16 genes encoding levansucrases and 5 genes that encode dextransucrases. Table 6: Summary of glycosyltransferase genes in Leuconostoc mesenteroides strains and the reference ATCC8293 strain.

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] ghf68p = glycoside hydrolase family 68 protein; ghf70p = glycoside hydrolase family 70 protein. EPS quantification

[0365] A preliminary EPS quantification was conducted for all strains in MRS supplemented with 2 % sucrose supplementation at 37°C for 20 hours. Overall correlated with the outcomes of the genomic analysis.

[0366] Further EPS quantification experiment was carried out for the top three strains that had the highest number of levansucrase and dextransucrase genes related to the extracellular biosynthesis pathway and C20 with 10 % sucrose supplementation to induce EPS production. These strains were / .. mesenteroides strains C18, C13, C12 and C20.

[0367] Among these three strains, the highest EPS production was by L. mesenteroides C13, followed by C18, C12 and C20 in that order. Their EPS yields were 1.67 mg / mL, 1.57 mg / mL, 1.34 and 0.82 mg / mL respectively, and the values were not statistically different from one other (p>0.05).

[0368] Discussion

[0369] Among homopolysaccharides, dextrans and levans are produced by the extracellular glycosyltransferase (dextransucrase and levansucrase also known as glucansucrase and fructansucrase) enzymes and are assembled from precursors obtained through the cleavage of sucrose molecules (Nicolescu et al., 2023; Poulsen et al., 2020; Schmid etal., 2015). Dextransucrase (sucrose: l,6-a-d-glucan-6-a-d-glucosyltransferase, EC 2.4.1.5) and levansucrase (sucrose:2,6-P-d-fructan-6-P-d-fructosyltransferase EC 2.4.1.10) enzymes catalyse transglycosylation reactions that result in the formation of EPS (van Hijum et al., 2006).

[0370] The higher counts of levansucrases and dextransucrases indicate that the studied strains have the greatest potential for dextran and levan production. Among the strains studied, L. mesenteroides C18 and C13 demonstrated the highest counts of genes linked to levansucrase and dextransucrase, followed by C12, C14 and C20. This suggests that these strains possess a greater potential for dextran and levan production compared to the other strains investigated. Moreover, the bioinformatics analysis revealed a higher prevalence of levansucrase genes compared to dextransucrase genes in general specially in the C strains. For example, L. mesenteroides Cl 8 strain contained 16 levansucrase gene hits and five dextransucrase gene hits. This finding indicates that levan is potentially the predominant EPS type produced by these strains, which is interesting considering the various reported health benefits of levan. Levan has been reported to have prebiotic effects, as well as anti-tumour and cholesterol reduction effects. Levan stimulates the growth of indigenous Bifidobacteria and other probiotic microorganisms and can be used as a prebiotic supplement among other benefits (Srikanth et al., 2015). In contrast, dextran is known to be completely digestible in the small intestine (Kokubo et al., 2022).

[0371] Earlier studies demonstrated that certain L. mesenteroides strains produce dextran and levan. For instance, L. mesenteroides N5 produced 14.53 g / L dextran with supplementation of 15 % sucrose (Ma’unatin et al., 2022). There are also studies in the literature on the structure of dextrans produced by L. mesenetroides as the basis of understanding their functions. For example, Wang et al. (2023) reported that the dextran produced by L. mesenteroides RSG7 had 5.47 x 106Da molecular weight and consisted of a-(l— >6) glycosidic linkages as backbone and a-(l— >2), a-(l— >3), a-(l— >4), and a-(1— >6) glycosidic linkages as side chains. The EPS produced by Lactobacillus sakei L3 consisted of a-(l— >6)-l inked dextran (Wang et al., 2019).

[0372] In contrast, levan that is produced by Leuconostoc mesenteroides DSM 20343 during faba bean fermentation consisted of mainly P-(2^6) linkages (Shi et al., 2019). These varied sizes and structural features of EPS are important characteristics that influence their bio-functional properties and their impact on the techno-functional properties of food systems. Hence, further studies are required on strains identified to have higher genomic capacity for EPS production to understand the structure of the EPS produced by these strains as a basis for predicting their function.

[0373] These novel strains have a significant potential for dextran and levan production through extracellular synthesis pathway. Particularly, L. mesenteroides strains C13, C18, C12, C14 and C20 have emerged as promising candidates, characterised by a high abundance of glycosyltransferase-related genes and distinctive genetic profiles, aligning with their experimentally demonstrated EPS production capabilities. Hence, these strains can be used for fermentative production of levan and dextran that can be used for a range of applications spanning from the in-situ modification of the techno-functional properties of food substrates through, sugar reduction in sugar rich substrates and fermentative production of bio-thickening, gelling, viscosifying, and emulsifying ingredients as well as prebiotic supplements.

[0374] Example 6 - Identification of Leuconostoc mesenteroides cultures with genes encoding mannitol dehydrogenase and mannitol-l-Phosphate 5-dehydrogenase The bioinformatics pipeline described in Example 5 was used to assess the presence of mannitol dehydrogenase and mannitol- 1 -phosphate 5- dehydrogenase genes in these cultures, which catalyse the conversion of fructose to mannitol and the reverse reaction. Data are summarised in Table 7. Table 7: Summary of number of genes encoding mannitol dehydrogenase, Mannitol-l-Phosphate 5-dehydrogenase and Malolactic enzyme in Leuconostoc mesenteroides strains.

[0375]

[0376] In terms of mannitol production enzymes, C12, C13, C15, and C18, have relatively higher potential, possessing two genes each encoding mannitol dehydrogenase and mannitol- 1 -Phosphate 5 -dehydrogenase enzymes.

[0377] Example 7: Identification of Leuconostoc mesenteroides cultures with genes encoding malolactic enzyme (MLE)

[0378] Malolactic fermentation is conducted in most red wine and some white and sparkling wine production after the primary yeast fermentation to stabilise the wine and reduce wine astringency by converting malic acid into lactic acid. This is mainly performed using oenococcus oeni bacteria, due to their ability to withstand the harsh condition in wine (low pH, alcohol and low nutrients). The presence of genes encoding malolactic enzymes were assessed in the Leuconostoc mesnteroides strains using the bioinformatics pipeline described above in Example 5. The data are presented in Table 8. As can be seen, all the strains except BF2 and RG5 possess genes encoding malolactic enzyme. On the other hand, C12, C13, C14, C18 and C20 have relatively higher potential for malolactic fermentation. Thus, they have the potential to enable both sugar reduction and malolactic fermentation during the sugar reduction fermentation step so that malolactic fermentation will not be needed post yeast fermentation as in the case of conventional wine making process. This was experimentally confirmed for C12 and C14 cultures. BF2 does not possess malolactic enzyme genes and does not convert malic acid into lactic acid as shown in several sugar reduction fermentation experiments.

[0379] Overall, Cl 8 stands out as the best strain for sugar reduction in grape juice or must prior to yeast fermentation since it possesses the highest number of genes for glycosyltransferase enzymes as well as mannitol production and malolactic fermentation. Table 8: Summary of malolactic enzyme genes in Leuconostoc mesenteroides strains and the reference ATCC8293 strain.

[0380]

[0381] Example 8: Proteomics study to evaluate the extracellular production of glycosyltransferase enzymes

[0382] In order to validate the genomics data and determine the best condition for extracellular production of glycosyltransferase enzymes, fermentation was conducted after prestressing the cultures at different conditions. This was followed by proteomics characterisation of the ferments. The cultures with the highest genomic potential for production of glycosyltransferase enzymes i.e. C13 and C18 were selected for this study.

[0383] Experiments to determine the best condition for glycosyltransferase production According to literature, the application of various stressors enhances the production of exopolysaccharides which are produced by microbial cells as the mechanism of protection against various stresses (Caggianiello et al., 2016; Degeest et al., 2002; Nguyen et al., 2020). It hypothesized that the synthesis of glycosyltransferase enzymes responsible for exopolysaccharides by microbial cells is also enhanced under stress conditions. In this study, to assess the impact of stress on glycosyltransferase production, a combination of acid and temperature stresses were applied on the selected cultures as summarised in Table 9. Accordingly, the cultures were subjected to treatments at a total of nine pH and temperature combinations. For each treatment, stock cultures stored at -80°C were defrosted, washed twice, and resuspended in MRS broth supplemented with 2 % sucrose at 107CFU / mL. Then, the pH of the culture broth was adjusted to the experimental pH using a benchtop pH meter. And the samples were incubated for 2 hours in a thermostated water bath or incubator at the experimental temperature. This was followed by harvesting the cells by centrifugation (8,000 x g for 10 minutes), re-inoculation into MRS broth supplemented with 2% sucrose (pH 6.0) and fermentation for 24 hours at 25°C. Following fermentation, the samples were centrifuged at 8,000 x g for 10 minutes to remove the microbial cells. The supernatants were analysed for their proteomics profile as described below. The treatment at pH 6.0 and 25°C for 2 hours, followed by fermentation under the same conditions (pH 6.0 and temperature 25°C) for 24 hours, was considered the control treatment, as it does not constitute a stress treatment.

[0384] Table 9: Stress treatment conditions applied for 2 hours prior to fermentation of L. mesenteroides C13 and L. mesenteroides C18.

[0385]

[0386]

[0387] Microbial enumeration

[0388] After 2 hours of incubation under stress conditions and a subsequent 24 hours fermentation period, 100 pL aliquots from each replicate sample were taken and a serial dilution was prepared in maximum recovery diluent. The lactic acid bacteria counts were then determined by plating the serially diluted samples on MRS agar plates and incubating for 48 hours at 30°C under anaerobic conditions (Sanders, 2012).

[0389] Proteomics characterisation of fermentation supernatants

[0390] Untargeted extracellular proteomics analysis was conducted to assess the synthesis of glycosyltransferases and other proteins by the selected two cultures after 24 hours of fermentation and understand the impact of the applied stress (detailed in Table 9) on protein synthesis.

[0391] Protein quantitation and extraction

[0392] After 24 hours of fermentation, the ferment was centrifuged at 8,000zg for 10 minutes and the bacterial cells were removed. The protein concentration of the supernatants was determined using a Bradford colorimetric assay (read at 595 nm) using Bovine Serum Albumin (BSA) as standard (Bradford, 1976).

[0393] Tryptic digestion

[0394] The fermentation supernatant samples (100 pg) were subjected to Filter- Aided Sample Preparation (FASP) using a 10 kDa molecular weight cut-off (MWCO) filter (Millipore, Australia). Protein was extracted and reduced on-filter using a urea-based buffer (UTC buffer: 8 M urea, 2 M thiourea, 4 % CHAPS, 50 mM DTT in 0.1 M Tris-HC1, pH 8). All samples were shaken at 600 rpm for 40 minutes at room temperature in a thermomixer. After extraction, the protein on the filter was washed with two 200 pL volumes of UA buffer (8 M urea in 0.1 M Tris-HCl, pH 8.0) with centrifugation (20,800 xg, 15 min). To alkylate the cysteine residues, iodoacetamide (50 mM, 100 mL in UA) was applied to the protein on the filter and incubated for 30 minutes at room temperature (25°C) in the dark. The filter was again washed with 200 pL of UA buffer with centrifugation (20,800 x g, 15 min). The buffer was exchanged using 50 mM ammonium bicarbonate (pH 8.0) by two consecutive wash / centrifugation steps (Cordwell et al., 2001; Vaezzadeh et al., 2010).

[0395] Sequencing grade porcine trypsin (Promega) was added at a ratio of 1:50 (0.01 pg / pL, i.e. 2 pg in 200 pL of 50 mM ammonium bicarbonate with 1 mM CaCh) to the samples and incubated for 16 hours at 37°C in a thermomixer at 300 rpm. The filters were transferred to fresh centrifuge tubes and the filtrate (digested peptides) was collected following centrifugation (20,800 x g, 10 min). The filters were washed twice with 200 pL of 50 mM ammonium bicarbonate and the filtrates were combined and lyophilised. The resultant peptides were re-suspended in 50 pL of 0.1 % formic acid (Yu et al., 2018).

[0396] Pooled biological quality control (PBQC)

[0397] Proteomics data were acquired using discovery (Sequential Window Acquisition of All Theoretical Mass Spectra) SWATH-MS approach on a SCIEX 6600 QqTOF mass spectrometer. Data acquisition was randomised and a pooled biological quality control (PBQC), comprising 3 pL from each solubilised sample (n, 54) was included during the batch acquisition. Measurements incorporated external controls to monitor batch and continuous effects during acquisition (Tahmasian et al., 2022).

[0398] LC-MS analysis DDA mode

[0399] Proteolytically digested proteins from the PBQC sample (4 pL, equivalent to 8 pg) were analysed with chromatographic separation on an Ekspert nanoLC415 (Eksigent, Dublin, CA, U.S.A.) directly coupled to the OptiFlow ion source of a TripleTOF 6600 LC-MS / MS (SCIEX, Redwood City, CA, USA). The peptides were desalted for 3 minutes on a Trajan ProteCol C18 (3 pm, 120 A, 10 mm x 0.3 mm) trap column at a flow rate of 10 pL / min 0.1% formic acid and separated on a ChromXP C18 (3 pm, 120 A, 150 mm x 0.3 mm) column at a flow rate of 5 pL / min at 30°C.

[0400] The solvents for LC-M S were: (A) 5 % DMSO, 0.1 % FA, 94.9 % water; (B) 5% DMSO, 0.1% FA, 90% acetonitrile, 4.9% water. Sample injections were carried out to compare a 55 minute method (linear gradient from 3 to 25 % solvent B (mobile phase component of water and methanol) over 38 minutes was employed, followed by 5 minutes from 25 % B to 32 % B; 2 minutes 32 % B to 80% B; 3 minutes at 80 % B; 1 minute at 80 to 3 % B ; and 8 minutes reequilibration) to an 85 minute method (linear gradient from 3-25% solvent B over 68 minutes was employed, followed by: 5 minutes from 25% B to 35% B; 2 minutes 35% B to 80% B; 3 minutes at 80% B, 80-3% B, 1 minute; and 8 minutes re-equilibration).

[0401] The instrument parameters were: ion spray voltage at 4500 V; curtain gas at 30 psi; GSl(gas source 1) at 30 psi and GS2 (gas source 2) at 30 psi; and the heated interface at 150°C. Data were acquired in information-dependent acquisition (DDA) mode comprising a time-of-flight (TOF)-MS survey scan followed by 30 MS / MS product ion scans. First stage MS analysis was performed in positive ion mode, mass range m / z 400-1250 and 0.25 second accumulation time. Tandem mass spectra were acquired on precursor ions >150 counts / s with charge state 2-5 and dynamic exclusion for 15 seconds with a 100 ppm mass tolerance (Schmidt et al., 2011).

[0402] Proteomic profiling

[0403] Raw spectral data files were processed using ProteinPilotTM 5.0 software (AB SCIEX) with integrated false discovery rate analysis and the Paragon Algorithm was used for protein identification. Tandem mass spectrometry data was searched against a custom-built database comprising Uniprot L. mesenteroides (20230901) and a database of contaminant proteins known as the common repository of adventitious proteins (cRAP2). The total number of proteins when redundant sequences were removed in the custom database was 56,781 proteins. The search parameters were defined as: iodoacetamide modified for cysteine alkylation and trypsin as the digestion enzyme. The identification of proteins was recorded in the Supplemental Excel Document if MS / MS spectral scores were achieved at the p < 0.01 confidence level, i.e. at a 1% global false discovery rate (FDR).

[0404] SWATH-MS data acquisition

[0405] The individual digested protein extracts (4.0 pL of sample equivalent to 8 pg of peptide) were chromatographically separated as mentioned earlier. In order to monitor and evaluate the stability of the system (a representative sample) was inserted in the sample cohort (10 ng injections), and the data consistency of this pooled biological quality control (PBQC) was evaluated. The MS source conditions were also identical. The TOF-MS survey scan was collected over the mass range of m / z 400-1250 with a 50 millisecond accumulation time and the product ion mass spectra were acquired over the mass range of m / z 100-1400 with 30 millisecond accumulation time. Variable window SWATH ranges were determined using the SWATH variable window calculator 1.0 (SCIEX) to identify 100 optimal ranges (including 1 Da overlap) spanning m / z 100-1400, resulting in a 3.1 second cycle time. Collision energy (CE) was determined using each window centre as the input m / z for CE equations and a CE spread of 5 eV was used to allow for m / z variance across each SWATH window. Spectra were acquired using Analyst TF vl.8.1 software. Preliminary spectral analysis visualising the total ion chromatograms was performed using PeakView (SCIEX).

[0406] Protein quantitation using DIA-NN software

[0407] For the protein and peptide identification, raw data files from the DIA (data independent acquisition) based experiments were processed using the neural networkbased processing with inference correction in the DIA-Neural Network (DIA-NN) software suite 3. First, a spectral library was generated from the pooled samples processed against the Uniprot / .. mesenteroides using the following settings: N-terminal methionine excision was enabled; in-silico digest-cuts were at K (lysine), R (arginine) with maximum number of missed cleavages was set to 1. The analysis specified the use of cysteine carbamidomethylation as a fixed modification.

[0408] Data analysis

[0409] The multivariate statistical analysis of the proteomics data was conducted using Metaboanalyst 6.0 software (https: / / www.metaboanalyst.ca).

[0410] Results

[0411] Proteomics analysis of the fermentation supernatant identified four levansucrases and 4 dextransucrases in both C13 and C18 samples pre-treated at different stress conditions (Table 10) compared to the 14 levansucrases and 6 dextransucrases predicted for C13 and the 16 levansucarses and 5 dextransucrases predicted for Cl 8 by the genomic analysis. Only Leuconostoc mesenteroides proteome database was used in the proteomics data analysis and as such more levansucrases and dextransucrases could potentially be identified in the samples if a more comprehensive database was used in the analysis. Table 10: Glycosyltransferases identified in C13 and C18 ferments pre-treated at different stress conditions.

[0412]

[0413] *: The numbers are arbitrarily given to differentiate the glycosyltransferases.

[0414] Chemometric analysis was conducted on the data focusing on glycosyltransferase enzymes. Principal component analysis showed all the samples clustering together except samples pre-treated at 40 and pH 3.0 (Figure 11), which produced much less glycosyltransferase, except in the case of dextransucrase in Cl 8 ferments, compared to the other samples. This could be related to the impact of the stress treatments on microbial growth, which resulted in one log less (10 times less) microbial biomass after fermentation in samples that were subj ected to pH 3.0 stress treatment at the three studied temperatures (Figure 12). Interestingly, only samples that were subjected to acid stress (pH 3.0) at 40°C showed significantly lower production of glycosyltransferase s (Figures 13-16). Interestingly, significantly higher glycosyltransferase production per cell was observed in samples that were subjected to pH 3.0 stress at all temperatures in the case of C13 (Figures 13 and 14). In the case of Cl 8, the production per cell of dextransucrases was significantly higher in pH 3.0 treated samples at all temperatures (Figure 16) whereas that was the case for levansucrases only for samples pre-treated at 10°C and 25°C and pH 3.0 (Figure 15). In the case of samples pre-treated at pH 3.0 and 40°C, the total production of levansucrases per unit cell was comparable to control and samples treated at pH 4.5, although levansucrase 2 was absent in the samples (Figure 15). The relative abundance of levansucrases was higher than that of dextransucrases at all conditions as can also be predicted from the genomics data. On the other hand, lower abundance of both glycosyltransferase were observed in C18 ferments compared to C13, although Cl 8 possess more glycosyltransferase encoding genes compared to C13. Overall, the result of this study indicates that production of glycosyltransferase enzymes and subsequently exopolysaccharides is a mechanism by which both C13 and Cl 8 protects themselves against acid stress. Example 9: Evaluation of pre-fermentation using lactic acid bacteria for production of reduced alcohol wine in a winery environment

[0415] The objective of the current study was to assess the feasibility of the process for production of reduced alcohol Shiraz, Grenache and Sauvignon Blanc wines at small pilot scale in a setting that simulates industrial wine making environment. The study was conducted at Charles Sturt University (CSU) research wine facility.

[0416] Materials and methods

[0417] Grape juices, chemical and biochemical reagents

[0418] Grape juice samples were purchased from Australian Vintage LTD (AVL), Australia. The samples used in this study were pasteurised Shiraz (Batch number BX31242-BJR, 2023) and Sauvignon Blanc (Batch number, BX3111-CPJW, 2023) and unpasteurised Grenache juices (BX4106-BJRN, 2024). Wine de-acidifier Blanco V® composed of 95% potassium bicarbonate and 5% potassium tartrate was sourced from Vason Enologica (Verona, Italy). Yeast extract was sourced from Cell Biosciences (Melbourne, Australia). Food grade DE MAN, ROGOSA, SHARPE (MRS) media for starter culture production was purchased from Amyl media (Melbourne, Australia). All wine making ingredients were purchased from winery suppliers and the sources of the ingredients are specified in the method sections. Similarly, all other chemical and biochemical reagents for microbial and chemical analyses were analytical grade or better and were sourced from major reagent suppliers and details are provided on them in the method sections.

[0419] Lactic acid bacteria culture (LAB)

[0420] The lactic acid bacteria (LAB) cultures for this study were selected based on an earlier proof of concept study conducted at CSIRO on Shiraz and Pinot gris grape juices. A strain of Leuconostoc mesenteroides (C12) from CSIRO’s lactic acid bacteria collection was used in the trial. The master culture was kept at -80°C, in MRS with a cryoprotectant (15% glycerol). The strain was activated and propagated in an MRS broth CN0359 (Oxoid Ltd., Basingstoke, Hampshire, England) at 30°C for 48-72 hours. The colony forming unit (CFU) of cells and purity was confirmed by optical density (OD) and by plating on MRS agar CM0929 (Oxoid Ltd., Basingstoke, Hampshire, England) at 30°C under anaerobic condition for 48-72 hours. The culture was then grown in MRS broth for 24 hours at 30°C, the broth removed by centrifugation and the cell pellet was resuspended in fresh MRS broth containing 15% glycerol at ~5xl09CFU / mL cell concentration. The cell suspension was then subsampled into 1 mL stock cultures in 2 mL Eppendorf tubes, which were stored frozen at -80°C until needed. The starter culture for the trial was prepared in a two-step process whereby the frozen stock culture was used as inoculum to produce the pre-culture in a small volume, which was subsequently used as seed culture to produce the starter culture at a larger scale. Accordingly, 3 vials of 1 mL frozen C12 stock cultures were thawed by keeping them for one hour at ambient temperature. Three mL of the stock culture was used to inoculate 800 mL of MRS broth (~107CFU / mL culture dose) to prepare the preculture. The inoculated MRS broth was incubated for ~17 hours at 30°C. The pre-culture was harvested by centrifugation for 10 min at 4000 g at 4°C and was used to inoculate 18 L MRS broth for starter culture production at larger scale. The 18 L MRS broth was incubated for ~17 hours at 30°C, followed by harvest of the cultures by centrifugation of the broth (4000 g, 10 min, 4°C) and resuspension in smaller volume of fresh MRS broth with 15% glycerol to achieve 5xl010CFU / mL culture concentration. Fifty mL of the culture suspension was subsampled into 50 mL falcon tubes and the tubes were kept frozen at -80°C for use as starter culture during the primary lactic acid bacteria fermentation of grape juice samples.

[0421] Two step wine making trials at small pilot scale (20L)

[0422] Two step fermentation experiments involving primary lactic acid bacteria fermentation followed by standard wine fermentation were conducted on three varieties of grape juice: Sauvignon Blanc, Grenache and Shiraz. Standard wine fermentations were also conducted on the same juices without primary lactic acid bacteria fermentation to produce untreated control samples as reference. All treatments were conducted in triplicates. The experimental design is summarised in Table 11. Variable capacity tanks (200 L) were used for the fermentation of the Sauvignon Blanc and Grenache wines whereas 25 L demijohn wine tanks were used for Shiraz fermentation. The wine making trials were conducted at Charles Sturt University (CSU) Winery.

[0423] Table 11: Description of experimental samples.

[0424]

[0425] Juice sample preparation Grape juice samples in drums were transferred from cold room (2°C) to a 30°C room a day before the trial. After temperature equilibration overnight, the pH of the juices was adjusted to pH~6.0 by adding de-acidifier (BlancoV®) powder after dissolving in the respective juices at 10.8 g / L, 8.1 g / L and 9.3 g / L for Sauvignon Blanc, Grenache and Shiraz juices respectively. Following pH adjustment, 20 L juice samples were transferred to the respective fermentation tanks and yeast extract (2 g / L) was added to the samples. The untreated control was directly fermented into wine without prior temperature incubation to 30°C or pH adjustment.

[0426] Lactic acid bacteria fermentation

[0427] LAB fermentation was conducted by inoculating the grape juice samples with Leuconostoc me senter oides C12 culture after pH adjustment. The frozen starter culture tubes (50 mL) prepared as described above were thawed by keeping them for ~1 hour at room temperature and were centrifuged (4000 g, 10 min, 4°C) to remove the liquid (MRS broth containing 4 g / L glycerol) in which the culture was suspended. The pellets containing the cells were resuspended in the same volume of the respective juices and were used to inoculate the juices. Two culture tubes were used per 20 L juice to achieve ~2.5xl08CFU / mL culture dosage. The samples were incubated at 30°C until the pH decreased to ~4.3. Samples were taken immediately after inoculation (t=0) and at various time points for pH measurement, microbiological and chemical analyses.

[0428] Wine fermentation

[0429] At the end of the LAB fermentation, 80 mg / L potassium metabisulphite (PMS) was added to the fermented samples to inactivate lactic acid bacteria and other contaminating microorganisms in the samples prior to wine fermentation. Both fermented and control juice samples were analysed for pH, titratable acidity (TA), Baume, free SO2, total SO2, acetic acid, malic acid, glucose, fructose and ammonia contents. The LAB fermented samples were kept chilled (3°C) for up to 16 hours after the end of fermentation prior to yeast inoculation for alcoholic fermentation.

[0430] Red wine fermentation

[0431] Untreated and LAB fermented Grenache and Shiraz samples were warmed to 18°C and were inoculated with 250 mg / L of EnartisFerm WS yeast (Enartis Pacific, Melbourne, Australia) after rehydration in the respective juice together with the activator Laffort superstart Rouge (LAFFORT® Australia, Nuriootpa, Australia). Fermentation was conducted at temperature ranging from 20 to 24°C. The Baume and temperature were measured daily and Diammonium phosphate (DAP) and proprietary yeast nutrient preparations (Go Ferm Protect Evolution (GFPE), Fermaid O and Nutristart™ Org) were added to the ferments at different stages of fermentation. When the samples reached 9 °Be, 100 ppm GFPE (Lallemand, Adelaide, Australia) and 50 ppm DAP were added to the ferments followed by 100 ppm Fermaid O (Lallemand oenology, Adelaide, Australia) and 50 ppm DAP addition at 7 °Be. Lastly 100 ppm Nutristart™ Org (LAFFORT® Australia, Nuriootpa, Australia) was added to the ferments when they reached 5 °Be. The wine fermentation was conducted until dry i.e. the sugar (G / F: glucose / fructose) concentration of the samples reached less than 2 g / L. At the end of fermentation, the gross lees were racked off and malolactic fermentation (MLF) were conducted at 21 °C where required to reduce the malic acid content of the wines to less than 0.2 g / L. Lalvin Mcbb Oenococcus oeni culture (Lallemand oenology, Adelaide, Australia) was used for the MLF fermentation. Once the MLF were completed, the wines were racked, and PMS was added at 80 mg / L and the ferments were moved to chilled (3 °C) storage for two weeks for cold stabilisation. Following that, the sulphite level of the wines was adjusted where needed to free SO2 and molecular SO2 levels of 40 ppm and 0.5 to 0.8 ppm respectively followed by racking, filtration and bottling.

[0432] White wine fermentation

[0433] Untreated and LAB fermented Sauvignon Blanc juices were equilibrated to 15°C. Then they were inoculated with 250 mg / L of Enartis EZ Ferm44 yeast (Enartis Pacific, Melbourne, Australia) after rehydration in the juice with the activator Laffort superstart Blanc (LAFFORT® Australia, Nuriootpa, Australia). Fermentation was conducted at 15°C until dry. As in the case of red wine fermentation, 100 ppm GFPE and 50 ppm DAP were added to the ferments at 9 °Be, 100 ppm Fermaid O and 50 ppm DAP at 7 °Be and 100 ppm Nutristart™ Org at 5 °Be. At 2 °Be, ferments were warmed to 18°C to enable completion of fermentation to sugar level less than 2 g / L. At the end of fermentation, the molecular SO2 level were adjusted to 0.8 ppm. Samples were then cold settled at 4°C for three days. This was followed by bentonite trial. The samples were then racked, and 1.2 g / L of bentonite was added to the ferments. The ferments were then tested for heat stability. This was followed by chilled storage for 14 days at 3°C. Samples were then tested for cold stability and were racked, filtered and bottled.

[0434] Microbial analysis

[0435] The microbial quality of the samples was assessed prior to and following LAB fermentation in accordance with methods described in Cai et al. (2019). Samples were serially diluted as required using 9.5 g / L of sterilised maximum recovery diluent (MRD) (Oxoid, VIC, Australia). The enumeration of lactic acid bacteria was done by plating 0.1 mL of the diluted sample on DE MAN, ROGOSA, SHARPE (MRS) (Oxoid Ltd., Cat. No. CM0361) agar in duplicates and incubation in an anaerobic box with anaerobic sachets (Thermo Fisher Scientific, Carlsbad, CA, USA) for 48 h at 30°C. For the enumeration of yeast and mould, 0.1 mL of the diluted sample was plated on Dichloran Rose-Bengal Chloramphenicol (DRBC) Agar (Oxoid Ltd., Cat. No. CM0059) in duplicates. The plates were incubated at 25°C for 72 hours. Following incubation, the colony-forming units (CFU) were counted and the results were expressed in CFU / mL.

[0436] Chemical analysis

[0437] The pH and titratable acidity (TA) of the juice and wine samples were measured using Metrohm 855 Robotic Titrosampler (Metrohm, NSW, Australia). The free sulphur dioxide (FSO2), total sulphur dioxide (TSO2), acetic acid, malic acid, glucose, fructose and ammonia concentrations were measured using Thermo Scientific Arena 20XT Chemistry Analyser (Thermo Fisher Scientific, Australia). The Baume of the samples was analysed using Anton Paar Density Meter Analyser (DMA) 35 (Anton Paar, North Ride, Australia).

[0438] High Performance Liquid Chromatography (HPLC) for Sugar Analysis

[0439] The sugar content of the samples was analysed prior to and after fermentation as described in (Xu et al., 2020; Ye et al., 2019). In short, samples were mixed with three volumes of ethanol. This was followed by centrifugation to remove the alcohol insoluble residues. The supernatant was filtered and loaded to HPLC system for sugar analysis. HPLC analysis of the filtered samples was performed using an Arc HPLC system coupled with a 2414 refractive index detector (Waters Corp., Wilford, MA, USA) at 35°C. A Shodex Asahipak NH2P-504E (4.6 x 250 mm) and NH2P-50G column (4.6 x 50 mm) (Showa Denko K.K, Japan) was used to separate the sugars at temperature of 30°C with 68% acetonitrile as mobile phase. During operation, the samples and the sugar standard mixture for calibration were kept at 5°C. Chromatograms were recorded and compared with 5 mg / mL sugar standard mixture of fructose F0127 (Sigma), mannitol 291484B (BDH Reagents & Chemicals), glucose G8270 (Sigma) and sucrose VWRC27480.294 (AnalaR NORMAPUR®). Sugar content was calculated based on the sugar standard calibration curve. The sugar reduction of the samples was reported as percentage compared to initial sugar present in non-fermented grape juice. Data analysis

[0440] All the experiments were conducted in triplicates and the results were expressed as the mean ± SD in cases where data for individual samples are not presented. Statistical analysis of the experimental data was performed using OriginPro 2019 (OriginLab, Northampton, MA, USA). Data was analysed by one-way ANOVA to evaluate treatment effects at 0.05 significance level. Tukey post-hoc analysis was used to compare treatment means.

[0441] Chemical and microbiological characteristics of grape juice samples

[0442] The chemical and microbiological characteristics of the grape juice samples were determined prior to fermentation or incubation treatments. Data are presented in Tables 12 and 13.

[0443] Table 12: Chemical profile of grape juices samples.

[0444] <

[0445] <

[0446] <

[0447]

[0448] FSO2: Free SO2, TSO2: Total SO2, TA: Titratable acidity

[0449] Table 13: Microbial profile of grape juice samples.

[0450] < <

[0451] < <

[0452]

[0453] ND: Not detected, TNTC: too numerous to count, Y: Yeast like colonies on MRS

[0454] The chemical characteristics of the juices relevant to wine quality and alcohol content including °Be, pH, titratable acidity and sugar content were within the range expected for the grape varieties investigated (Table 12). The Shiraz and the Sauvignon Blanc juices were microbiologically clean as they were pasteurised, whereas the Grenache juice was highly contaminated with yeast (Table 13).

[0455] Primary fermentation of grape juice samples

[0456] Progress of the LAB fermentation process

[0457] The pH profile of the LAB fermented juices are presented in Figures 17 to 19. A steady decrease in pH was observed in all the samples as expected, indicating successful fermentation. A fast initial decrease in pH followed by slower decrease was observed in the Sauvignon Blanc and Grenache juices whereas pH decrease was slow and steady in the case of Shiraz following a slight increase in pH during the first two hours of fermentation. The fermentation of the Sauvignon Blanc and Grenache juices was relatively fast, with the samples reaching end point pH of ~4.3 within 48 hours of fermentation. In the case of Shiraz juice, the time to reach similar pH was 91 hours, which is almost twice as long. This is most likely due to the higher tannin content of Shiraz juice, which has inhibitory effect on the growth and metabolic activity of Leuconostoc mesenteroides and other lactic acid bacteria (Huang et al., 2022). Some lactic acid bacteria have tannin degrading ability (Shang et al., 2019; Ueda et al., 2016). However, the analysis of the whole genome sequence of the C12 strain that was used in this study showed that it does not possess the gene encoding tannase.

[0458] Changes in microbial profile of samples during primary fermentation

[0459] The microbial quality of the grape juice samples was assessed at the beginning, after 21 hours and at the end of fermentation. Data are presented in Table 14. In LAB fermented Sauvignon Blanc juice samples, the LAB count was ~108CFU / mL immediately after inoculation (t=0) as expected. Yeast and mould were not detected in the samples. After 21 hours of fermentation, there was one log increase in the LAB count, whereas no yeast and mould were detected in the samples indicating that the Leuconostoc mesenteroides culture inhibited the growth of contaminating microorganisms. Lactic acid bacteria such as Leuconostoc mesenteroides synthesise various compounds including organic acids such as lactic acid, acetic acid, and phenyllactic acid, and antimicrobial peptides such as bacteriocins, which suppress other microorganisms (Di Cagno et al., 2013; Terefe & Augustin, 2020). At the end of the 48 hours fermentation, there was a slight decline in the LAB count. Yeast was also detected in the samples at 102CFU / mL. Leuconostoc mesenteroides have relatively low acid tolerance and the decrease in pH during fermentation gives relative advantage for yeast contaminants to grow, which explains the higher number of yeast count at the end of fermentation and the slight decline in the LAB count (Plengvidhya et al., 2007).

[0460] The LAB fermented Grenache and Shiraz juices also had high level of LAB count (~107CFU / mL) and no detectable yeast count just after inoculation (t=0). However, the level of LAB count was one log less than the inoculation level. It seems that the tannins and other polyphenols in these juices may have inactivated some of the LAB inoculum. There was one log increase in LAB count after 21 hours and no yeast or mould was detected in these juices as in the case of Sauvignon Blanc. This is especially interesting in the case of the unpasteurised Grenache juice, which had high level of yeast contaminants before inoculation (Compare Table 13 with Table 14), indicating the efficacy of the starter culture for controlling yeast growth when the environmental condition such as pH favours them. There was one log reduction in LAB count at the end of fermentation in both Grenache and Shiraz juices. On the other hand, the yeast count increased substantially in both juices at the end of the LAB fermentation step (Table 14).

[0461] Table 14: Changes in microbial count (CFU / mL) of grape juice samples during primary fermentation.

[0462] <

[0463] < >

[0464] <

[0465]

[0466] TNTC: too numerous to count at the tested dilution, Y: yeast like colonies, *: accuracy dependent on sample dilution rate used in the analysis, values in red are number of yeast like colonies observed on MRS plate. Beginning of fermentation (t=0) is 24 hours after incubation of the untreated juice at the experimental temperature (30°C) and immediately after pH adjustment and inoculation of the samples. End of fermentation (t=end) is 48 hours for Sauvignon Blanc and Grenache samples and 91 hours for Shiraz samples.

[0467] Changes in the sugar content of grape juice samples

[0468] Significant reductions in the fermentable sugar contents of the grape juices were observed following LAB fermentation (Figure 20, Table 15). The extent of sugar reduction was dependent on the grape variety. The average total sugar reductions were 33%, 41% and 25% respectively in the LAB fermented Sauvignon Blanc, Grenache and Shiraz juices. In the case of LAB fermented Sauvignon Blanc and Shiraz samples, more reduction was observed in fructose content whereas similar level of fructose and glucose reduction was observed in the case of the LAB fermented Grenache juices. The fructose in the LAB fermented Sauvignon Blanc and Shiraz juices appears to be totally converted to mannitol, whereas only part of the fructose consumed during LAB fermentation appears to be converted to mannitol in the case of the LAB fermented Grenache juice (Table 15). This seems to be due to the greater effect of yeast growth in the case of the Grenache juices, which was not pasteurised. Heterofermentative bacteria such as Leuconostoc mesenteroides catalyse the reversible reduction of fructose to mannitol using the enzyme mannitol dehydrogenase (Wisselink et al., 2002). The C12 strain that was used in this study has genes encoding mannitol dehydrogenase.

[0469] Table 15: Sugar profile of grape juice in untreated samples compared to samples at the end of primary fermentation at 30°C (48 hours for Sauvignon Blanc and Grenache samples, 91 hours for Shiraz samples).

[0470]

[0471] Reported data are average of three independent replicates and standard deviation. Different superscript letters within the same juice group and the same column indicate significantly different (p<0.05) values.

[0472] Changes in the chemical profile of grape juice samples

[0473] The changes in some chemical attributes of the grape juice samples after primary LAB fermentation are presented in Table 16. Substantial increase in the titratable acidity and acetic acid content the LAB fermented samples (Compare Table 12 and Table 16).

[0474] The titratable acidity increased from 6.5, 4.8 and 5.4 g / L tartaric acid equivalent to 9.5, 8.6 and 7.8 g / L tartaric acid equivalent in the case of Sauvignon Blanc, Grenache and Shiraz juices respectively during LAB fermentation. The acetic acid also increased from close to zero to 4.9, 4.05 and 2.9 g / L after lactic acid bacteria fermentation for Sauvignon Blanc, Grenache and Shiraz juices. The malic acid concentration on the other hand substantially decreased from 3.15, 1.33 and 2.23 g / L to 0.08, 0.1 and 0.14 g / L for Sauvignon Blanc, Grenache and Shiraz juices respectively. A slight increase in the alcohol content of the samples was observed following LAB fermentation in the case of Sauvignon Blanc and Shiraz juices whereas substantial production of alcohol was observed in the case of Grenache juices. The small amount of alcohol produced during LAB fermentation is the consequence of sugar metabolism by heterofermentative bacteria including Leuconostoc me senter oides. The increase in the titratable acidity of the samples after LAB fermentation is also due to the metabolism of sugars into organic acids such as lactic acid and acetic acid utilising the same pathway. Heterofermentative lactic acid bacteria use the phosphoketolase pathway to metabolise glucose into lactic acid, CO2, and acetic acid and / or ethanol (Bintsis et al., 2018; Terefe & Augustin, 2020). The relatively high production of ethanol in the Grenache juice during LAB fermentation is most likely due to metabolism of sugar into ethanol by contaminating yeast, which partly explain the observed sugar reduction and the relatively different sugar profile compared to the other juices after LAB fermentation. In the case of Sauvignon Blanc and Shiraz juices, the reduction of fructose was higher than glucose whereas higher glucose reduction was observed in the Grenache juices, which indicate the contribution of yeast metabolism. The decrease in malic acid in the LAB fermented juices is due to the malolactic activity of the starter culture. Leuconostoc mesenteroides are known to have malolactic activity (Lonvaud-Funel & de Saad, 1982). The whole genome sequence data shows that the C12 strain that was used in this trial has two genes encoding malolactic enzyme.

[0475] Table 16: Chemical profile of grape juice samples at the end of primary LAB fermentation.

[0476]

[0477]

[0478] Reported data are average of three independent replicates and standard deviation.

[0479] Secondary yeast fermentation of juices into wine

[0480] Progress of the yeast fermentation process

[0481] The yeast fermentation of the LAB fermented Sauvignon and Grenache samples took 21 days to complete whereas the fermentation of the LAB fermented Shiraz samples were completed in 20 days. On the other hand, fermentation of the untreated control samples was completed in 15 days in all cases. Most of the LAB fermented samples were stuck and needed to be re-inoculated with yeast except the LAB fermented Sauvignon Blanc samples. The metabolites that were formed during LAB fermentation may have inhibited the growth and metabolic activity of the yeast inoculum.

[0482] Alcohol content and chemical profile of treated and control wines

[0483] The alcohol content and other relevant chemical attributes of the LAB fermented and untreated wine samples were analysed during and at the end of wine fermentation. Data corresponding to the end of fermentation are presented in Table 17. LAB fermentation resulted in a significantly lower alcohol (p<0.05) compared to untreated control for all wine varieties (Figure 21, Table 17). The average level of percentage alcohol reduction compared to untreated control wines were 42.5% (5%v / v lower alcohol) in the case of Sauvignon Blanc, 41.1% (5.5%v / v lower alcohol) in the case of Grenache and 24% (3.2%v / v lower alcohol) in the case of Shiraz wines.

[0484] Table 17: Chemical profile of control and LAB pretreated wines.

[0485]

[0486]

[0487]

[0488] Reported data are average of three independent replicates and standard deviation. Different superscript letters within the same juice group and the same column indicate significantly different (p<0.05) values. *: MLF is not completed for the untreated Grenache samples

[0489] Overall, the level of alcohol reduction was much lower in the case of Shiraz compared to the other two varieties perhaps due to the inhibitive effects of tannins in Shiraz juice on the lactic acid bacteria inoculum. If grape must was used in this trial as in conventional red wine making, the tannin issue could have been even more challenging. A better approach for implementing the LAB primary fermentation in the case of strong red varieties such as Shiraz is to separate the juice without maceration from the skin, conduct the LAB fermentation on the juice and reincorporate the skin at the alcoholic fermentation stage.

[0490] Pearson correlation analysis was conducted between the fermentable sugar content at the end of primary LAB fermentation and the alcohol content of the samples after alcoholic fermentation. There was a reasonable correlation between the two with correlation coefficient of 0.71. Ideally, a correlation coefficient of close to 1 is expected between fermentable sugar content at the beginning of alcoholic fermentation and the alcohol content at the end of the process. The deviation in this case can be attributed to the continuous activity of contaminating low alcohol producing yeast and conversion of some of the sugar into other metabolites such as acetic acid.

[0491] The result of this study indicates that primary fermentation by Leuconostoc mesenteroides enables significant fermentable sugar reduction and reduced alcohol wine after secondary alcoholic fermentation. However, this is accompanied by very high volatile acidity (acetic acid production). Very high concentration of acetic acid was observed in all the LAB fermented samples at the end of the LAB fermentation step, which increased during yeast fermentation despite the sulphite treatment to inactivate the Leuconostoc mesenteroides culture at the end of the lactic acid bacteria fermentation.

[0492] Table 18: Sugar profile of wine samples. Wine samples were analysed after post processing and bottling using HPLC.

[0493]

[0494]

[0495] Reported data are average of three independent replicates and standard deviation. Different superscript letters within the same juice group and the same column indicate significantly different (p<0.05) values. An asterix (*) in the superscript indicates significant difference with concentration after LAB fermentation.

[0496] At the end of LAB fermentation, the acetic acid concentrations were 4.89 g / L, 4.05 g / L and 2.8 g / L for the LAB fermented Sauvignon Blanc, Grenache and Shiraz samples respectively. That increased to 6.0 g / L, 5.5 g / L and 2.9 g / L after alcoholic fermentation into wine (Table 17). It has to be noted that the maximum acetic acid concentration acceptable for red and white wines are 1.4 g / L and 1.2 g / L respectively. Overall, the acetic acid concentration was proportional to the level of sugar and alcohol reduction in the samples. The main contributor to the formation of the excessive amount acetic acid in these samples is sugar metabolism by Leuconostoc mesenteroides via phosphoketolase pathway, which favours acetic acid formation instead of alcohol under aerobic condition. The increase in the acetic acid content specially in the Grenache and Sauvignon Blanc wines from their levels at the end of LAB fermentation indicates that the metabolic activities of the Leuconostoc mesenteroides culture continued during the secondary alcohol fermentation. Although sulphite treatment was applied on the samples post LAB fermentation, that didn’t seem to be fully effective for inactivating the LAB culture, perhaps due to the relatively high pH of the LAB fermented samples (4.25 to 4.33, see Table 17) and consequently lower free SO2 level responsible for microbial inactivation. There was also an increase in the mannitol content of the LAB fermented Sauvignon Blanc and Grenache samples (statistically significant only in the case of Grenache sample, Table 18), following wine fermentation, which confirms the continuous activity of the LAB cultures during wine fermentation albeit at slower rate (Table 18) This indicates that the LAB fermented grape juices need to be acidified to the natural pH level of grape juice for effective inactivation of the LAB starter culture prior to the wine fermentation step.

[0497] There was no increase in acetic acid or mannitol concentration in the LAB fermented Shiraz samples during wine fermentation perhaps due to the unfavourable condition for LAB fermentation created by the high tannin content of the Shiraz samples in addition to competition with yeast. Both the primary and secondary fermentations were conducted in closed tanks, although there was frequent opening of the sampling port of the tanks for sampling, which may have led to ingression of air. Under such conditions, chemical oxidation of alcohol to acetic acid is also possible, which may have contributed to the observed high acetic acid level in the samples post alcoholic fermentation.

[0498] The very high volatile acidity in the LAB treated wines is a major quality defect with negative consequence on the sensory acceptance of the alcohol reduced wines. Clearly, that will limit the general application of the LAB based technology for production of reduced alcohol wines. Nevertheless, if target sugar reduction is limited to lowering the alcohol content of wines by 1-2% (v / v) required for addressing climate change related increase in sugar content of grapes, the approach can be used without causing the excessive volatile acidity observed in this trial as the acetic acid production is largely proportional to the level of sugar reduction. Blending with untreated wine may also result in wine with acceptable volatile acidity and modest alcohol reduction of 1 to 2% (v / v) on absolute terms. Another strategy to overcome the challenge with volatile acidity is the application of adsorption process for selective removal of acetic acid. The challenge with such approaches is the possibility of simultaneous adsorption and removal of other wine components essential for wine quality such as pigments and volatile compounds. This can be potentially overcome by introducing the adsorption step just after the LAB fermentation of non-macerated juice (and incorporation of grape skin during the wine fermentation step) and through judicious selection of adsorbents.

[0499] The sugar analysis of the wine samples showed that the mannitol content of the LAB fermented wine samples slightly increased or remained the same following wine fermentation indicating that wine fermentation yeast can’t ferment mannitol into alcohol. Mannitol is a naturally occurring sugar alcohol with zero glycaemic index and 50-70% sweetness and 40% calorie relative to sucrose. It is commonly used in the food industry as sweetener, cooling agent, humectant, and thickener in baked goods, hard candies, and spreads (Chen et al., 2020). Nevertheless, excessive consumption may cause bloating and laxative effect, although 50 to 200 g of mannitol can be administered to patients in clinical applications. The presence of mannitol in the reduced-alcohol wines in this study may contribute positively to their sensory profile and mouthfeel. The risk of diarrhoea and bloating from these products is minimal as people are unlikely to consume more than 500 mL of these products at one go. On average, 10.9% (in Shiraz) to 26.7% (in Sauvignon Blanc) of the sugar in the LAB fermented samples was converted into mannitol, whereas 1.2% (Shiraz) to 2.9% (Sauvignon Blanc) was converted to acetic acid, with the rest converted to alcohol and other metabolites and used up for growth of the LAB and yeast cultures.

[0500] The results of this study showed that microbial contamination is unavoidable in a winery environment even when pasteurised juices are used as the starting material. Pasteurised Sauvignon Blanc and Shiraz juices were used in this trial. However, yeast contaminants eventually grew in these samples at the end of the LAB fermentation step. However, both in the case of the pasteurised juices and the non-pasteurised Grenache juice, the sugar profile of the LAB fermented samples indicates that the dominant microorganism that contributed to sugar reduction was the Leuconostoc mesenteroides starter culture. This shows that the technology can be used in a winery environment, where high sanitation standards cannot be maintained and unpasteurised grape juices / musts are normally used. Obviously, starting with clean grape juice treated either by microfiltration or heat as in the flash detent process enables better control of the process and ensure consistent outcome.

[0501] Conclusions

[0502] This study successfully demonstrated the feasibility of producing reduced alcohol wine through primary fermentation using Leuconostoc mesenteroides cultures prior to alcohol fermentation in a winery environment. The starter cultures overcame competition from contaminating microbes, which are unavoidable in a winery environment, and redirected sugar metabolism into metabolites such as mannitol, resulting in a significant (p<0.05) alcohol reduction in all treated samples. Nevertheless, the trials were conducted in a research winery at small scale (20 L) and additional trials at larger scale and in a commercial winery are required to further confirm the scalability of the technology.

[0503] Substantial alcohol reduction of 42.5%, 41.1 % and 24% compared to control were observed in Sauvignon Blanc, Grenache and Shiraz wines respectively. Clearly, the approach was more effective for Sauvignon Blanc and Grenache varieties which have lower content of tannins compared to Shiraz. Conducting the primary fermentation in non-macerated juice and introducing the skin and pomace during the wine fermentation step may help to increase the level of sugar and alcohol reduction in strong red varieties such as Shiraz.

[0504] Example 10: Evaluation of reducing acetic acid content of wines using weak anionic adsorbents

[0505] The objective of this study was to evaluate whether weak anionic adsorbents can be used to selectively adsorb acetic acid from reduced alcohol wine produced using the two-step fermentation process. Experiments were conducted on reduced alcohol wines produced as described in Example 9. Experiments were conducted on three varieties of reduced alcohol wine namely: Sauvignon Blanc, Grenache and Shiraz.

[0506] Materials and methods

[0507] Resin preparation

[0508] The polyanionic resin Amberlite IRA67 was purchased from Dupont Water solutions (Midland USA). The resin was rinsed to remove shipping preservatives using deionised water and was soaked overnight to fully hydrate. Then it was treated with 1 M NaOH solution for 1 hour to convert the resin to its free base form. This was followed by rinsing using Milli-q water. This was followed by treatment with 0.5 M HCL to condition the resin and rinsing with Milli-q water until the effluent reached neutral pH.

[0509] Static adsorption experiments

[0510] Three grams of wet resin were added to 50 mL falcon tubes. Then 15 g of reduced alcohol wine were added to the test tube to maintain 1:5 wet resin to wine ratio. The mixture was incubated for 18 hours at room temperature. Samples were gently shaken using Ratek RM4 orbital shaker (Ratek Instruments, Australia) during incubation at a speed setting of 15 to ensure uniform contact between the wine and the resin. Following incubation, samples were centrifuged (4000 g, 5 minutes, 21 °C) to separate the resin as the residue. The supernatant, which is the treated reduced alcohol wine, was analysed for acetic acid concentration and colour. The acetic acid analysis was conducted in external laboratory (Winechek, Melbourne). The colour of the samples was measured using Minolta Chroma Meter (CR-400, Konica Minolta Inc, Japan) as CIE L*, a* and b* values. The total colour change was calculated in accordance with equation 1 :

[0511]

[0512] Where Lo, a0and bo are the average values for the untreated samples. The acetic acid content and colour of untreated samples were also analysed for use as controls. Experiments were conducted on three biological replicates per grape variety.

[0513] Results and discussion

[0514] The effects of resin treatment on acetic acid content of reduced alcohol wine

[0515] The treatment of the reduced alcohol wine samples using the resin Amberlite IRA67, resulted in substantial reduction of the acetic acid contents of the reduced alcohol wine samples, although some degree of sample to sample variation was observed (Table 19). Overall, the percentage reduction was above 60% except in one of the Sauvignon Blanc samples which had the lowest acetic acid content. The treatment reduced the acetic acid content of Shiraz wine to the accepted acetic acid level of less than 1.5 g / L. Further optimisation of the process with better selection of adsorbents as well as processing conditions is possible to enable higher level of acetic acid reduction. The result indicates that adsorption treatment using weak anionic resins is a promising technique for reducing acetic acid in reduced alcohol wines produced using the two-step fermentation process.

[0516] The effect of resin treatment on wine colour and pH

[0517] The adsorption treatment was accompanied by substantial pH increase (Table 19), which would require pH adjustment following treatment using acids such as tartaric acid. There was also substantial change in the colour of the samples (Table 19), which could be due to the adsorption of pigment compounds including anthocyanins and other polyphenolic compounds onto the resin. This affects not only the colour of the wines but also taste and flavour as these compounds contribute to the taste and flavour of wines. The change in colour was observed in all the samples. The highest colour change was observed in Shiraz samples followed by Grenache and at distance by Sauvignon Blanc samples. The colour changes were in the ‘great changes in visually perceptible colour’ range for all samples. The total colour change (AE) ranges for ‘not noticeable’, ‘slightly noticeable’, ‘noticeable’, ‘well visible’ and 'great changes in visually perceptible colour' are 0 to 0.5, 0.5 tol.5, 1.5 to 3, 3 to 6, and 6 to 12 respectively (Terefe et al., 2016).

[0518] Table 19: The effect of resin treatment on acetic acid content, pH and colour of wine samples.

[0519]

[0520]

[0521] Conclusion

[0522] The results of this study showed that adsorption treatment using weak polyanionic resins can effectively remove acetic acid from wines. This is accompanied by substantial change in colour due to absorption of pigments, which negatively affect the acceptability of the wine from both visual quality and taste and flavour perspectives. Alternatively, the resin adsorption treatment may be conducted after the first fermentation step on grape juices (lactic acid bacteria fermentation) and prior to wine fermentation as shown schematically in Figure 22. Accordingly, the modified process involves 1) grape crushing to separate the grape skin from the juice for both red and white wines, 2) storage of the skin, 3) conducting lactic acid bacteria fermentation on the juice after pH adjustment to pH 5.0 to 7.0 (and adding yeast extract etc. as nitrogen source), 4) conducting resin adsorption step using resins such as Amberlite IRA 67, 5) pH adjustment of the juice to pH 3.0 to 3.6 using for instance tartaric acid and 6) Combining the treated juice with the stored skin and conducting yeast fermentation to wine using wine yeast cultures and additional nutrients to support fermentation.

[0523] Example 11: Evaluation of reducing the acetic acid content of lactic acid bacteria fermented juice using weak anionic adsorbents

[0524] The objective of this study was to evaluate the feasibility of removing acetic acid from lactic acid fermented juice using weak anionic adsorbents so as to implement it in the modified reduced alcohol wine production process described in Figure 22.

[0525] Materials and methods

[0526] Fermented grape juice preparation

[0527] Pasteurised Sauvignon Blanc grape juice (Batch number, BX3111-CPJW, 2023) was purchased from Australian Vintage LTD (AVL, Australia). The juice pH was adjusted to 5.5 using 3 M potassium bicarbonate. Yeast extract (Flavex, Cell Biosciences, Melbourne, Australia) was added to the juice sample at 4 g / L as a supplementary nitrogen source. The juice sample was then inoculated with Leuconostoc mesenteroides C12 culture at 107CFU / mL and the sample was incubated for 48 hours at 30°C. Once fermentation was completed, samples were centrifuged at 9600 g for 15 minutes to remove the culture cells. The pH and microbial count of the samples were assessed following fermentation. Fermentation experiments were conducted in triplicates.

[0528] Adsorbent resin preparation

[0529] Amberlite IRA67 resin was used in the adsorption experiments after pre-treating the resin as described in Example 10.

[0530] Static adsorption experiments

[0531] Static adsorption experiments on juice samples were conducted as described in Example 10. However, in this case, the resin to juice ratio was 1 to 10 and for that 2 grams of wet resin were mixed with 20 grams of fermented juice. Following incubation, the juice was separated from the resin by centrifugation at 4000 g for five minutes at 21 °C. Samples were then analysed for acetic acid concentration as described in Example 10. Adsorption experiments were conducted on three biological replicate samples.

[0532] Results and discussion The pH of the Sauvignon Blanc juice samples dropped to ~3.8 after 48 hours fermentation. The lactic acid bacteria count at the end of fermentation was ~108CFU / mL. As in the case of the wine samples, adsorption treatment with Amberlite IRA67 resulted in substantial acetic acid reduction of 44% to 47% (Table 20) The sample to sample variation was minimal perhaps due to the similar initial acetic acid concentration of the fermented juice samples. The level of acetic acid reduction was significantly lower than what was observed for the wine samples. This is most likely due to the lower resin to sample ratio. The weight of the wet resin used in the wine treatment was twice that was used in the juice treatment. The level of acetic acid removal is expected to improve with process optimisation including resin to liquid ratio and selection of a more suitable resin and optimised processing conditions. The resin caused substantial increase in pH due to the removal of acetic acid and perhaps other organic acids. Thus, reacidification of the juice is needed prior to the wine fermentation step.

[0533] Table 20: The effect of adsorption treatment using amberlite IRA67 on acetic acid content and pH of fermented lactic acid bacteria Sauvignon Blanc grape juice.

[0534]

[0535] Conclusion

[0536] The results of this study showed that adsorption treatment using weak anionic resin could be implemented after lactic acid bacteria fermentation and prior to wine fermentation to reduce acetic acid concentration and improve the quality of reduced alcohol wine produced using the two-step fermentation process as envisaged in the modified process described in Figure 22. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0537] This application claims priority from Australian Provisional Application No.

[0538] 2024903699 entitled “Method for producing low alcohol or reduced alcohol beverages” filed on 12 November 2024, the entire contents of which are hereby incorporated by reference.

[0539] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0540] 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 of each claim of this application. REFERENCES

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Claims

CLAIMS1. A method of producing a low alcohol or a reduced alcohol product from a base alcohol production biomass the method comprising:i) primary fermentation of the base alcohol production biomass with lactic acid bacteria to reduce fermentable sugar;ii) secondary alcohol fermentation of the material from i) with yeast forming a low alcohol or reduced alcohol product.

2. The method of claim 1, wherein the base alcohol production biomass is selected from one or more of: must, juice, juice concentrate, puree, wort, reconstituted fruit or vegetable powder, rehydrated dried fruit pieces and sugary fraction of fruit and vegetable processing.

3. The method of claim 2, wherein the juice is fruit juice.

4. The method of claim 3, wherein the must or juice is derived from grapes selected from: Shiraz, Chardonnay, Sauvignon Blanc, Grenache, Pinot gris, Kyoho, Cabernet Sauvignon, Sultanina, Merlot, Tempranillo, Airen, Syrah, Pinot Noir / Blauer Burgunder, Trebbiano Toscana / Ugni Blanc Semilion, Riesling and Muscat of Alexandria.

5. The method of any one of claims 1 to 4, wherein the base alcohol production biomass is juice separated from the skin of the biomass, wherein at least 80% of the skin is separated from the juice.

6. The method of any one of claims 1 to 5, wherein lactic acid bacteria are removed or deactivated from the fermented material produced by step i) and the fermented material, an extract thereof, or dried powder produced from the fermented material or an extract thereof, is incubated with unfermented base alcohol production biomass before step ii).

7. The method of claim 6, wherein the ratio of fermented material to unfermented base alcohol production biomass is about 1 : 100 to about 1:5.

8. The method of claim 7, wherein the ratio of fermented material to unfermented base alcohol production biomass is about 1:5.

9. The method of any one of claims 6 to 8, wherein incubation is for about 2 to about 50 hours at about 25°C to about 50°C.

10. The method of any one of claims 6 to 9, wherein incubation is for about 24 to about 48 hours at about 30°C.

11. The method of any one of claims 1 to 10, wherein a low alcohol or reduced alcohol product comprises about 0.1 to about 1.15% alcohol by volume.

12. The method of any one of claims 1 to 11, wherein alcohol in the product is reduced by about 5% to about 50% compared to a control product.

13. The method of claim 12, wherein alcohol is present at about 0.1% to about 15% alcohol by volume, or about 0.1% to about 9% alcohol by volume, or at about 4.5% to about 9% alcohol by volume, or about 4.5% alcohol by volume.

14. The method of any one of claims 1 to 13, wherein fermentable sugar comprises one or more of: fructose, glucose and sucrose.

15. The method of any one of claims 1 to 14, wherein fermentable sugar in the product is reduced by about 30% to about 70% compared to a control product.

16. The method of any one of claims 1 to 15, wherein mannitol in increased in the product compared to a control product.

17. The method of any one of claims 1 to 16, wherein sucrose is added before step i), during step i) or before step ii).

18. The method of any one of claims 1 to 17, wherein the primary fermentation comprises malolactic fermentation.

19. The method of any one of claims 1 to 18, wherein the lactic acid bacteria are selected from one or more of the genera selected from: Leuconostoc, Oenococcus, Lactobacillus, Weissela, Pediococcus, Lactococcus, Streptococcus, Aerococcus,Carnobacterium, Enterococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, Weissella, Bacillus and Zymomonas.

20. The method of claim 19, wherein the lactic acid bacteria are selected from one or more of: Leuconostoc mesenteroides, Oenococcus Oeni, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactococus lactis, Pediococcus pentosaceus, Lactobacillus rhamnosus, Pedicoccus acidilacti, Bacillus subtilis, Bacillus subtilis natto, Bacillus circulans and Zymomonas mobilis.

21. The method of claim 20, wherein the lactic acid bacteria are a Leuconostoc mesenteroides.

22. The method of any one of claims 1 to 21, wherein the lactic acid bacteria comprise an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme oxalacetate dehydrogenase, and glycosyltransferase.

23. The method of any one of claims 1 to 22, wherein the glycosyltransferase is selected from one or more of: dextransucrase, levansucrase and altemansucrase.

24. The method of claim 23, wherein the lactic acid bacteria comprise malolactic enzyme.

25. The method of any one of claims 1 to 24, wherein the lactic acid bacteria produce greater than 1 mg / mL of exopolysaccharides when cultivated at 37°C for 20 hours with 10% sucrose supplementation.

26. The method of claim 25, wherein the exopolysaccharides are predominantly homopolysaccharides (HoPS).

27. The method of claim 20 or claim 21, wherein the Leuconostoc mesenteroides is selected from one or more of:i) Leuconostoc mesenteroides BF deposited under VI 7 / 021729 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof;ii) Leuconostoc mesenteroides BF2 deposited under V17 / 021730 on 25 September 2017 at the National Measurement Institute Australia or a derivative thereof;iii) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;iv) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof;v) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;vi) Leuconostoc mesenteroides Cl 8 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and vii) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

28. The method of any one of claims 1 to 27, wherein the base alcohol production biomass is adjusted to a pH of about 5 to about 7 before step i).

29. The method of claim 28, wherein the pH is adjusted with the addition of a food grade salt.

30. The method of claim 29, wherein the food grade salt is selected from one or more of: CaCCh, Ca(OH)2, KHCCh or KHPC .

31. The method of any one of claims 1 to 30, wherein before step i) the base alcohol production biomass is pre-treated to reduce the microbial load.

32. The method of claim 31, wherein pre-treatment comprises one or more of: addition of a sulphite, addition of dimethyl decarbonate or heat treatment.

33. The method of claim 32, wherein the sulphite is selected from one or both of: sulphur dioxide or potassium metabisulphite.

34. The method of any one of claims 1 to 33, wherein primary fermentation is for about 10 hours to about 17 days.

35. The method of any one of claims 1 to 34, wherein primary fermentation is for about 10 to about 24 hours.

36. The method of any one of claims 1 to 35, wherein primary fermentation occurs till the pH is reduced to about 4.

37. The method of any one of claims 1 to 36, wherein primary fermentation is at about 22°C to about 34°C.

38. The method of any one of claims 1 to 37, wherein primary fermentation is at about 30°C.

39. The method of any one of claims 1 to 38, wherein the lactic acid bacteria are deactivated or removed after step i).

40. The method of claim 39, wherein the lactic acid bacteria are deactivated by the addition of a bactericidal agent or removed by microfiltration.

41. The method of claim 40, wherein the bactericidal agent is selected from sulphur dioxide, potassium metabisulphite or dimethyl decarbonate.

42. The method of any one of claims 1 to 41, wherein after step i) the pH is adjusted to a pH of about 3.4 to about 3.7.

43. The method of claim 42, wherein the pH is adjusted with the addition of tartaric acid.

44. The method of any one of claims 1 to 43, wherein before step ii) the level of acetic acid in the ferment from i) is reduced.

45. The method of any one of claims 1 to 44, wherein the skin of the biomass is added before step ii).

46. The method of any one of claims 1 to 45, wherein the yeast is selected from one or more of: Saccharomyces cerevisiae, Pichia stipitis and Candida tropicalis.

47. The method of claim 46, wherein the yeast is Saccharomyces cerevisiae.

48. The method of any one of claims 1 to 47, wherein secondary fermentation is for about 5 to about 40 days.

49. The method of any one of claims 1 to 48, wherein secondary fermentation is at about 15°C to about 30°C50. The method of any one of claims 1 to 49, wherein the secondary fermentation is anaerobic.

51. The method of any one of claims 1 to 50, comprising post-treating after step ii) with one or more of: microfiltration, fining, the addition of preservatives, addition of further biomass, reverse osmosis and reducing the level of acetic acid.

52. The method of any one of claims 1 to 51, wherein post-treating comprises reducing the level of acetic acid.

53. The method of claim 52, wherein the post-treating reduces the level of acetic acid to about 0.01 g / L to about 1.5 g / L.

54. The method of claim 52 or claim 53, wherein the post-treating reduces the level of acetic acid in the ferment from ii) by about 40 to about 99%.

55. The method of claim 51, wherein the further biomass is juice.

56. A lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product obtained, or obtainable, by the method of any one of claims 1 to 55.

57. A lactic acid bacteria enzyme composition for producing a low alcohol or reduced alcohol product, wherein the composition comprises fermentation enzymes and exopolysaccharides.

58. The lactic acid bacteria enzyme composition of claim 56 or claim 57, wherein the fermentation enzymes comprise one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme oxalacetate dehydrogenase and a glycosyltransferase.Ill59. The lactic acid composition of any one of claims 56 to 58, where the composition comprises one or more of the following components:i) exopolysaccharides;ii) an enzyme selected from one or more of: malic enzyme, L-lactate dehydrogenase, malate dehydrogenase, mannitol dehydrogenase, malolactic enzyme oxalacetate dehydrogenase and glycosyltransferase.

60. The lactic acid composition of claim 59, wherein the glycosyltransferase is selected from one or more of: dextransucrase, levansucrase and altemansucrase.

61. A product comprising low alcohol or reduced alcohol product obtained, or obtainable, by the method of any one of claims 1 to 55.

62. A double fermented low alcohol or reduced alcohol product.

63. The product of claim 61 or claim 62, wherein the product comprises one or more of: mannitol, fructan and glucans.

64. The product of claim 63, wherein the fructan is selected from one or more of: levan, inulin and a fructooligosaccharide.

65. The product of claim 63, wherein the glucan is selected from one or more of: dextran, mutan, reuteran, alteman and a glucooligosacchaide.

66. The product of any one of claims 61 to 65, wherein the product is a low alcohol beverage67. The product of any one of claims 61 to 66, wherein the product is a reduced alcohol beverage and wherein the product comprises about 0.5% to about 9% alcohol.

68. The product of any one of claims 61 to 67, wherein the product is a low sugar beverage comprising between about 0 g / L to about 30 g / L total sugar.

69. The product of any one of claims 61 to 68, wherein the product comprises no detectable level of malic acid or a low level of malic acid.

70. The product of any one of claims 61 to 69, wherein the beverage is selected from: wine and cider.

71. An isolated strain of Leuconostoc mesenteroides selected from:i) Leuconostoc mesenteroides C12 deposit number V24 / 007038 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;ii) Leuconostoc mesenteroides C13 deposit number V24 / 015136 dated 10 October 2024 at the National Measurement Institute Australia or a derivative thereof.iii) Leuconostoc mesenteroides C14 deposit number V24 / 007039 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof;iv) Leuconostoc mesenteroides Cl 8 deposit number V24 / 007040 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof; and v) Leuconostoc mesenteroides C20 deposit number V24 / 007041 dated 16 April 2024 at the National Measurement Institute Australia or a derivative thereof.

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