WO2024108254A1

WO2024108254A1 - Baking yeasts able to utilise glycerol

Info

Publication number: WO2024108254A1
Application number: PCT/AU2023/051184
Authority: WO
Inventors: Philip John Livingston Bell; Sophia MANDARAKAS
Original assignee: Microbiogen Pty Ltd
Priority date: 2022-11-22
Filing date: 2023-11-21
Publication date: 2024-05-30

Abstract

The present invention relates to baking strains of Saccharomyces cerevisiae which grow efficiently utilising glycerol as a sole carbon source, and uses thereof.

Description

YEAST FIELD OF THE INVENTION The present invention relates to baking strains of Saccharomyces cerevisiae, and uses thereof. BACKGROUND Yeast of the genus Saccharomyces are widely used in industry for the production of baked goods, various beverages and fuel ethanol. For most applications, particularly traditional applications such as baking and beverage production, strains of Saccharomyces have been bred and selected over many years for phenotypes which make them highly suited to a specific application. As a consequence, strains of Saccharomyces for specific applications are highly specialised for those applications, but are not well suited for other applications. Thus, for example, strains of Saccharomyces that are suitable for use in beverage or fuel ethanol production are not suitable for baking purposes. In the baking industry, strains of Saccharomyces that are used for baking purposes, also referred to as baking yeast or baker’s yeast, possess a complex combination of stable phenotypes that make these strains of yeast ideal and highly specialised for baking purposes. Minor changes in the combination of phenotypes of baker’s yeast can result in a yeast strain with inferior or unsuitable baking properties. Moreover, as baking protocols have been extensively developed around the performance of established baking strains, minor changes to the phenotypic profile of baking strains of Saccharomyces could be significantly disruptive to the baking industry. As a consequence, the complex combination of phenotypes characteristic of baker’s yeast has remained largely unchanged over several decades. Due to the widespread consumption of baked goods worldwide, there is a need to produce very large quantities of baker’s yeast cost effectively. To produce such large quantities of baker’s yeast cost effectively, the yeast is grown on substrates which provide a relatively inexpensive carbon source. A substrate that is commonly used in the industrial production of Saccharomyces for baking purposes is molasses derived from sugar production such as cane molasses and beet molasses. Molasses is commonly used because it is a relatively complete medium capable of supplying many of compounds required for yeast growth, including a carbon source in the form of sucrose, and other nutrients such as minerals and vitamins. In addition to molasses, glucose or corn syrups can be used to produce bakers’ yeast biomass, but apart from the carbon present in the form of sugars, these substrates do not provide many of the compounds required for yeast growth, and therefore require supplementation with compounds such as vitamins, amino nitrogen and minerals such as phosphate. The aerobic production of yeast converts approximately 40-50% of the carbon in a substrate into yeast biomass and thus the price of molasses, glucose and corn syrup is a major cost to the production of baker’s yeast. Also, as the price of molasses and other sugars varies according to the laws of supply and demand, the price of growth substrates can fluctuate which impacts on the cost of production of the bakers’ yeast biomass. It would therefore be advantageous to produce strains of Saccharomyces for baking purposes that are capable of growing on alternative, more cost effective, substrates. While some non-baking strains of Saccharomyces exhibit the ability to grow on some alternative (e.g., non-sugar) substrates, introducing such phenotypes into baking strains of Saccharomyces while retaining the complex combination of phenotypes which makes them uniquely suitable for baking purposes has not been possible. Owing to the complexity of the phenotypes associated with baking strains, the introduction of new phenotypes into baking strains inevitably results in loss of phenotypes that that are important for the strain’s baking capabilities. Thus, previous attempts to produce new baking strains of Saccharomyces that have additional growth capabilities have compromised the baking capabilities of the yeast. It would be advantageous to develop baking strains of Saccharomyces that can be produced more cost effectively without compromising the baking capabilities of the strain. SUMMARY The inventors have developed baking strains of Saccharomyces cerevisiae which grow efficiently utilising glycerol as a sole carbon source, while demonstrating the suite of characteristics required for baking purposes. Prior to the present invention, baking strains of Saccharomyces have not been capable of any significant growth utilising glycerol as a sole carbon source. Moreover, as noted above, prior to the present invention, it was considered not possible to introduce glycerol utilisation capabilities into baking strains of Saccharomyces without losing or diminishing the phenotypes that that are essential for baking performance. The ability to utilise glycerol as a sole carbon source is advantageous as glycerol is a component of waste streams from various industries, including the bioethanol and biodiesel industries, and therefore represents a cost-effective source of carbon. A first aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain produces at least a 25-fold increase in biomass when grown on glycerol as a sole carbon source in test T1, and comprises the following further characteristics: (i) produces between 80 and 130% of the gas produced by a conventional baking yeast in test T2; (ii) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3. In one embodiment, the strain retains activity at similar rates as the conventional baking yeast when stored at 4 deg C for 14 days. A second aspect provides a Saccharomyces yeast strain selected from: (a) a Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007968 (strain MBG5050); (b) Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007967 (strain MBG5049), (c) a derivative of Saccharomyces strain V22/007968; and (d) a derivative of Saccharomyces strain MBG5049. A third aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain comprises the following characteristics: (i) produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as strain V22/007968 or strain V22/007967 when grown under the same conditions; (ii) produces about the same amount of gas as strain V22/007968 when fermenting a bread dough with no added sugar; (ii) produces about the same amount of gas as strain V22/007968 or strain V22/007967 when fermenting a bread dough with 30% sucrose added; and (iii) retains activity at similar rates as strain V22/007968 or strain V22/007967 when stored at 4 C for 14 days. A fourth aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain produces at least a 3-fold greater increase in biomass than a conventional baking yeast when grown under the same conditions with glycerol as a sole carbon source, and comprises the following further characteristics: (i) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T2; (ii) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3. A fifth aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain comprises the following characteristics: (i) produces an increase in biomass in Test T1 that is in the range of from a 3-fold increase to an increase that is about the same as that of strain V22/007968 or strain V22/007967: (ii) Produces between 80 and 130% of the gas produced by a conventional baking yeast in test T2; and (iii) Produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3. In one embodiment, the strain retains activity at similar rates as the conventional baking yeast when stored at 4 deg C for 14 days. A sixth aspect provides a method of producing baker’s yeast, comprising incubating a Saccharomyces yeast strain of any one of the first to fifth aspects with a growth substrate under conditions which promote growth of the Saccharomyces yeast strain. A seventh aspect provides a method of producing baker’s yeast, comprising incubating a Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007967 (MBG5049) or V22/007968 (MBG5050), or a derivative of Saccharomyces strain V22/007967 or strain V22/007968, with a growth substrate, under conditions which promote growth of the Saccharomyces yeast strain. In one embodiment, the growth substrate comprises glycerol. In one embodiment, the growth substrate comprises molasses. In one embodiment, the growth substrate comprises glycerol and molasses. An eighth aspect provides baker’s yeast produced by the method of the sixth or seventh aspect. A ninth aspect provides a composition comprising the Saccharomyces strain of any one of the first to fifth aspects or the baker’s yeast of the eighth aspect. A tenth aspect provides a method of producing a baked good, comprising incubating the Saccharomyces strain of any one of the first to fifth aspects, or the baker’s yeast of the eighth aspect, or the composition of the ninth aspect, with a dough under conditions for baking. An eleventh aspect provides use of a strain of any one of the first to fifth or eighth aspects, or the composition of the ninth aspect, in the production of a baked good. A twelfth aspect provides a method of producing a baked good, comprising introducing into a baking substrate the strain of any one of the first to fifth aspects, the bakers yeast of the eighth aspect, or the composition of the ninth aspect. A thirteenth aspect provides a baked good produced by the method of the tenth or twelfth aspect. DETAILED DESCRIPTION The inventors recognized that it would be advantageous to grow baker’s yeast on a readily available, cheap substrate, such as a by-product of large-scale industrial processes. Glycerol is an alcohol that is increasingly produced at scale in both the biodiesel and bioethanol industries. In the case of the bio-diesel industry, crude glycerol is produced as a result of the transesterification of triacylglycerides with alcohols such as ethanol or methanol. In the case of bioethanol production, approximately 1 litre of glycerol is produced for every 10 litres of ethanol produced. Thus, biodiesel production and large scale corn ethanol production results in an abundant supply of crude glycerol rich syrups as a by-product. The inventors recognized that it would be advantageous to be able to grow baker’s yeast on glycerol as a cost-effective abundant substrate. However, the dominant strains of bakers’ yeast used in the baking industry grow on glycerol too slowly to allow glycerol to be used as a cost effective alternative substrate or adjunct for the industrial production of bakers’ yeast. The inventors therefore sought to develop a bakers’ yeast that can grow on glycerol whilst simultaneously possessing the characteristics required for a yeast to perform under modern industrial baking processes. The inventors have produced Saccharomyces strains NMI V22/007967 (also referred to herein as strain MBG5049) and V22/007968 (also referred to herein as strain MBG5050), which are each non- genetically engineered strains of Saccharomyces cerevisiae capable of rapidly growing on glycerol as a sole carbon source, and that also display the characteristics required to perform effectively at industrial scale in baking of high and low sugar doughs. The invention therefore relates in one aspect to a strain of Saccharomyces cerevisiae selected from: (a) a Saccharomyces yeast strain deposited under the Budapest Treaty at the National Measurement Institute (NMI) on 26 April 2022 and having NMI accession no. V22/007968 (strain MBG5050); (b) Saccharomyces yeast strain deposited under the Budapest Treaty at the National Measurement Institute (NMI) on 26 April 2022 and having NMI accession no. V22/007967 (strain MBG5049). The ability to grow on or in glycerol as a sole carbon and energy source makes it possible to totally or partially replace sugars such as sucrose, glucose and fructose in the production of yeast biomass for use by the baking industry. In this regard, strains V22/007967 and V22/007968 can be grown on a growth medium in which glycerol is the sole carbon source, and other macronutrients such as phosphate, nitrogen and salts such as magnesium are provided in an inorganic form. Importantly, in addition to its ability to grow efficiently on glycerol, strains V22/007967 and V22/007968 also possess the characteristics required to make them suitable baking strains. In this regard, strains MBG5049 and MBG5050 comprise the baking characteristics of a conventional baker’s yeast. Strain nos. V22/007967 and V22/007968 are non-recombinant Saccharomyces cerevisiae strains which have the following characteristics: (a) produces more than a 25-fold increase in biomass when grown on glycerol as a sole carbon source under the conditions specified in Test T1; (b) have equivalent gassing activity to a conventional baking yeast in test T2 which predicts performance in a lean dough where maltose metabolism is important; and (c) have equivalent gassing activity to a conventional baking yeast in test T3 which predicts performance a high sugar dough where osmotic resistance is important. As used herein, a “conventional baking yeast” is a strain of Saccharomyces cerevisiae that is a strain of baker’s yeast conventionally used in the baking industry for baking purposes. Conventional baking yeast grows poorly on glycerol, and in this regard produce less than a 3-fold increase in biomass under the conditions specified in Test T1. Examples of conventional baking yeasts include the following commercially available baking yeasts: Mauripan Red (available from AB Mauri, Australia); Fermipan Brown (available from AB Mauri, Australia); Fermipan Red (available from AB Mauri, Australia); Saf-Instant Red (available from LeSaffre Yeast Corporation, USA),; and Saf- Instant Gold (available from LeSaffre, Yeast Corporation, USA). In one embodiment, the conventional baking yeast is Mauripan Red. One aspect provides a Saccharomyces yeast strain which produces at least a 25-fold increase in biomass when grown on glycerol as a sole carbon source, typically under the conditions specified in test T1, and comprises at least one of the following further characteristics: (i) produces between 80 and 130% of the gas produced by a conventional baking yeast in test T2 which is corresponds to the activity of yeast fermenting a bread dough with no added sugar (ii) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3 which corresponds to the activity of yeast fermenting a bread dough with 30% sucrose added (iii) retains activity at similar rates as the conventional baking yeast when stored at 4 deg C for 14 days. In one embodiment, the conventional baking yeast is Mauripan Red. In various embodiment, the Saccharomyces yeast strain produces at least a 30-fold, a 35-fold, a 40-fold, a 45-fold, a 50-fold, a 55-fold, a 60-fold, a 65-fold, a 70-fold, a 75-fold, a 80- fold, a 85-fold, a 90-fold, a 95-fold, or at least a 100-fold, increase in biomass when grown on glycerol as a sole carbon source, typically when grown under the conditions specified in test T1. In one embodiment, the Saccharomyces yeast strain produces at least a 100-fold increase in biomass when grown on glycerol as a sole carbon source, typically when grown under the conditions specified in test T1. Another aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain produces an increase in biomass utilising glycerol as a sole carbon source that is at least 3- fold greater than a conventional baking yeast when grown under the same conditions, and comprises the following further characteristics: (i) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T2; (ii) produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3. In one embodiment, the strain retains activity at similar rates as the conventional baking yeast when stored at 4 deg C for 14 days. In various embodiments, the strain produces an increase in biomass utilising glycerol as a sole carbon source that is at least 5-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, or at least 100-fold, greater than the conventional baking yeast when grown under the same conditions. Another aspect provides a Saccharomyces yeast strain for baking purposes, wherein the strain comprises the following characteristics: (i) produces an increase in biomass in Test T1 that is in the range of from a 3-fold increase to an increase that is about the same as that of strain V22/007968 or strain V22/007967: (ii) Produces between 80 and 130% of the gas produced by a conventional baking yeast in test T2; and (iii) Produces between 80 and 130% of the gas produced by the conventional baking yeast in test T3. In one embodiment, the strain retains activity at similar rates as the conventional baking yeast when stored at 4 deg C for 14 days. In various embodiments, the strain produces an increase in biomass in Test T1 that is in the range from a 5-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or a 100-fold, increase to an increase that is about the same as that of strain V22/007968 or strain V22/007967. In some embodiments, the Saccharomyces strain produces between 90 and 110% of the gas produced by the conventional baking yeast in test T2. In some embodiment, the Saccharomyces strain produces between 90 and 110% of the gas produced by the conventional baking yeast in test T3. In some embodiments, the Saccharomyces strain produces between 90 and 110% of the gas produced by the conventional baking yeast in test T2, and between 90 and 110% of the gas produced by the conventional baking yeast in test T3. In one embodiment, the conventional baking yeast is Mauripan red. Another aspect provides a Saccharomyces yeast strain which produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as strain V22/007968 or strain V22/007967 when grown under the same conditions, and which has about the same baking characteristics of strain V22/007968 or strain V22/007967. Another aspect provides a Saccharomyces yeast strain which comprises the following characteristics: (i) produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as strain V22/007968 or strain V22/007967 when grown under the same conditions; (ii) produces about the same amount of gas as strain V22/007968 or strain V22/007967 when fermenting a bread dough with no added sugar; (ii) produces about the same amount of gas as strain V22/007968 or strain V22/007967 when fermenting a bread dough with 30% sucrose added; and (iii) Retains activity at similar rates as strain V22/007968 or strain V22/007967 when stored at 4C for 14 days. The increase in biomass utilising glycerol as a sole carbon source may be determine using any methods known in the art. In one embodiment, the increase in biomass utilising glycerol as a sole carbon source is determined using Test T1. The gas produced by Saccharomyces during fermentation of bread dough is carbon dioxide. Accordingly, a reference to “gas” herein is a reference to carbon dioxide. The amount of gas produced when fermenting a bread dough with no added sugar may be determined using any methods known in the art. In one embodiment, the amount of gas produced when fermenting a bread dough with no added sugar is determined using Test T2. The amount of gas produced when fermenting a bread dough with 30% added sugar may be determined using any methods known in the art. In one embodiment, amount of gas produced when fermenting a bread dough with 30% added sugar is determined using Test T3. The ability to grow strains V22/007967 and V22/007968 utilizing glycerol as a sole carbon source paired with the strains characteristics necessary for baking applications allows for the cost-effective production of large amounts of baker’s yeast biomass. Accordingly, one aspect provides a method of producing baker’s yeast biomass, the method comprising incubating a Saccharomyces yeast strain selected from: (a) a Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007968 (strain MBG5050); (b) Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007967 (strain MBG5049), (c) a derivative of Saccharomyces strain V22/007968; and (d) a derivative of Saccharomyces strain V22/007967, with a growth substrate under conditions which promote growth of the Saccharomyces yeast strain. In one embodiment, the growth substrate comprises glycerol. In one embodiment, the growth substrate comprises molasses. In one embodiment, the growth substrate comprises glycerol and molasses. Strains V22/007967 and V22/007968 produce more than a 100-fold increase in biomass when grown under the conditions specified in Test T1. The ability of strains V22/007967 and V22/007968 to produce more than a 100-fold increase in biomass under the conditions specified in Test T1 is a characteristic which distinguishes these strains from other baking strains of Saccharomyces. As current baking strains of Saccharomyces are not capable of growth on glycerol at the rate at which strains V22/007967 and V22/007968 grow on glycerol, strains V22/007967 and V22/007968 are readily differentiated from current baking strains of Saccharomyces. The invention also relates to a derivative of Saccharomyces strain V22/007967, and a derivative of Saccharomyces strain V22/007968. As used herein, a “derivative of strain V22/007967” is a strain derived from strain V22/007967, and a “derivative of strain V22/007968” is a strain derived from strain V22/007968. A yeast strain may be derived from another yeast strain through, for example, mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. In one embodiment, the strain derived from strain V22/007967 or strain V22/007968 is a direct progeny (i.e., the product of a mating between strain V22/007968 and another strain, or with itself; or the product of a mating between strain V22/007967 and another strain, or with itself). In one embodiment, a derivative of strain V22/007967 is a hybrid strain produced by culturing a first yeast strain with strain V22/007967 under conditions which permit combining of DNA between the first yeast strain and strain V22/007967. In one embodiment, a derivative of strain V22/007968 is a hybrid strain produced by culturing a first yeast strain with strain V22/007968 under conditions which permit combining of DNA between the first yeast strain and strain V22/007968. In one embodiment, the derivative of strain V22/007967 produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as strain V22/007967 when grown under the same conditions, and exhibits one or more of the following further characteristics: (i) produces about the same amount of gas as strain V22/007967 when fermenting a bread dough with no added sugar; (ii) produces about the same amount of gas as strain V22/007967 when fermenting a bread dough with 30% sucrose added; and (iii) retains activity at similar rates as strain V22/007967 when stored at 4C for 14 days. In one embodiment, the derivative of strain V22/007968 produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as strain V22/007968 when grown under the same conditions, and exhibits one or more of the following further characteristics: (i) produces about the same amount of gas as strain V22/007968 when fermenting a bread dough with no added sugar; (ii) produces about the same amount of gas as strain V22/007968 when fermenting a bread dough with 30% sucrose added; and (iii) retains activity at similar rates as strain V22/007968 when stored at 4C for 14 days. In one embodiment, the derivative of strain V22/007967 exhibits all of the characteristics of strain V22/007967. In one embodiment, the derivative of strain V22/007968 exhibits all of the characteristics of strain V22/007968. In one embodiment, the derivative of strain V22/007967 or V22/007968 may be prepared by culturing a first yeast strain with strain V22/007967 or V22/007968, under conditions which permit combining of DNA between the first yeast strain and strain V22/007967 or V22/007968. In one embodiment, culturing a first yeast strain with a second strain, under conditions which permit combining of DNA between the first yeast strain and the second yeast strain, comprises: (i) sporulating the first yeast strain and the second yeast strain; (ii) germinating and hybridizing spores produced by the first yeast strain with spores produced by the second yeast strain. Methods for sporulating, germinating and hybridising yeast strains, and in particular, Saccharomyces strains, are known in the art and are described in, for example, Ausubel, F. M. et al., (1997) Current Protocols in Molecular Biology, Volume 2, pages 13.2.1 to 13.2.5 (John Willey & Sons Inc); Chapter 7, “Sporulation and Hybridisation of yeast” by R.R. Fowell, in “The Yeasts” vol 1, A.H. Rose and J.S. Harrison (Eds), 1969, Academic Press. In one embodiment, the yeast strains may be cultured under conditions which permit cell fusion. Methods for the generation of intraspecific or interspecific hybrids using cell fusion techniques are described in, for example, Spencer et al. (1990) in, Yeast Technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York. In another embodiment, the yeast strains may be cultured under conditions which permit cytoduction. Methods for cytoduction are described in, for example, Inge-Vechymov et al. (1986) Genetika 22: 2625-2636; Johnston (1990) in, Yeast technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York. In one embodiment, a derivative of strain V22/007967 or V22/007968 may be a mutant of these strains. Methods for producing mutants of Saccharomyces yeast, and specifically mutants of Saccharomyces cerevisiae, are known in the art and described in, for example, Lawrence C.W. (1991) Methods in Enzymology, 194: 273-281. A further aspect provides a composition comprising a Saccharomyces strain selected from strain V22/007967 and V22/007968 or a derivative of strain V22/007967 or strain V22/007968. The composition may be, for example, cream yeast, compressed yeast, wet yeast, crumble yeast, stabilized liquid yeast or frozen yeast. Methods for preparing such yeast compositions are known in the art. In one embodiment, the composition is cream yeast or compressed yeast. The composition comprising yeast may contain additional components, such as for example, fresh medium, buffering agents, water, or other agents known in the art for preparing yeast compositions. Also described herein is a baked good. A baked good is a food product made from dough (e.g., comprising flour of wheat, maize, rice, oats, rye, or other cereal crops and water or another liquid) that is leavened and then baked, typically in an oven or other source of heat. Examples of baked goods include bread (e.g., a loaf, roll, bun, etc.), bagels, pretzels, brioche, and the like. The practice of the present invention employs, unless otherwise indicated, conventional microbiology and classical genetics. Such techniques are known to the skilled worker, and are explained fully in the literature. See, for example, Sherman et al. "Methods in Yeast Genetics" (1981) Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, New York; European Patent number EP 0511108 B. As used herein, the singular forms “a”, “an” and “the” include plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes a plurality of such cells. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the term “about” means a slight variation of the value specified, preferably within 10 percent of the value specified. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. All publications mentioned in this specification are herein incorporated by reference. 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. As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. Test T1 Yeast strains are streaked onto Glucose Yeast extract Bacteriological Peptone medium solidified with 2% Agar using standard microbiological techniques. After incubation for 72 hr at 30 deg Celsius, yeast cells are taken from plates using a sterile microbiological loop and inoculated to an OD (Optical Density at 600 nm) of between 0.1 and 0.2 units (OD at T) in 50 ml of broth. The broth contains glycerol (3% w/v), Difco Yeast Nitrogen Base w/o amino acids (0.67% w/v) in distilled water in a 250 ml Erlenmeyer flask. Cultures are incubated at 30 deg Celsius with shaking at 220 rpm (10 cm orbital diameter) for 48 hr prior to measuring OD (OD at T ). The fold increase in biomass is determined by the equation: OD at T / OD at T. Test T2 Yeast is prepared at 30% solids. 0.029 g is resuspended in 2 ml of syndough with the following formulation (all in g/L) Sorbitol 20g KH2PO4 2.07 (NH4)2SO4 0.67 MgSO4.7H2O 2.76 Casamino acids 2.31 Citric acid 4.42 Tri sodium citrate 48.68 Maltose 30 Sucrose 8 The syndough is supplemented with a vitamin stock solution to a final level in the syndough of mg l : thiamine HCL 10.1, pyridoxine HCL 10.1, nicotinic acid 50.6, Ca pantothenate 20.2 and biotin 0.00025. To assay the fermentative power of the yeast, the yeast/syndough suspensions are transferred into a commercially available 24 well multidish microtitre tray. Fermentation activity is measured over a 2-hour period at 30 deg C using a Multi Fermentation Screening System (Dunas 1988). The Multi Fermentation Screening System (MFSS) is a 24-channel computerized instrument for the study of yeast anaerobic fermentation. The evolved carbon dioxide is registered as the increasing pressure at a constant volume. This low sugar syndough allows the activity of yeast to be evaluated for their ability to rapidly produce carbon dioxide gas for dough leavening, and requires the yeast to switch from fermentation of glucose and fructose derived from sucrose hydrolysis to the fermentation of maltose derived from the action of amylases on the starch component of flour. Activities approximating the gassing activity of Mauripan Red translate into proofing times in the range of 45 to 60 minutes in a dough with no added sugars. Test T3 Yeast is prepared at 30% solids. 0.029 g is resuspended in 2 ml of syndough with the following formulation (all in g/L) Sorbitol 20g KH2PO4 2.07 (NH4)2SO4 0.67 MgSO4.7H2O 2.76 Casamino acids 2.31 Citric acid 4.42 Tri sodium citrate 48.68 Sucrose 300 g The syndough is supplemented with a vitamin stock solution to a final level in the syndough of mg l : thiamine HCL 10.1, pyridoxine HCL 10.1, nicotinic acid 50.6, Ca pantothenate 20.2 and biotin 0.00025. To assay the fermentative power of the yeast, the yeast/syndough suspensions are transferred into a commercially available 24 well multidish microtitre tray. Fermentation activity is measured over a 2-hour period at 30 deg C using a Multi Fermentation Screening System (Dunas 1988). The Multi Fermentation Screening System (MFSS) is a 24-channel computerized instrument for the study of yeast anaerobic fermentation. The evolved carbon dioxide is registered as the increasing pressure at a constant volume. This high sugar syndough allows the activity of yeast to be evaluated for their ability to rapidly produce carbon dioxide gas for dough leavening under osmotically stressful conditions resulting from high sugar additions. Activities approximating the gassing activity of Mauripan Red translate into proofing times in the range of 90 to 120 minutes in a dough with with high sugar additions such as a brioche dough. In order to exemplify the nature of the present invention such that it may be more clearly understood, the following non- limiting examples are provided. EXAMPLES Strains MBG5049 and MBG5050 and

with industrial strains Strain MBG5050 and MBG5049 are products of a breeding program to introduce Saccharomyces cerevisiae strains that are useful for baking and which can grow on glycerol as a sole carbon source. Such a combination of phenotypes is not found in naturally occurring Saccharomyces strains. Strain MBG5050 was deposited under the Budapest Treaty at the National Measurement Institute (NMI), 1/153 Bertie Street, Port Melbourne, Victoria, Australia 3207 on 26 April 2022 having NMI deposit accession no. V22/007968. Strain MBG5049 was deposited under the Budapest Treaty at the National Measurement Institute (NMI), 1/153 Bertie Street, Port Melbourne, Victoria, Australia 3207 on 26 April 2022 having NMI deposit accession no. V22/007967. The growth of MBG 5050 and MBG 5049 on glycerol as a sole carbon source was compared to that of several industrial baker’s yeast strains. The industrial baker’s yeast strains included Mauripan Red (available from AB Mauri, Australia), Fermipan Brown (available from AB Mauri, Australia), Fermipan Red (available from AB Mauri, Australia), Saf-Instant Red (Saf Red in Table 1) (available from LeSaffre Yeast Corporation, USA), and Saf- Instant Gold (Saf Gold in Table 1) (available from LeSaffre, Yeast Corporation, USA). The results are shown in Table 1. As shown in Table 1, when grown under the conditions of Test T1 (with glycerol as a sole carbon source), MBG 5050 and MBG 5049 both increased in biomass by over 100-fold, whilst none of the industrial baker’s yeasts increased by more than two-fold. Table 1

Example 2: Carbon dioxide production in test T2 when grown on sucrose The ability to rapidly produce carbon dioxide via sugar fermentation is an important industrial feature of commercial baker’s yeasts. Yeast used in the industrial baker’s yeast market, such as Mauripan Red, display an exceptional ability to rapidly ferment sugars such as sucrose, and maltose into carbon dioxide for bread leavening. The exceptional ability of yeast such as Mauripan Red to ferment maltose is of particular importance when bread is made using no exogenous sugar additions since maltose is the predominant sugar present in these ‘plain doughs’ during later stages of fermentation. To prepare yeast biomass for assessing gassing activity, Mauripan Red, MBG 5049 and MBG 5050 were grown aerobically in 50 ml of a sucrose based medium for 24 hr at 32 deg C with orbital shaking at 120 rpm using sucrose as a sole carbon source. Growth medium contained as g l : sucrose 5, KHPO 0.25, (NH)SO 1.2, CaCl·2HO 0.1, MgSO·7HO 0.5, citric acid 3.42 and tri-sodium citrate 8.9. Composition of the mineral supplement was mg l : ferric citrate 6.05, CuSO·5HO 0.2, ZnSO·7HO 0.5, MnSO·4HO 1.0, NaMoO·2HO 0.5 and NaBO·10HO 0.5. The medium was supplemented with a vitamin stock to a final concentration of mg l : Ca-pantothenate 1.0, thiamine HCL 1.0, pyridoxine HCL 1.0, inositol 2.0, nicotinic acid 0.5 and biotin 0.2. The initial inoculation density at OD600 is ~2. After 24 hr incubation the yeast is harvested by centrifugation, and water is removed from the harvested yeast until it reaches a concentration of 30% w/v solids. Fermentation activity of the yeast is evaluated as described in test T2 and expressed relative to the activity of the Mauripan Red yeast grown and tested under the same conditions. A value close to 1 corresponds to the proof time of between 45 to 60 minutes observed when Mauripan Red is used to leaven a dough with no added sugar. Production of carbon dioxide in test T2 predicts the performance of yeast leavening in a bread made from only flour, water, salt and yeast. In the plain syndough, maltose is the predominant sugar available after 1 hour of fermentation, and thus a high activity in this test indicates good maltose fermentation characteristics. Mauripan Red yeast is used as a control in test T2 and all activities are expressed relative to the Mauripan Red yeast grown and tested under the same conditions As shown in Table 2, the MBG 5049 and MBG 5050 are equivalent in gassing activity to the Mauripan Red yeast in a plain syndough. Table 2:

Example 3: Carbon dioxide production in Test T3 when grown on sucrose The ability to rapidly produce carbon dioxide via sugar fermentation is an important industrial feature of commercial baker’s yeasts. Yeast used in the industrial baker’s yeast market, such as Mauripan Red ferment rapidly over a relatively broad range of sugar concentrations and are thus suited to manufacturing various bread styles ranging from bread with no added sugars to bread with up to 30% added sugars. To prepare yeast biomass for assessing gassing activity, Mauripan Red, MBG 5049 and MBG 5050 were grown aerobically in 50 ml of a sucrose based medium for 24 hr at 32 deg C with orbital shaking at 120 rpm using Sucrose as a sole carbon source. Growth medium contained as g l : sucrose 5, KHPO 0.25, (NH)SO 1.2, CaCl·2HO 0.1, MgSO·7HO 0.5, citric acid 3.42 and tri-sodium citrate 8.9. Composition of the mineral supplement was mg l : ferric citrate 6.05, CuSO·5HO 0.2, ZnSO·7HO 0.5, MnSO·4HO 1.0, NaMoO·2HO 0.5 and NaBO·10HO 0.5. The medium was supplemented with a vitamin stock to a final concentration of mg l : Ca-pantothenate 1.0, thiamine HCL 1.0, pyridoxine HCL 1.0, inositol 2.0, nicotinic acid 0.5 and biotin 0.2. The initial inoculation density at OD is ~2. After 24 hr incubation the yeast is harvested by centrifugation, and water is removed from the harvested yeast until it reaches a concentration of 30% w/v solids. 0.029 g of yeast is resuspended in 2 ml of high sugar syndough. High sugar syndough contains in g l : sorbitol 20, KHPO 2.07, (NH)SO 0.67, MgSO 2.76, casamino acids 2.31, citric acid 4.42 and tri-sodium citrate 48.68, with sucrose 300. The high sugar syndough is supplemented with a vitamin stock solution to a final level of mg l : thiamine HCL 10.1, pyridoxine HCL 10.1, nicotinic acid 50.6, Ca pantothenate 20.2 and biotin 0.00025. Production of carbon dioxide in the high sugar syndough is predictive of the performance of yeast leavening in a highly sugared bread dough such as Brioche. Fermentation activity is measured over a 2-hour period at 30 deg C using a Multi Fermentation Screening System (Dunas 1988, Multi Fermentation Screening System (MFSS): computerised simultaneous evaluation of carbon dioxide production in twenty yeasted broths or doughs. J. Microbiol. Methods 8:303-314). The Multi Fermentation Screening System (MFSS) is a 24-channel computerized instrument for the study of yeast anaerobic fermentation. The evolved carbon dioxide is registered as the increasing pressure at a constant volume. Fermentation activity of the yeast is evaluated as described in test T2 and expressed relative to the activity of the Mauripan Red yeast grown and tested under the same conditions. A value close to 1 corresponds to the proof time of between 90 to 120 minutes observed when Mauripan Red is used to leaven a highly sugared dough such as a brioche. As shown in Table 3, the MBG 5049 and MBG 5050 are similar to or better than the Mauripan Red yeast in fermentation performance in a high sugar syndough. Table 3:

Example 4: Relative carbon dioxide production performance in test T2 and T3 of Mauripan Red, MBG 5049 and MBG 5050 when grown on sucrose compared to when grown on glycerol Preparation of biomass using sucrose as a carbon source To prepare yeast biomass for assessing gassing activity, Mauripan Red, MBG 5049 and MBG 5050 were grown aerobically in 50 ml of a sucrose based medium for 24 hr at 32 deg C with orbital shaking at 120 rpm using sucrose as a sole carbon source. Growth medium contained as g l : sucrose 5, KHPO 0.25, (NH)SO 1.2, CaCl·2HO 0.1, MgSO·7HO 0.5, citric acid 3.42 and tri-sodium citrate 8.9. Composition of the mineral supplement was mg l : ferric citrate 6.05, CuSO·5HO 0.2, ZnSO·7HO 0.5, MnSO·4HO 1.0, NaMoO·2HO 0.5 and NaBO·10HO 0.5. The medium was supplemented with a vitamin stock to a final concentration of mg l : Ca-pantothenate 1.0, thiamine HCL 1.0, pyridoxine HCL 1.0, inositol 2.0, nicotinic acid 0.5 and biotin 0.2. The initial inoculation density at OD is ~2. Preparation of biomass using glycerol as a carbon source To prepare yeast biomass for assessing gassing activity, Mauripan Red, MBG 5049 and MBG 5050 were grown aerobically in 50 ml of a glycerol based medium for 24 hr at 32 deg C with orbital shaking at 120 rpm using glycerol as a sole carbon source. Growth medium contained as g l : glycerol 5, KHPO 0.25, (NH)SO 1.2, CaCl·2HO 0.1, MgSO·7HO 0.5, citric acid 3.42 and tri-sodium citrate 8.9. Composition of the mineral supplement was mg l : ferric citrate 6.05, CuSO·5HO 0.2, ZnSO·7HO 0.5, MnSO·4HO 1.0, NaMoO·2HO 0.5 and NaBO·10HO 0.5. The medium was supplemented with a vitamin stock to a final concentration of mg l : Ca-pantothenate 1.0, thiamine HCL 1.0, pyridoxine HCL 1.0, inositol 2.0, nicotinic acid 0.5 and biotin 0.2. The initial inoculation density at OD is ~2. After biomass production the yeast was harvested and washed by centrifugation. Water was removed from the harvested yeast until it reached a concentration of 30% w/v solids. The activity of yeast grown on glycerol and sucrose was compared to the activity of the Mauripan yeast grown on sucrose as a carbon source in both test T2 and test T3. The production of carbon dioxide in the test T2 predicts the performance of yeast leavening in a bread made from only flour, water, salt and yeast. Production of carbon dioxide in test T3 is predictive of the performance of yeast leavening in a highly sugared bread dough such as Brioche. Activity in test T2 Mauripan Red only grew on sucrose and did not grow on glycerol. The Mauripan Red yeast could therefore only be assayed in test T2 when grown on sucrose. By contrast, MBG 5049 and MBG 5050 grew on both sucrose and glycerol as sole carbon sources. When grown on sucrose both MBG 5049 and MBG 5050 were as active as Mauripan Red grown on sucrose in test T2. Similarly, when grown on glycerol, both MBG 5049 and MBG 5050 were as active as Mauripan Red grown on sucrose in test T2. The results are shown in Table 4. Table 4: Activity relative to Mauripan Red in test T2

Activity in test T3 Mauripan Red only grew on sucrose and did not grow on glycerol. The Mauripan Red yeast could therefore only be assayed in test T3 when grown on sucrose. By contrast, MBG 5049 and MBG 5050 grew on both sucrose and glycerol as sole carbon sources. When grown on sucrose both MBG 5049 and MBG 5050 were as active as Mauripan Red grown on sucrose in test T3. Similarly, when grown on glycerol, both MBG 5049 and MBG 5050 were as active as Mauripan Red grown on sucrose in test T3. The results are shown in Table 5. Table 5: Activity relative to Mauripan Red in test T3

These results demonstrate the unique ability of strains MBG 5049 and MBG 5050 to grow on alternative substrates such as glycerol whilst maintaining the fermentation performance required in the bread making industry. Example 5: Performance of MBG 5049 and MBG 5050 in tests T2 and T3 when grown in aerated bioreactors using cane molasses as a carbon and nutrient source The industrial production of Baker's yeast mostly uses molasses using large scale aerated bioreactors. To prepare yeast biomass for assessing gassing activity when biomass is produced using a protocol typical of industrial production, Mauripan Red, MBG 5049 and MBG 5050 were each separately grown in an aerobic bioreactor with 5 L working volume using cane molasses as a source of sugar using a fed- batch fermentation protocol. Fed-batch fermentations were carried out in a continuously stirred, aerated bioreactor controlled at 30C, and pH 5.0 ±0.5 using NaOH and HSO. Yeast cells were inoculated to a density of 20 g l in a set water containing vitamins (B1 at 24mg/L, B5 at 6mg/L, B6 at 6mg/L) and cane molasses diluted to a concentration of 1% sucrose. Aeration was maintained by supplying air at flow rate of 2.0 v v m , with stirring increasing to a maximum of 1500 rpm, based on culture demand. An initial batch phase of fermentation was allowed to proceed until ethanol concentration peaked at which point the fed-batch stage was started by pumping cane molasses medium into the vessel to maintain supply of nutrients. Cane molasses was prepared at a concentration of 30% sugars. Urea and Monoammonium phosphate stocks were fed into the fermenters over the first 6 hours to allow yeast to reach targeted protein levels of 48%, and phosphate of 2.5% respectively. Ethanol concentration in off-gas was monitored and the fed-batch phase was controlled using an ethanol feed-back loop maintaining concentration at 0.8 g l until 1.9kg of molasses wort had been added. After biomass production the yeast is harvested and washed by centrifugation. Water is removed from the harvested yeast until it reaches a concentration of 30% w/v solids. The activity of yeast grown in aerated bioreactors using cane molasses as a carbon source was evaluated both test T2 and test T3. The production of carbon dioxide in the test T2 predicts the performance of yeast leavening in a bread made from only flour, water, salt and yeast. Production of carbon dioxide in test T3 is predictive of the performance of yeast leavening in a highly sugared bread dough such as Brioche. Activities are expressed relative to the Mauripan yeast. As shown in Table 6, the performance of MBG 5049 and MBG 5050 are similar to the Mauripan Red yeast in a plain syndough and high sugar syndough, indicating that these strains display a similar performance to Mauripan Red over a broad range of sugar concentrations. Table 6:

Example 6: Comparison of performance of MBG 5049 and MBG 5050 in test T2 and test T3 when grown on molasses and on a 50:50 mix of molasses derived sugars and glycerol To prepare yeast biomass for assessing gassing activity after growth on a blend of cane molasses and glycerol, MBG 5049 and MBG 5050 were each separately grown in aerobic bioreactor with 5 L working volume using a blend of cane molasses and glycerol as a substrate using a fed-batch growth protocol. The blended feed contained 150g/L sugars from cane molasses and 150g/L glycerol. Fed-batch growth was carried out in a continuously stirred, aerated bioreactor controlled at 30oC, and pH 5.0 ±0.5 using NaOH and H2SO4. Yeast cells were inoculated to a density of 20 g l in a set water containing vitamins (B1 at 24mg/L, B5 at 6mg/L, B6 at 6mg/L) and cane molasses diluted to a concentration of 1% sucrose. Aeration was maintained by supplying air at flow rate of 2.0 v v m , with stirring increasing to a maximum of 1500 rpm, based on culture demand. An initial batch phase of fermentation was allowed to proceed until ethanol concentration peaked at which point the fed-batch stage was started by pumping cane molasses/glycerol medium into the vessel to maintain supply of nutrients. A cane molasses/glycerol feed was prepared at a concentration of 15% sugars derived from cane molasses, supplemented with 15% glycerol. Urea and Monoammonium phosphate stocks were fed into the fermenters over the first 6 hours to allow yeast to reach targeted protein levels of 48%, and phosphate of 2.5% respectively. Ethanol concentration in off- gas was monitored and the fed-batch phase was controlled using an ethanol feed-back loop maintaining concentration at 0.8 g l until the propagation was completed. After biomass production the yeast is harvested and washed by centrifugation. Water is removed from the harvested yeast until it reaches a concentration of 30% w/v solids. The activity of yeast grown in aerated bioreactors was evaluated both test T2 and test T3. The production of carbon dioxide in the test T2 predicts the performance of yeast leavening in a bread made from only flour, water, salt and yeast. Production of carbon dioxide in test T3 is predictive of the performance of yeast leavening in a highly sugared bread dough such as Brioche. Fermentation activity is measured over a 2-hour period at 30 deg C using a Multi Fermentation Screening System (Dunas 1988). The Multi Fermentation Screening System (MFSS) is a 24-channel computerized instrument for the study of yeast anaerobic fermentation. The evolved carbon dioxide is registered as the increasing pressure at a constant volume. Activities were compared between yeast that had been produced on molasses and yeast that had been produced on a blend of Molasses and glycerol. As shown in Table 7, the fermentation activity of the yeast produced using the molasses/glycerol blend ranged between 90 and 99% of the activities observed in test T2 and T3 when produced purely on cane molasses. Table 7

Dunas, F. (1988) Multi Fermentation Screening System (MFSS): computerised simultaneous evaluation of carbon dioxide production in twenty yeasted broths or doughs. Journal of Microbiological Methods 8, 303-314. The present application claims priority from Australian provisional application no. 2022903539, the entirety of which is incorporated herein by reference.

Claims

CLAIMS: 1. A Saccharomyces yeast strain which comprises the following characteristics: (i) produces an increase in biomass utilising glycerol as a sole carbon source that is about the same as Saccharomyces strain V22/007968 or strain V22/007967 when grown under the same conditions; (ii) produces about the same amount of gas as Saccharomyces strain V22/007968 or strain V22/007967 when fermenting a bread dough with no added sugar under the same conditions; and (ii) produces about the same amount of gas as Saccharomyces strain V22/007968 or strain V22/007967 when fermenting a bread dough with 30% sucrose added under the same conditions.

2. A Saccharomyces yeast strain which comprises the following characteristics: (i) produces at least a 25-fold increase in biomass when grown on glycerol as a sole carbon source; (ii)produces about 80 to about 130% of the gas produced by a conventional baking yeast in test T2; and (ii) produces about 80 to about 130% of the gas produced by the conventional baking yeast in test T3.

3. The Saccharomyces strain of claim 1 or 2, wherein the increase in biomass on glycerol as a sole carbon source is as determined using Test T1.

4. The Saccharomyces strain of any one of claims 1 to 3, wherein the strain retains gassing activity at similar rates as the conventional baking yeast when stored at 4C for 14 days.

5. The Saccharomyces strain of any one of claims 2 to 4, wherein the conventional baking yeast is Mauripan Red.

6. The Saccharomyces yeast strain of any one of claims 1 to 5, wherein the strain is selected from: (a) a Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007968 (strain MBG5050); (b) Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007967 (strain MBG5049), (c) a derivative of Saccharomyces strain V22/007968; and (d) a derivative of Saccharomyces strain V22/007967.

7. A Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007968.

8. A Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V22/007967.

9. A method of producing baker’s yeast, comprising incubating the Saccharomyces yeast strain of any one of claims 1 to 8 with a substrate comprising glycerol, molasses, or molasses and glycerol, under conditions which promote growth of the Saccharomyces yeast strain.

10. A baker’s yeast produced by the method of claim 9.

11. A composition comprising the Saccharomyces strain of any one of claims 1 to 8, or the baker’s yeast of claim 10.

12. A method of producing a baked good, the method comprising incubating the Saccharomyces strain of any one of claims 1 to 8, or the baker’s yeast of claim 10, or the composition of claim 10, with a dough under conditions for baking.

13. Use of a strain of any one of claims 1 to 8, or the baker’s yeast of claim 10, or the composition of claim 11, in the production of a baked good.

14. A method of producing a baked good, comprising introducing into a baking substrate the strain of any one of claims 1 to 8, or the baker’s yeast of claim 10, or the composition of claim 11.

15. A baked good produced by the method of claim 12 or 14.