WO2007136280A1

WO2007136280A1 - Dairy product and process

Info

Publication number: WO2007136280A1
Application number: PCT/NZ2007/000121
Authority: WO
Inventors: Shao Quan Liu; Vaughan Leslie Crow
Original assignee: Fonterra Co-Operative Group Limited
Priority date: 2006-05-23
Filing date: 2007-05-23
Publication date: 2007-11-29

Abstract

A dairy-based flavouring ingredient comprising methionol as a dominant flavour compound is prepared. The method comprises providing a dairy stream with 0.001% to 3.0% w/v of L-methionine or L-methionine present in peptides having 2-6 amino acid residues, followed by fermentation of a sugar already present in or added to the starting material selected from (a) lactose, (b) glucose or galactose resulting from lactose hydrolysis, (c) galactose produced resulting from treatment of the starting material with a lactose-fermenting and galactose non-fermenting bacterium, or (d) an added sugar, with production of methionol.

Description

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DAIRY PRODUCT AND PROCESS

Technical Field

The invention relates to a dairy product and a process for preparing it. The product is a dairy-based, intense flavour building block. This invention enables more economical production of other dairy- products such as natural and processed cheeses, enzyme modified cheeses and cheese analogues.

Background Art

Sulphur-containing flavour compounds are desired in a variety of fermented dairy products, particularly in cheese. The common sulphur flavour compounds found in cheese include hydrogen sulphide (rotten eggs), methanethiol (cooked cabbage), dimethyl sulphide (cabbage), dimethyl disulphide (cauliflower, garlic), dimethyl trisulphide, methional (3-methylthio-l-propanal, boiled potato), J^'-methyl thioacetate (cooking cauliflower), j'-methyl thiopropanoate (cheesy) and ^-methyl thiobutanoate (chives) (Weimer B, Seefeldt K and Bias B, Antonie van Leeuwenhoek, 76: 247-161, 1999).

The flavour threshold values of sulphur flavour compounds are generally very low, although they are also present in trace amounts in cheese (both at ppb levels) (MoHmard P and Spinnler H E, J Dairy Sci, 79: 169-184, 1996; Sable S and Cottenceau G, J Agric Food Chem, 47: 4825-4836, 1999). The natural process of developing sulphur flavour compounds in cheese is long and complex, and their exact mechanism of formation is not completely understood. This makes it difficult to direct and to control development of sulphur flavour compounds in cheese.

Methionol (3-methylthio-l-propanol) is another potent volatile sulphur flavour compound. The odour of methionol can be described as raw potato, soup or meat-like, cauliflower, cooked cabbage and garlic savoury or toasted cheese. The flavour threshold value of methionol is low, ranging from <1 ppm to up to 4.5 ppm, which is dependent upon the medium in which the detection limit is tested.

Methionol is a known off-odour compound in beer and wine (Hill P G and Smith R M, J

Chromatogr A, 872: 203-213, 2000; Mestres M, Busto, O and GuaschJ, J Chromatogr A, 881: 569- 581, 2000). Methionol is not commonly found in fermented dairy products according to the literature, although this sulphur compound has been found in trace amounts in premium quality Cheddar and Camembert cheeses (Urbach G, Intl J Dairy Technol, 50: 79-89, 1997). A recent study has indicated that the presence of rnethionol in cheese is more common than previous studies have revealed. This study has identified the presence of methionol in a number of cheeses including Cheddar, Blue cheeses, hard cheeses (Parmesan, Pecorino and Grana Padano); the relative concentration of methionol in Blue cheeses is particularly higher than that in other cheese varieties (Frank D C, Owen C M and Patterson J, Lebensm-Wiss u-Technol, 37: 139-154, 2004). The exact mechanism of methionol formation in fermented dairy products is not known, which is complicated by the very complex nature of the microbial flora involved in the cheese fermentation process, especially on the surface (smear)-ripened and mould-ripened cheeses (e.g. Blue cheeses and Camembert cheeses).

The role of dairy yeasts in the formation of sulphur flavour compounds has been investigated. These so-called dairy yeasts include Kluyverotnyces lactis, Geotrichum candidum, Debaryomjces hansenii and Yarmwia lipojl tica. The dairy yeasts can produce a range of sulphur flavour compounds, including methanethiol, dimethyl sulphide, dimethyl disulphide, dimethyl trisulphide, and some thioesters, from a sulphur-containing substrate such as L-methionine (Bonnarme P, Lapadatescu C, Yvon M and Spinnler H E, J Dairy Res, 68: 663-674, 2001; Spinnler H E, Berger C, Lapadatescu C and Bonnarme P, Intl Dairy J, 11: 245-252, 2001). These studies were conducted under conditions not relevant to the dairy environment. Further, there is no evidence to suggest that these dairy yeasts can produce methionol from either L-methionine or other sulphur-containing substrates.

In spite of the failure of the above-mentioned studies to show the production of methionol by dairy yeasts, some other yeasts are known to produce methionol from L-methionine. For example, Zygosaccharomyces rouxii (a salt-tolerant yeast important in soy sauce fermentation) is known to produce methionol from L-methionine (Aoki T and Uchida K, Agric Biol Chem, 55: 2113-2116, 1991; van der Sluis C, Rahardjo Y S P, Smit B A, Kroon P J, Hartmans S, ter Schure E G, Tramper J and Wijffels R H, J Biosci Bioeng, 93: 117-124, 2002). Another example is the production of methionol from L-methionine by Saccharomyces cereυisiae (Perpete, P, Duthoit O, de Maeyer S, Imray L, Lawton A I, Stavropoulos K E, Gitonga V W, Hewlins MJ E and Dickinson J R, FEMS Yeast Res, 6: 48-56, 2006). In addition, methionol can be produced from L-methionine by some basidiomycetous yeasts such as Biilleromjces albus, Ciyptococcus spp and Rhodosporidium tomloides (Buzzini P, Romano S, Turchetti B, Vaughan A, Pagoni U M and Davoli P, FEMS Yeast Res, 5: 379-385, 2005). These studies were conducted under conditions that are not applicable to the dairy environment such as milk or cream. There is no evidence to indicate that these yeasts grow in milk or cream. US patent (US 6,753,022 Bl) describes a method for preparing a flavourful dairy product using a lactose-negative microorganism (yeast and/ or bacterium). The yeast is Candida ^elanoides, Debaryomyces hansenii, Saccharomyces cerevisiae, Candida robusta or Tygosaccharomyces rouxii. The bacterium is Micrococcus Intern, Arthrobacter spp or Corymb acterium spp. This process necessitates the addition of nutrients (e.g. carbon and/ or nitrogen sources) to the milk; otherwise, the lactose-negative microorganism will not grow in milk. In this patent, there is no evidence for the presence of methionol in the flavourful dairy product, nor is there any evidence for the formation of methionol by any of the microorganisms used. Thus, the execution of this method will not result in the production of an intense sulphur flavour building block in which methionol is a key flavour component.

Three patents (EP 1186244A2, US6, 562,383 Bl, US 20050112238A1 and US 20070048404A1) are directed to a similar process for preparing a cheese flavouring system comprising a sulphury-cheddar flavour component, a creamy-butter flavour component, and a cheesy flavour component. The sulphury-cheddar flavour component is prepared by firstly treating a milk concentrate or a whey protein concentrate with a lactic acid bacterial culture, a lipolytic enzyme and a highly proteolytic culture or a proteolytic enzyme. This is followed by adding to the mixture a sulphur-containing substrate (L-methionine, L-cysteine, L-glutathione, or mixtures thereof) and a Brepibacterium linens culture or a yeast from the genera Debaryomyces or Kluyveromyces. Then the mixture is allowed to ferment for up to 10 days. The sulphury-cheddar flavour is attributed to sulphur-containing volatile compounds methanethiol, dimethyldisulphide and dimethyltrisulphide. There is no evidence to demonstrate the presence of methionol in the sulphury-cheddar flavour component, nor is there any evidence to show the production of methionol by any of the cultures used in the process, as described in the patent documents.

Natural development of sulphur flavour in cheese is a long and complex process, and the flavour intensity developed as such is often weak. Thus, natural process of sulphur flavour development in cheese is often uneconomical. There is a need for a process that can accelerate the development of sulphur flavour either in cheese or through the provision of sulphur flavour building block that can be applied to cheese or other dairy products. The object of the present invention is to provide an economical, dairy-based, intense sulphur (methionol) flavour building block for flavouring cheese and other dairy products and/or to provide the public with a useful choice.

Disclosure of the Invention

In one aspect the invention provides a method for preparing a dairy-based flavouring ingredient comprising methionol as a dominant flavour compound, the method comprising providing a dairy stream with 0.001% to 3.0%, preferably 0.001% to 2.0% w/v of L-methionine or L-methionine present in peptides having 2-6 amino acid residues, followed by fermentation of a sugar already present in or added to the starting material selected from

(a) lactose;

(b) glucose or galactose resulting from lactose hydrolysis;

(c) galactose produced resulting from treatment of the starting material with a lactose- fermenting and galactose non-fermenting bacterium; or (d) an added sugar, with production of methionol.

Preferably, methionol is the predominant flavour compound of the dairy-based flavouring ingredient.

The methionine may be added for example as methionine itself or as free methionine included within a protein hydrolysate produced by acidic or enzymic hydrolysis or by fermentation.

Preferably, the methionine added is 0.005-1.0% (w/v), more preferably 0.01-1.0% (w/v), even more preferably 0.01-0.5% (w/v), most preferably 0.05-0.3% (w/v).

The methionine may alternatively be provided in the form of peptides having 2-6 amino acid residues provided that the fermentation conditions allow the conversion of the methioninyl residues to methionol.

The term "dairy stream" refers to a liquid dairy product that is milk, cream, whey, skim milk, whey permeate, milk protein concentrate or another dairy product that comprises at least 0.1% (w/v) milk solids, preferably at least 0.5%, more preferably at least 1%, most preferably at least 2%. Dairy streams used in the invention typically contain up to 40% (w/v) solids, preferably 10-20%. In the case of cream, a cream comprising 5-50%, preferably 20-40% milk fat is preferred. Dairy streams used in the invention may be naturally liquids or be prepared by reconstitution from dry solids. The dairy streams used in the invention contain sufficient nutrients (for example naturally present or added sugars, minerals and nitrogenous compounds for the yeast to grow.) Cream, whole milk and 5-15% (w/v) reconstituted skim milk are preferred dairy streams for use in the invention.

In another embodiment, the starting material may be any milk or cream or another dairy stream suitable for use as a cheese ingredient. For example, in addition to milk or cream, the starting material may comprise modified milks or creams including skim milk, and modified milks or creams including those with altered protein, lipid or mineral composition. While non-dairy ingredients may be added, it is currently preferred to use mostly or only ingredients obtained from milk.

The term "dominant flavour compound" means that the flavour compound contributes significantly to the flavour of the product.

The term "predominant flavour compound" means that the compound is the strongest flavour of the product. The presence of methionol as a dominant or predominant flavour compound can be detected by experienced testers or by detecting the presence of methionol at levels above 15 ppm.

The term 'comprising' as used in this specification means 'consisting at least in part of, that is to say when interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present.

Fermentation is carried out by microorganisms. Preferably, the conditions are selected to allow growth of the microorganism converting methionine to methionol. For example, addition of esterases and/ or lipases would generally be avoided to avoid production of free fatty acids and acetic acid that will generally inhibit yeast growth and metabolism.

In a preferred embodiment, the method comprises:

(a) treating the dairy stream with an enzyme having lactase activity; (b) subsequently or simultaneously fermenting the dairy stream with lactose non-fermenting yeast.

In a further preferred embodiment, the method comprises: (a) treating the dairy stream with a lactose fermenting and galactose non-fermenting lactic acid bacterium (or bacteria);

(b) subsequently or simultaneously fermenting the dairy stream with lactose non-fermenting and galactose fermenting yeast.

In another preferred embodiment the method comprises:

(a) treating and fermenting the dairy stream with a lactose fermenting yeast.

In another preferred embodiment the method comprises: (^a) adding a fermentable substrate for example sugar (typically 0.01% to 3% w/v) (can be added together with L-methionine) to the dairy stream;

(b) subsequently or simultaneously fermenting the dairy stream with lactose non-fermenting yeast. Glucose and sucrose are examples of fermentable substrates that may be added in this embodiment.

In preferred embodiments, the fermentation to produce methionol is carried out by organisms, preferably yeasts, preferably selected from species and strains of Brettanomyces sp, Candida sp, Ciyptococcus sp, Dekera sp, Debaryomyces sp, Endomycopsis sp, Galactomyces sp, Geotrichum sp, Hanseniospora sp, Kloeckera sp, KJuyveromyces sp, Lodderomyces sp, Pichia sp, Tkhodotorula sp, Saccharomyces sp, Schisζosaccharomyces sp, Tondaspora sp, Triώosporon sp, Williopsis sp, Yarrowia sp and Zygosaccharomyces sp. Other genera may also produce methionol from L-methionine and thus, are also included.

In preferred embodiments of the invention, the organism strain to be used is selected from a range of strains for its ability to convert methionine to methionol. Alternatively, a strain may be used without such selection provided that it is effective in converting methionine to methionol. A strain suitable for use is one that ferments the methionine-supplemented dairy stream with production of more than 15 ppm methionol. Typically a single strain is used, but a mixture of strains may also be used.

One preferred lactose fermenting yeast that may be used includes Kluyveromyces sp and Candida kefyr.

Currently preferred lactose non-fermenting and galactose-fermenting yeasts that may be used include S acchammyces ceremsiae Laffort-Zymaflore VLl, Saccharomyces cerevisiae Lalvin L2056 and Saccharomyces bayanus Lalvin CVC-NF74 sold under the name Lalvin by Lallemand, Inc. Yeasts for use in the invention are available from other commercial sources and from culture collections such as ATCC, CBS and NCYC.

In further preferred embodiments, the lactose fermenting lactic acid bacteria that produce galactose and do not ferment it are preferably selected from strains and species of Streptococcus thermophilic and Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis The lactose fermenting and galactose non-fermenting lactic acid bacteria may also be selected from variants and mutants of other lactic acid bacteria of the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Oenococcus, Enterococcus, and Bifidobacterium. Genetically modified lactose fermenting lactic acid bacteria that produce galactose may also be included.

The product preferably has a methionol concentration of at least 25 ppm of methionol by weight, preferably at least 50 ppm, more preferably at least 100 ppm.

In one embodiment the dairy stream is pre-treated with the lactase (also known as beta- galactosidase).

In other embodiments the lactase enzyme may be added along with the yeast or subsequent to the addition of the yeast.

In another embodiment L-methionine and/ or the fermentable substrate such as sugar may be added before or along with the yeast and/ or lactose fermenting lactic acid bacteria or subsequent to the addition of yeast and/ or lactose fermenting lactic acid bacteria.

In a further embodiment a lactose-fermenting and galactose non-fermenting lactic acid bacterium (or bacteria) may be added before or along with the yeast or subsequent to the addition of yeast.

The fermentation step is generally carried out at a temperature of 15-35⁰C (preferably at 25⁰C), for 10-48 hours (preferably 24 hours) without deliberate aeration but with gentle stirring for large volumes. Generally the pH is in the tange 4-7. The pH used will depend on the stability and activity of the lactase enzyme and/or the yeast and/or lactic acid bacteria. Preferably the pH is maintained in the range 5.0-6.0, most preferably at pH of approximately 5.5.

Optionally NaCl (up to 5% w/v) and/or Nisaplin (Danisco) can be added to help control growth of potential spoilage microorganisms.

In a preferred embodiment an appropriate amount of enzyme (lactase) is prepared aseptically (preferably by aseptic filtration) and added to the sterile cream or milk at the time of yeast inoculation. In another embodiment, sterile cream milk is pre-treated with lactase before yeast inoculation. The fermentation is conducted at 15 to 35⁰C (preferably 20 to 30⁰C) for 10 to 48 hours, preferably without shaking. After fermentation, the fermented cream or milk is generally either pasteurised or concentrated or dehydrated.

The currently preferred lactase is a fungal lactase from Aspergillus niger (Validase Fungal Lactase

Concentrate, Valley Research, Indiana, USA). A range of commercial lactases (beta-galactosidases) such as those from Aspergillus ory^ae (Enzidase Fungal Lactase 100,000, Genencor Kyowa Co., Tokyo, Japan) and Klujperomjces lactis (Enzidase Yeast Lactase, Genencor Kyowa Co., Tokyo, Japan) are also suitable for use in the process of the invention.

The dairy stream used may be from any mammalian species. Preferably the cream or milk is from cows, goats or sheep. Cows' cream or milk is preferred. The cream used in the Examples is cows' cream.

In another aspect the invention provides a cream or milk product obtainable by a process of the invention.

In a furdier aspect, the invention provides a method of preparing cheese comprising adding a cheese flavouring ingredient, prepared by a method of the invention, to a cheese-making mixture, and processing the cheese-making mixture to prepare a cheese or process cheese.

For example, a cheese flavouring ingredient prepared by a method of the invention may be added to a cheese milk that is subsequently treated with proteolytic enzymes or acidification to produce a curd, and then processed to prepare cheese or process cheese. Alternatively, a cheese-making ingredient according to the invention tnay be mixed with a cheese curd or molten cheese to add flavour to the cheese end product.

In preferred embodiments, no curing of cheese is required as the flavour is similar to that of a mature cheese without curing.

The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

Brief Description of the Drawings

Figure 1 shows the effect on methionol production of variation of (a) temperature, (b) sodium chloride concentration, (c) pH, (d) aeration, (e) methionine and (£) glucose concentration in cream with 0.05% (w/v) added L-methionine and 3% (w/v) glucose (except where the glucose concentration is being varied).

Examples

The following examples serve to illustrate preferred practices of the present invention and are illustrative only and do not limit the present invention.

Media, culturing and analysis

Yeasts are generally cultured in a broth (pH 5.0) consisting of glucose (2% w/v, Merck), yeast extract (0.25% w/v, BBL), malt extract (0.25% w/v, Merck) and Bacto peptone (0.25% w/v, Difco). Lactic acid bacteria are generally grown in Ml 7 broth (Difco) or 10% w/v reconstituted skim milk in the case of Streptococcus sp and JLactococcus sp, or MRS broth (Difco) in the case of Lactobacillus sp, Leuconostoc sp and Pediococcus sp. The culture media are sterilised either by autoclaving at 121⁰C for 15 minutes or by aseptic filtration through a 0.22 μm or 0.45 μm membrane. Both yeasts and lactic acid bacteria are incubated at 30⁰C for 24-48 hours. Aeration at 150 rpm is required for the cultivation of aerobic (respiratory/non-fermentative) yeasts such as Debaryomyces hansenii, Geotrichum candidum and Yarroma lipolytica.

Volatile compounds including methionol are analysed using the procedure of solid-phase microextraction (SPME) and gas chromatography (GC)-mass spectrometry (MS), as described elsewhere (Liu S-Q, Holland R, Mcjarrow P and Crow V L, Intl J Food Microbiol, 89: 265-273, 2003).

Example 1 100 mL of cream (or reconstituted skim milk) is sterilised at 115°C for 15 minutes, followed by cooling to room temperature. Lactase (Validase Fungal Lactase Cone, Valley Research, Indiana, USA) is then added at a rate of 0.1 g per 100 g lactose, followed by the addition of sterile L- methionine to a final concentration of 0.1% w/v. L-Methionine can also be added to the cream or milk before sterilisation. A yeast culture grown overnight in a broth at 3O⁰C is then added (1% v/v). The yeast culture is lactose non-fermenting and galactose fermenting S accharoniyces cerevisiae Laffort- Zymaflore VLl or S accharomyces bayanus Lalvin CVC-NF74. The inoculated cream or milk is then incubated anaerobically and statically at 3O⁰C for 24 hours. Results are shown in Table 1.

As shown Table 1, both yeasts produced considerable amounts of methionol in both cream and milk. There was no formation of methionol in the uninoculated cream or milk (data not shown). S. cereviήae produced similar amounts of methionol in cream and milk, whereas S. bayanus produced significantly more methionol in cream than in milk. A number of volatiles, such ^"as esters, ethanol, branched-chain alcohols and branch-chain fatty acids, were also produced, which add complexity to the methionol flavour note of the ferment. Ketones did not increase and are naturally present in cream.

Table 1 Volatile compounds in cream and milk fermented for 24 h with Saccharomjces cereviάae Laffort-Zymaflore VLl or Saccharomjces bayanus Lalvin CVC-NF74

Example 2

100 HiL of cream containing 0.05% w/v L-methionine and 3% w/v glucose is sterilised at 115°C for 15 minutes, followed by cooling to room temperature. A yeast culture grown overnight in a broth at 30⁰C is then added (1% v/yj. The yeast culture is lactose non-fermenting and galactose fermenting Saccharotnyces cerevisiae Lalvin L2056. The inoculated cream is then incubated anaerobically and statically at 3O⁰C for 72 hours. Results are shown in Table 2.

As shown in Table 2, the concentration of methionol reached a maximum at 24 h. There was no formation of methionol in the uninoculated cream, nor in the cream inoculated with yeast but not added methionine (data not shown). The yeast also produced other volatiles such as branched-chain alcohols. The fermented cream also contained other volatiles such as ketones and free fatty acids. Ketones did not increase and are naturally present in cream. All of these volatiles add complexity to the methionol flavour note.

Table 2 Volatile compounds in cream fermented with Saccharotnyces cerevisiae Lalvin L2056

Example 3

100 mL of cream (or reconstituted milk) is sterilised at 115⁰C for 15 minutes, followed by cooling to room temperature. This is followed by the addition of sterile L-methionine to a final concentration of 0.1% w/v. L-Metlxionine can also be added to the cream or milk before sterilisation. A yeast culture grown overnight in a broth at 30⁰C is then added (1% v/vj. The yeast culture is lactose fermenting Kluyveromjces marxiamis ATCC 8460 or Candida kefyrNCYC 143. The inoculated cream or milk is then incubated at 3O⁰C for 24 hours with or without shaking (150 rpm). Results are shown in Table 3.

As shown Table 3, both yeasts produced considerable amounts of methionol in cream and/ or milk. There was no formation of methionol in the uninoculated cream or milk (data not shown). K. marxianus produced twice as much methionol in cream than in milk. A number of volatiles, such as esters, ethanol, branched-chain alcohols and branch-chain fatty acids, were also produced, which add complexity to the methionol flavour note of the ferment. Ketones did not increase and are naturally present in cream.

Table 3 Volatile compounds in cream and milk fermented with Klujveromyces marxianus ATCC 8460 or Candida ^rNCYC 143

Example 4

100 InL of reconstituted skim milk (10% w/v milk solids) is sterilised at 115°C for 15 minutes, followed by cooling to room temperature. This is followed by the addition of sterile L-methionine to a final concentration of 0.1% w/v. L-Methionine can also be added to the cream or milk before sterilisation. A lactose fermenting and galactose non-fermenting lactic acid bacterium {Streptococcus thermophilus Fonterra B2522 pre-cultured in reconstituted skim milk) is inoculated (1% v/v) to the sterilised, cooled cream or milk. This is followed by the inoculation (1% v/vj of a yeast culture pre- grown in a broth at 3O⁰C. The yeast culture is lactose non-fermenting and galactose fermenting Saccharomyces «r«rø^ Laffort-Zymaflore VLl oi S accharomyces £<2jw/zw Lalvin CVC-NF74. The inoculated milk is then incubated at 30⁰C for 24 hours without shaking. Results are shown in Table 4.

As shown Table 4, the two yeasts produced considerable and similar amounts of methionol in milk. There was no formation of methionol in the uninoculated milk (data not shown). A number of volatiles, such as esters, ethanol, branched-chain alcohols and branch-chain fatty acids, were also produced, which add complexity to the methionol flavour note of the ferment.

Table 4 Volatile compounds in milk co-fermented with Streptococcus thennophilus Fonterra B2522 and Saccharomyces cereviήae Laffort-Zymaflore VLl or Saccharomyces bayanus Lalvin CVC-NF74

Example 5

100 InL of cream (or reconstituted skim milk) is sterilised at 115°C for 15 minutes, followed by cooling to room temperature. Lactase (Validase Fungal Lactase Cone, Valley Research, Indiana, USA) is then added at a rate of 0.1 g per 100 g lactose, followed by the addition of sterile L- methionine to a final concentration of 0.1% w/v. L-Methionine can also be added to the cream or milk before sterilisation. A yeast culture grown overnight in a broth at 30⁰C is then added (1% v/vj. The yeast culture is lactose non-fermenting Geotήchutn candidum CMICC 335426, Debaryomyces hansenii Fonterra B9010 or Ymrowia lipolytica Fonterra B9014. The inoculated cream or milk is then incubated at 3O⁰C for 24 hours with shaking (150 rpm). Results are shown in Table 5.

As shown Table 5, the three yeasts produced considerable amounts of methionol in cream and/ or milk. There was no formation of methionol in the uninoculated cream or milk (data not shown). D. hansenii produced much higher amounts of methionol in cream than in milk. A number of volatiles, such as esters, ethanol, branched-chain alcohols and branch-chain fatty acids, were also produced, which add complexity to the methionol flavour note of the ferment. Ketones did not increase and are naturally present in cream.^"

Table 5 Volatile compounds in cream and milk fermented with Geotήchum candidum CMICC 335426, Debaryomjces hansenii Fonterra B9010 or Yarrowia lipojl tica Fonterra B9014

Example 6

50 mL of cream is sterilised at 115⁰C for 15 minutes, followed by cooling to room temperature. This is followed by the addition of 50 mL of aseptically filtered 0.1% w/v L-methionine and 6% w/v glucose solution. To optimise the fermentation conditions, sometimes methionine and glucose levels are altered. On other occasions the pH of the cream is adjusted before autoclaving and the pH of methionine and glucose solution adjusted accordingly. The final concentrations of methionine and glucose are 0.05% w/w and 3% w/v, respectively, unless indicated otherwise. Incubations are also conducted at different temperatures and with or without shaking. The effect of NaCl is also examined by adding different levels of NaCl to the cream before autoclaving. The details of the parameters of the example are indicated in Figure 1. A yeast culture grown overnight in a broth at 30⁰C is then added (1% v/vj. The yeast culture is lactose non-fermenting and galactose fermenting Sac. cerevisiae Lalvin L2056. The inoculated cream is then incubated anaerobically and statically at 30⁰C for 24 hours. Results are shown in Figure 1.

As indicated in Figure 1, aeration has no beneficial effect on methionol production by this yeast. Methionol production increases as pH decreases from 7 to 5. NaCl from 2% to 5% w/v showed inhibition of methionol formation. The yeast produced considerably more methionol at both 25°C and 3O⁰C. Significant amounts of methionol were produced with the addition of as low as 0.05% w/v methionine and 0.5% w/v glucose. On balance, the selected conditions for producing methionol in full or diluted cream by this yeast are pH 5.5, 25°C, 0.05% w/v methionine and 3% glucose, without aeration and no NaCl.

Example 7 Five grams of food grade L-methionine and 300 g of food grade glucose are dissolved in a final volume of 5 L of water. This solution is combined with 5 L of cream (40% fat) in a 14 L vessel (e.g. starter pot) to give a total volume of 10 L. The pH of the mixture is then adjusted to 5.5 with food grade lactic acid. This is followed by heating the mixture at 9O⁰C for 20 minutes. After cooling to 25°C, the cream-L-methionine-glucose mixture is inoculated with 250 mL of Saccharomyces cerevisiae Lalvin L2056, followed by gentle stirring and incubation at 25°C for 24 hours. After 24 hours, the cream ferment is heated at 85°C for 15 minutes to terminate the fermentation. Samples are taken for analysis using SPME and GC-MS as described above. Alternatively the L-methionine and glucose solution can be sterilised by aseptic filtration (or by autoclaving each solution separately) before being added to the sterilised cream. Optionally nisaplin can be added to the cream mixture.

Approximately 400 ppm of methionol is produced in the final cream ferment before heat inactivation, regardless whether L-methionine and glucose are added to the cream before or after heat treatment. Heat inactivation after fermentation can reduce the methionol concentration by up to 20%. The cream ferment contains other volatile flavour compounds, in addition to methionol. The results are given in Table 7.

Table 7 Volatile compounds in diluted cream (2-fold) fermented with Saccharomyces cereυisiae Lalvin L2056

Example 8

The ferment prepared in Example 7 was subjected to sensory evaluation. This concentrate was diluted in a 20% fat cream, mixed at various dilutions and tasted. At 1% addition rate of the concentrate, there was a savoury note along with a slight potato flavour note. At 5% addition rate of the concentrate the creaminess was enhanced with a strong potato and savoury flavour. At the 10% addition rate of the concentrate the strong flavour was described as both cooked cheese and potato. At 20% addition rate of the concentrate the strong flavour was described as acrid, fishy, oily and vegetable.

Any discussion of documents, acts, materials, devices or the like that 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 forms 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.

The above examples are illustrations of the practices of the invention. It is not the intention to limit the scope of the invention to the above-mentioned examples only. It will be appreciated by those skilled in the art that the invention may be carried out with numerous modifications and variations. For example, the fermentation conditions may be varied in both temperature and time and different fermenting organisms may be used.

Claims

1. A method for preparing a dairy-based flavouring ingredient comprising methionol as a dominant flavour compound, the method comprising providing a dairy stream with 0.001% to 3.0% w/v of L-methionine or L-methionine present in peptides having 2-6 amino acid residues, followed by fermentation of a sugar already present in or added to the starting material selected from

(a) lactose;

(b) glucose or galactose resulting from lactose hydrolysis; (c) galactose produced resulting from treatment of the starting material with a lactose- fermenting and galactose non-fermenting bacterium; or

(d) an added sugar,

with production of methionol.

2. A method as claimed in claim 1 wherein methionol is the predominant flavour compound of the dairy-based flavouring ingredient.

3. A method as claimed in claim 1 or claim 2 wherein the methionine is added as methionine itself or as free methionine included within a protein hydrolysate produced by acidic or enzymic hydrolysis or by fermentation.

4. A method as claimed in any one of claims 1-3 where methionine is added.

5. A method as claimed in claim 4 wherein 0.01-1.0% (w/w) L-methionine is added.

6. A method as claimed in claim 1 or claim 2 wherein L-methionine is provided in the form of peptides having 2-6 amino acid residues and the fermentation conditions allow the conversion of the methioninyl residues to methionol.

7. A method as claimed in any one of claims 1-6 wherein the dairy stream is selected from milk, cream, whey, skim milk, whey permeate, milk protein concentrate or another dairy product that comprises at least 2% (w/v) milk solids.

8. A method as claimed in any one of claims 1-6 wherein the dairy stream is selected from the group consisting of cream, whole mirk and 5-15% (w/v) reconstituted skim milk. 2

9. A method as claimed in claim 8 wherein the dairy stream is cream comprising 5-50% (w/v) milk fat.

10. A method as claimed in any one of claims 1-6 wherein the dairy stream is a cheese ingredient.

11. A method as claimed in any one of claims 1-10 wherein the method comprises: (a) treating the dairy stream with an enzyme having lactase activity;

(b) subsequently or simultaneously fermenting the dairy stream with lactose non-fermenting yeast.

12. A method as claimed in any one of claims 1-10 wherein the method comprises:

(a) treating the dairy stream with a lactose fermenting and galactose non-fermenting lactic acid bacterium (or bacteria);

13. A method as claimed in any one of claims 1-10 wherein the method comprises: (a) treating and fermenting the dairy stream with a lactose fermenting yeast.

14. A method as claimed in any one of claims 1-10 wherein the method comprises:

(a) adding a fermentable substrate to the daky stream;

(b) subsequendy or simultaneously fermenting the dairy stream with lactose non-fermenting yeast.

15. A method as claimed in claim 14 wherein the fermentable substrate comprises glucose or sucrose.

16. A method as claimed in any one of claims 1-15 wherein the fermentation to produce methionol is carried out by yeasts.

17. A method as claimed in claim 16 wherein the yeasts are selected from species and strains of Brettanomyces sp, Candida sp, Cryptococcus sp, Dekera sp, Debaryomyces sp, Endomycopsis sp, Galactomjces sp, Geotrichum sp, Hanseniospora sp, Kloeckera sp, KJuyveromyces sp, ljodderomyces sp,

Piώia sp, R ^" hodotortila sp, S accharomyces sp, Schi^psaccharomyces sp, Tomiaspora sp, Trichosporon sp, Williopsis sp, Yarrowia sp and Zygosacώaromyces sp.

18. A method as claimed in any one of claims 1-17 wherein an organism strain to be used for the fermentation is selected from a range of strains for its ability to convert methionine to methionol.

19. A method as claimed in any one of claims 1-18 wherein the fermentation is carried out by a lactose fermenting yeast selected from the gtoup Kluyveromyces sp and Candida kejyr.

20. A method as claimed in any one of claims 1-18 wherein the fermentation is carried out by lactose non-fermenting and galactose-fermenting yeasts selected from the group S accharomyces cerevisiae and S accharomyces bayanus.

21. A method as claimed in any one of claims 1-15 wherein the fermentation is carried out by lactose fermenting lactic acid bacteria that produce galactose and do not ferment it.

22. A method as claimed in claim 21 wherein the bacteria are selected from strains and species of Streptococcus thennophilus and Lactobacillus delbnickii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis

23. A method as claimed in claim 21 wherein the bacteria are selected from variants and mutants of lactic acid bacteria of the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus,

Oenococcus, Enterococcus, and Bifidobacterium.

24. A method as claimed in any one of claims 1-23 wherein the product has a methionol concentration of at least 25 ppm of methionol by weight.

25. A method as claimed in any one of claims 1-10 and 14-23 wherein the dairy stream is pre- treated with lactase.

26. A method of preparing cheese comprising adding a flavouring ingredient, prepared by the method of claim 1, to a cheese-making mixture, and processing the cheese-making mixture to prepare a cheese or process cheese.

27. A method of preparing a cheese comprising mixing a flavouring ingredient prepared by the method of claim 1 with a cheese curd or molten cheese to add flavour to the cheese end product.