DAIRY PRODUCT AND PROCESS

Technical Field

The invention relates to the preparation of dairy products and to extending the shelf life of dairy products.

Background Art

Various microbes cause spoilage of dairy products and thus, reducing product shelf life and incurring economic losses to the manufacturer and retailer. Yeasts and moulds are the major spoilage microorganisms in a range of dairy products such as cheeses and yoghurts. Chemical preservatives such as sodium benzoate, potassium sorbate and various salts of propionate are commonly used to inhibit growth of yeasts and moulds. However, public health concerns have driven the need to use natural alternatives. One such alternative is to use protective cultures as a way of natural preservation.

US patent 5,378,458 and European patent application 1308506A1 describe methods of using bacteria (certain species and strains of lactobacilli and propionibacteria) as protective cultures to control yeasts and moulds. The mechanism of inhibition is attributed to the production of organic acids such as lactic acid, acetic acid and propionic acids by these cultures. These cultures do not always have a sufficient controlling effect, especially at elevated temperatures (above refrigerated temperatures). Further, these cultures can grow in the product, thus imparting potentially undesirable flavours to the product.

Although yeasts can cause food spoilage, yeasts (especially Saccharomyces cerevisiae) have a long history of proven safe use in the making of numerous fermented foods and beverages such as bread, beer, wine and some fermented dairy products (kefir, surface ripened cheeses, etc)

(Jakobsen M and Narvhus J, 6: 755-768, 1996). Infections caused by the few, known pathogenic yeasts {Candida albicans or Cryptococcus neoformans) are not transmitted through foods and consequently, the public health significance of yeasts in foods is considered by most health authorities as minimal, if not negligible (Fleet GH, J Appl Bacteriol, 68: 199-211 , 1990).

Certain yeasts possess antagonistic property toward other microorganisms such as moulds and other yeasts. This property has been exploited in the biological control of postharvest diseases of fruits, resulting in the application of yeasts as biocontrol agents (Fleet GH, Ih: Yeasts in Food,

page 267-280, 2003; Spadaro D and Gullino ML, Intl J Food Microbiol, 91: 185-194, 2004). Some non-antagonistic yeasts can also inhibit growth of other microbes by competing for nutrients and space, and can effectively function as biocontrol agents (Spadaro D and Gullino ML, Intl J Food Microbiol, 91: 185-194, 2004).

Despite the commercial utilisation of yeasts as biocontrol agents on fruits to control postharvest diseases, there has been no reported use of yeasts as protective cultures in and/or on dairy products. Nevertheless, antagonistic yeasts have been used to control undesirable yeasts in brewing (Vaughan A, O'Sullivan T and van Sinderen D, 111: 355-371, 2005).

The nutritional and health benefits accorded to humans by live lactic acid bacteria and probiotics are well documented, such as control of intestinal infection (e.g. diarrhoea), relief of lactose intolerance, reduction in serum cholesterol level and modulation of immune response (see Lourens-Hattingh A and Viljoen BC, Intl Dairy J, 11:1-17, 2000; Doleyres Y and Lacroix C, Intl Dairy J, 15:973-988, 2005).

The consumer increasingly demands live lactic acid bacteria and probiotics in food and beverages, especially in fermented dairy products such as yoghurts in order to obtain health and nutritional benefits. For a product to be probiotic, it must contain live bacteria and the bacteria must be viable at a significant level at the end of shelf life or at the time of consumption. Regulatory requirements for the population of live lactic acid bacteria and probiotics present in food matrix vary from country to country, generally requiring > 10 ⁶ counts per g of foods or per mL of beverages, although a therapeutic minimum dose of > 10 ⁵ counts per g of foods or per mL of beverages is also proposed (see Roy D, Lait, 85:39-56, 2005). However, the specific dose required for a probiotic effect may be dependent upon the food matrix and the species and strains within the species of probiotics.

It is essential that lactic acid bacteria and probiotics retain their viability and activity in the food matrix so as to meet the recommended therapeutic minimum dose at the time of consumption. However, lactic acid bacteria and probiotics are unstable and die gradually even during refrigerated storage; they die rapidly under ambient conditions. Maintaining a reasonably high number of lactic acid bacteria and probiotics has been a challenge relative to product shelf-life, storage conditions and sensory properties (flavour and texture). Various methods have been developed to improve the survival of lactic acid bacteria and probiotics in food matrix under

refrigerated conditions and examples of such methods include addition of bifidogenic factors (also known as prebiotics) and nutrients, stress adaptation (acid-, cold- and heat shock), use of protectants and microencapsulation (see Shah NP, J Dairy Sci, 83: 894-907, 2000; Champgne C and Gardner NJ, Crit Rev in Food Sci and Nutri, 45:61-84, 2005; Doleyres Y and Lacroix C, see above; Ross PP, Desmond C, Fitzgerald GF and Stanton C, J Appl Microbiol, 98: 1410-1417, 2005). There is no evidence to suggest that these methods are applicable to enhancing the viability of lactic acid bacteria and probiotics under ambient conditions.

Yeasts (especially Saccharomyces cerevisiae) have a long history of proven safe use in the making of numerous fermented foods and beverages such as bread, beer, wine and some fermented dairy products (kefir, surface ripened cheeses, etc). With the exception of beneficial fermentation, yeasts often cause spoilage of foods and beverages, especially dairy products (see

Fleet GH, J Appl Bacteriol, 68: 199-211, 1990). The microbial ecosystem of fermented foods and beverages is complex, consisting of not only yeasts, but also bacteria, lactic acid bacteria in particular. Yeasts can interact with bacteria in three different ways: stimulation, inhibition or no impact and the specific mode of interaction is dependent upon the yeast-bacterium combination

(see Jakobsen M and Narvhus J, Intl Dairy J, 6: 755-768, 1996).

An early study indicates that milk cultures of Lactobacillus bulgaricus maintain viability for several months when grown in association with certain yeasts (see Graham VE, J Bacteriol,

45:51, 1943), but there is no information on viability of other lactic acid bacteria. Another early report shows that a synergism between yoghurt bacteria and certain yeasts (Torulopsis sp, now

Candida sp) significantly extends the viability of yoghurt bacteria for many months; however, these yeasts are highly proteolytic and cause dramatic pH increases that are commercially undesirable (see Soulides DA, Appl Microbiol, 3:129-131, 1955). A more recent study shows the biostabilisation of kefir (a fermented milk beverage) with a non-lactose fermenting yeast

{Saccharomyces cerevisiae) in terms of ethanol formation and sugar utilisation, not survival of lactic cultures (Kwak HS, Park SK and Kim DS, J Dairy Sci, 79:937-942, 1996). None of these studies demonstrates the population of cell survival, temperatures of storage and nature of yeasts

Another recent study indicates that the interaction between naturally occurring (contaminant) yeasts and lactic acid bacteria results in the stabilisation of lactic acid bacterial population in yoghurt (see Viljoen BC, Lourens-Hattingh A, Ikalafen B and Peter G, Food Res Intl, 36:193- 197, 2003). The conclusion of this study is compromised by the methodology of enumerating

lactic acid bacteria, because the MRS agar used to enumerate lactic acid bacteria contained no yeast inhibitor and consequently, yeasts and lactic acid bacteria would have grown simultaneously on the MRS agar plate. Further, the yeasts in this study were not deliberately added and were a mixture of contaminating flora.

US patent 6,294,166 describes a method for stabilising the vitality of dried viable probiotic and lactic acid bacteria using dried non-viable yeast and protein under ambient conditions. The matrix of this dry mixture differs substantially from that of high moisture foods and beverages and it is difficult to envisage that this method can be directly applied to high moisture food matrix to maintain high cell viability.

A published PCT patent application (WO 03/090546) describes a process for producing a fermented product such as yoghurt, butter and cheese using lactobacilli and lactose fermenting and/or galactose fermenting yeast for pharmaceutical applications. Another published PCT patent application (WO 00/60950) describes a process for manufacturing dietary supplements for the treatment of osteoporosis by means of a natural fermentation involving essentially Kefir bacteria and lactose fermenting and/or galactose fermenting yeasts. However, both processes entail an active role of yeasts (e.g. fermentation of lactose and/or galactose) during fermentation. Further, neither processes demonstrated the stabilising effect of yeasts on cell populations of lactic acid bacteria and probiotics under ambient conditions.

There remains a need for a method maintaining high viability of lactic acid bacteria and probiotics in high moisture food matrix or in beverages for longer periods of time under ambient conditions.

An object of the present invention is to provide a method of extending the shelf life of dairy products and/or to provide dairy products with improved shelf life and/or to provide the public with a useful choice. An object of certain embodiments of the present invention is to provide a method of maintaining high viability of lactic acid bacteria and probiotics in high moisture food and beverage systems for longer periods of time under ambient conditions, and/or to provide products containing lactic acid bacteria and probiotics with increased stability and/or to provide the public with a useful choice.

Disclosure of the Invention

The present invention overcomes the drawbacks of the prior art described above by providing a novel way of exploiting yeasts to extend the shelf life of dairy products. The present invention also provides a method of selecting yeasts for such purposes.

In one aspect, the invention provides a method for preparing a dairy product comprising adding a non-lactose fermenting and non-galactose fermenting yeast or a non-fermentative yeast to a dairy starting material or a dairy product,j^an amount sufficient to extend the shelf life of the resulting mixture or products prepared from it and if required further processing the mixture to obtain a further dairy product; wherein said yeast is weakly proteolytic or non-proteolytic and also weakly lipolytic or non-lipolytic.

In another aspect, the invention provides a method of extending the shelf life of a dairy product comprising including on or within the dairy product a non-lactose fermenting and non-galactose fermenting yeast or a non-fermentative yeast in an amount sufficient to reduce the growth of spoilage yeasts and moulds; wherein said yeast is weakly proteolytic or non-proteolytic and also weakly lipolytic or non-lipolytic

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 which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present.

The terms "weakly proteolytic" and "weakly lipolytic" mean that the degree of proteolysis and lipolysis is insufficient to alter the taste, aroma or texture to a human consumer and insufficient to raise the pH by more than 1.0 pH unit, preferably insufficient to raise the pH by more than 0.5 pH units. Preferred yeasts for use in the invention are non-proteolytic and non-lipolytic.

In this specification the term "ambient conditions" is used to refer to temperatures in the range 0°-35°C, preferably 10°-30°C. The term "ambient conditions" also generally is used to refer to non-refrigerated conditions but may sometimes include refrigerated conditions above O ⁰ C.

The term "food matrix" is used to refer preferably to fermented dairy products derived from mammal's milk such as cow's milk, goat's milk and sheep milk. The food matrix may also refer

to non-dairy foods and beverages such as fermented soy milk. The total milk solids content can be up to 70% w/v (preferably up to 50% w/v).

The term "high moisture" as used in this specification is defined as having a moisture content of at least 30% (w/w). Products for which the invention is useful often have moisture contents of at least 60% w/w or 70% w/w and sometimes greater than 90% (w/w).

In a preferred method the starting material is milk or skim milk and the product is yoghurt, or fermented milk drink or a cheese or analogue cheese. The yeasts may be added to a dairy product substantially ready for distribution to consumers. Alternatively the yeast-containing mixture may be further processed. For example, the yeast and a yoghurt starter culture may be added to milk or skim milk and incubated to form yoghurt or the yeast may be added before, during and/or after yoghurt fermentation. Likewise a milk or skim milk containing the yeast and an appropriate microorganism may be used to prepare a fermented milk. Cheesemilk containing the yeast may be processed by adding coagulating enzyme and collection of the curd with subsequent milling, salting and pressing. Alternatively cheeses and analogue cheeses can be prepared using acidification. The invention is generally more applicable to cheese manufacture where yeasts are not traditionally used. The invention is generally used with non-surface- ripened cheese.

Preferably the yeast is present within the dairy product.

The present invention is about the deliberate use of specific yeasts (single or mixed) as protective and/or stabilising cultures for shelf life extension. This use of the yeasts can allow storage of the fermented products for longer periods of time than when the yeasts are not used. The specific storage time/temperature required is dependent on consumers' needs and acceptance. The invention does not include use of a single or mixture of yeasts that include yeasts that ferment lactose or galactose or cause significant lipolysis and proteolysis.

The yeasts used in the invention do not grow in the food matrix and thus, do not play a role in fermentation. The use of such yeasts may require product reformulation in the case of yoghurts and other fermented milks, such as exclusion of fermentable sugars as sweeteners, instead, using non-fermentable sweeteners, in the case of a fermentative yeast being selected as the preferred yeast. Product reformulation may not be necessary if a non-fermentative yeast is selected or the

product is cheese or analogue cheese. In the case of fermented milks such as yoghurts and drinking yoghurts, lactose non-fermenting and galactose non-fermenting yeasts are preferred, or non-fermentative yeasts are preferred.

Active selection of appropriate yeasts is imperative in order to obtain protective and/or stabilising effects, as not all yeasts have protective and/or stabilising effects.

In preferred embodiments, the yeasts may be selected from species and strains of Brettanomyces sp, Candida sp, Cryptococcus sp, Dekera sp, Debaryomyces sp, Endomycopsis sp, Galactomyces sp, Geotrichum sp, Hansenula sp, Hanseniospora sp, Kloeckera sp, Kluyveromyces sp, Lodderomyces sp, Pichia sp, Rhodotorula sp, Saccharomyces sp, Schizosaccharomyces sp, Torulaspora sp, Trichosporon sp, Williopsis sp, Yarrowia sp and Zygosaccharomyces sp. Other genera may also have a protective effect and thus, are also included.

Yeasts of the genus Williopsis especially those of the species Williopsis saturnus are particularly suitable.

Yeasts of the genus Debaryomyces especially those of the species Debaryomyces hansenii are also particularly suitable.

The yeasts used are lactose non-fermenting and galactose non-fermenting, and include non- fermentative yeasts, and are preferably live. Further, the yeasts used are only weakly proteolytic (preferably non-proteolytic) and are only weakly lipolytic (preferably non-lipolytic). Preferably, the yeasts used do not catabolise lactic acid under normal fermentation conditions (semi- anaerobic and/or anaerobic).

In a further aspect, the invention provides a method for identifying a yeast as a protective culture against yeasts and moulds. The method comprises adding yeast(s) to a milk product, adding a potential spoilage yeast and/or mould and determining the growth of the potential spoilage yeast or mould after a period of time, preferably 1 or more weeks, at a temperature in the range 1- 3O ⁰ C.

In another aspect of the invention, the yeast extends the viability of lactic acid bacteria and probiotics in food and beverages for longer periods of time under ambient conditions.

Specifically, this aspect of the invention provides a method for preparing a high moisture milk product having a pH of less than 5.5 comprising adding one or more types of lactic acid bacteria or probiotics to a dairy starting material wherein a stabilising non-lactose fermenting and non- galactose fermenting live yeast or a stabilising dead yeast or yeast extract is added before, during or after the bacteria or probiotic adding step to extend the survival of the lactic acid bacteria or probiotic at counts of > 10 ⁵ per g or ml, preferably at > 10 ⁶ cells per g or mL. The method may include a step in which the lactic acid bacteria or probiotics are allowed to ferment the milk starting material.

In a preferred method of this aspect, the starting material is milk or skim milk and the product is a yoghurt, or fermented milk drink or a cheese.

In this aspect, the present invention involves the deliberate use of specific yeasts to maintain high viability of lactic acid bacteria and probiotics in high moisture-low pH (pH 5.5 or below, preferably 4.6 or below) food matrix. This use of the yeasts can allow storage of the fermented products for longer periods of time under ambient conditions than when the yeasts are not used. The specific storage time/temperature required is dependent on consumers' needs and acceptance.

In preferred embodiments, the present invention enables the maintenance of high cell counts of lactic acid bacteria and probiotics in high moisture food matrix (>10 ⁵ cell count per g or mL of food or beverage, preferably at > 10 cells per g or mL) at 35 ⁰ C or below (preferably 3O ⁰ C or below) for at least 2 weeks (see Examples). The maintenance of a specific cell count is also dependent upon the nature of a food matrix (e.g. pH), temperature range of storage, packaging and the species and strain of lactic acid bacteria and probiotics. The invention is particularly useful where refrigeration is not readily available, allowing storage for longer periods at temperatures greater than 10°C, or even 20°C or 30°C for example.

These yeasts do not grow in the food matrix and thus, do not play a role in fermentation. The use of such yeasts may require product reformulation, such as exclusion of fermentable sugars as sweeteners, instead, using non-fermentable sweeteners, in the case of a fermentative yeast being selected as the preferred yeast. Product reformulation may not be necessary if a non- fermentative yeast is selected. In the case of fermented milks such as yoghurts and drinking

yoghurts, lactose non-fermenting and galactose non-fermenting yeasts are preferred, or non- fermentative yeasts are preferred.

In preferred embodiments, the lactic acid bacteria and probiotics are selected from strains and species of Lactobacillus sp, Lactococcus sp, Leuconostoc sp, Pediococcus sp, Streptococcus Sp ₅

Oenococcus sp, Enterococcus sp, and Bifidobacterium sp. Other genera of bacteria such as

Propionibacterium sp may also be stabilised by the present invention and thus, are also included.

Not all yeasts have an equal stabilising effect on the viability of lactic acid bacteria and some yeasts such as Yarrowia lipolytica can have a detrimental impact on product quality, causing spoilage (flavour and texture) (see Examples 9 and 13). Further, the same yeast does not necessarily have an equal stabilising effect on the viability of different lactic acid bacteria and probiotics (see Example 10). Active selection of appropriate yeasts is imperative in order to maintain high viability of lactic acid bacteria and probiotics and to keep product quality. Use of yeasts without careful selection and avoidance of contaminating strains can have a deleterious effect on cell viability and product quality.

In preferred embodiments, the yeasts are selected from species and strains of Brettanomyces sp, Candida sp, Cryptococcus sp, Dekera sp, Debaryomyces sp, Endoniycopsis sp, Galactomyces sp, Geotrichum sp, Hanseniospora sp, Kloeckera sp, Kluyveromyces sp, Lodderomyces sp, Pichia sp, Rhodotorula sp, Saccharomyces sp, Schizosaccharomyces sp, Torulaspora sp, Trichosporon sp, Williopsis sp, Yarrowia sp and Zygosaccharomyces sp. Other genera may also have a stabilising effect and thus, are also included.

Yeasts of the genus Williopsis especially those of the species Williopsis saturnus are particularly suitable.

Yeasts of the genus Debaryomyces especially those of the species Debaryomyces hansenii are also particularly suitable.

The yeasts used are non-lactose fermenting and non-galactose fermenting, and include non- fermentative yeasts, and are preferably live. Further, the yeasts used are only weakly proteolytic (preferably non-proteolytic) and are only weakly lipolytic (preferably non-lipolytic). Furthermore, the yeasts used do not cause dramatic increases in pH of the final product (<5.5,

preferably <4.6). Preferably, the yeasts used do not catabolise lactic acid under normal fermentation conditions (semi-anaerobic and/or anaerobic).

In a further embodiment, live yeasts may be replaced with dead yeasts or products and/or substances derived from yeasts such as yeast extracts (see Examples 8 and 15). When dead yeasts or yeast extracts are used, these need not necessarily be derived from yeasts that do not ferment lactose or galactose.

Optionally, flavourings, colourings and texturisers commonly used in food and beverage manufacturing processes may be employed in the present invention. However, non-fermentable sweeteners such as artificial sweeteners and non-fermentable polyol sweeteners (e.g. xylitol) must be used, instead of using fermentable sugars as sweeteners (e.g. sucrose), in the case of a fermentative yeast being used. Any sweetener may be used in the case of non-fermentative yeast being used.

In another aspect, the invention provides a high moisture milk product including live lactic acid bacteria or probiotics and a yeast or yeast extract stabiliser, stabilising the lactic acid the bacteria or probiotics.

In a further aspect, the invention provides a method for identifying a yeast or yeast extract stabiliser for lactic acid bacterial or probiotics in a high moisture milk product. The method comprises adding bacteria or probiotics to a milk product, adding an uncontaminated yeast strain and determining the number of surviving bacteria or probiotics after a period of at least one week, preferably 2 or more weeks, at a temperature in the range 10-30°C preferably 10-35°C. Preferably a plurality of determinations of the numbers is made at different times.

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

Brief Description of Drawings:

Figure 1 shows inhibition by protective yeast Williopsis saturnus subsp. saturnus B9043 (CBS 254) of galactose fermenting yeast Saccharomyces cerevisiae B9030 (Laffort Zymaflore VL1) in cheese incubated at 2O ⁰ C. Strain B9043 was inoculated into the milk at a level of 10 ⁵ cfu/ml and strain B9030 was inoculated at levels of 10 ¹ and 10 ²

cuf/ml. The initial yeast cell count in the cheese curd increased by approximately 10- fold after the cheese was made. The y-axis shows yeast cell count in cheese in units of l0 ⁴ cfu/g.

Figure 2 shows inhibition by protective yeast Williopsis saturnus subsp. saturnus B9043 (CBS 254) of lactose and galactose fermenting yeast Kluyveromyces marxianus B9052 (ATCC 8640) in cheese incubated at 2O ⁰ C. Strain B9043 was inoculated into the milk at a level of 10 ^s cfu/ml and strain B9052 was inoculated at levels of 10 ¹ and 10 ² cuf/ml. The initial yeast cell count in the cheese curd increased by approximately 10- fold after the cheese was made. The y-axis shows yeast cell count in cheese in units of 10 ⁴ cfu/g.

Figure 3 illustrates a survival of yoghurt bacteria in set yoghurt with 20% w/v milk solids in the presence of added yeast extract (1% w/v) during storage at 3O ⁰ C. Yoghurt bacteria = Streptococcus thermophilus and Lactobacillus bulgaricus (MY-900,

Danisco). The triangles show the results with yeast extract, the circles show the results with controls.

Figure 4 illustrates a survival of yoghurt bacteria in set yoghurt with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cfu/g) during storage at 3O ⁰ C.

Yoghurt bacteria = Streptococcus thermophilus and Lactobacillus bulgaricus (MY- 900, Danisco).

Live yeasts added are: B9030 = Saccharomyces cerevisiae (Laffort Zymaflore VLl) ■

B9035 = Saccharomyces bay anus (Lalvin CVC-NF74, Lallemand) 0

B9028 = Saccharomyces cerevisiae (Lalvin R2, Lallemand) D

B9043 = Williopsis saturnus (CBS 254) *

B9010 = Debaryomyces hasenii (Fonterra) A B9014 = Yarrowia lipolytica (Fonterra) o

B9006 = Candida kefyr (NCYC 143) δ

B9052 = Kluyveromyces marxianus (ATCC 8640) x

The control values are shown with filled circles.

Figure 5 illustrates a survival of lactic acid bacteria in set fermented milk with 20% w/v milk solids in the presence of added live yeasts (~10 ⁶ cell counts/g) during storage at 3O ⁰ C.

B9043 = yeast added, Williopsis saturnus (CBS 254); Yoghurt bacteria were not added. Lb. = Lactobacillus

Figure 6 illustrates a survival of Lactobacillus rhamnosus DR20 (Fonterra) in drinking yoghurt with 5% w/v milk solids in the presence of added live yeast (~10 ⁶ cell count/g) during storage at 3O ⁰ C. Yoghurt bacteria (MY-900, Danisco) were added along with DR20. B9043 = yeast added, Williopsis saturnus (CBS 254).

Figure 7 illustrates a survival of Lactobacillus rhamnosus DR20 (Fonterra) in fermented milk with 5% w/v milk solids in the presence of added live yeast (~10 cell count/g) during storage at 3O ⁰ C. Yoghurt bacteria were not added. Lb. = Lactobacillus B9043 = yeast added, Williopsis saturnus (CBS 254)

Figure 8 illustrates a survival of Lactobacillus rhamnosus DR20 in set fermented milk with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cell counts/g) during storage at 3O ⁰ C. Yoghurt bacteria were not added.

Live yeasts added are: B9043 = Williopsis saturnus (CBS 254)

B9001 = Geotrichum candidum (CMICC 335426)

B9041 - Schizosaccharomyces pombe

B9049 = Hansenula subpecullosa

B9010 = Debaryomyces hasenii (Fonterra) B9042 = Pichia membranifaciens

B9014 = Yarrowia lipolytica (Fonterra)

B9046 = Zygosaccharomyces bailii

B9050 = Kloekera apiculata

B9053 = Torulaspora delbrueckii B9040 = Candida krusei

Figure 9 illustrates a survival of Lactobacillus rhamnosus DR20 (Fonterra) in stirred yoghurt with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cell count/g) during storage at different temperatures. Yoghurt bacteria were added along with

DR20. B9043 = yeast added, Williopsis saturnus (CBS 254).

Figure 10 illustrates a survival of Lactobacillus rhamnosus DR20 (Fonterra) in set fermented milk with 20% w/v milk solids in the presence of added dead yeast (equivalent to

~10 ⁶ live cell counts/g) during storage at 3O ⁰ C. Yoghurt bacteria were not added. The yeast B9043 was autoclaved before being added to milk.

Figure 11 illustrates a survival of Bifidobacterium lactis DRlO (Fonterra) in set fermented milk with 20% w/v milk solids in the presence of added live yeast Williopsis saturnus

B9043 (CBS 254) during storage at 3O ⁰ C. Yoghurt bacteria were not added.

Figure 12 illustrates a survival of Bifidobacterium lactis DRlO (Fonterra) in set yoghurt with 20% w/v milk solids in the presence of added live yeast during storage at different temperatures. Yoghurt bacteria were added along with DRlO. B9043 = yeast added,

Williopsis saturnus (CBS 254).

Examples

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

EXAMPLE 1

The following culture media are used when practising the invention:

YEPD broth: YEPD is comprised of Bacto-yeast extract (Difco), 1 %; Bacto-peptone (Difco), 1%; Dextrose (Merck), 2%; pH 5.0; it is autoclaved at 121°C for 15 minutes. This medium is used to grow yeasts at 30°C for up to 48 hours with or without aeration (shaking at 150 rpm).

YEPD agar: YEPD agar is comprised of Bacto-yeast extract (Difco), 1%; Bacto-peptone (Difco), 1%; Dextrose (Merck), 2%; Bacto-agar (Difco), 2%; pH 4.5 (buffered with 0.1 M citrate- 0.2 M phosphate buffer); it is autoclaved at 121 °C for 15 minutes and kept in 45°C water bath before use.

RSM: Reconstituted skim milk, 10%; it is autoclaved at 115 °C for 15 minutes. This medium is used for preparing yoghurt starter cultures and for preparing plain yoghurt.

EXAMPLE 2

Detection of inhibitory activity of whole cells of yeasts by agar diffusion method using YEPD agar (see Example 1)

10 μl of a culture of potential spoilage yeast is mixed into 20 ml of molten YEPD agar maintained at 45° C. This mixture is then poured into a sterile Petri dish. After cooling down to room temperature, 3 spots of 50μl of a culture of protective yeast are inoculated onto the agar plate surface. After drying, the plates are incubated for up to 7 days at 30°C and are checked daily for clear zones surrounding the protective yeast spots. The inhibitory activity is recognized by the inhibition of growth (clear zones) around the protective yeast spots.

Detection of inhibitory activity of filtrate of protective yeasts by well diffusion method using YEPD agar (see Example 1)

10 μl of a culture of potential spoilage yeast is mixed into 30 ml of molten YEPD agar maintained at 45 °C. This mixture is then poured into a sterile Petri dish. After cooling down to room temperature, 3 separate wells (10 mm diameter) are cut into the agar on each plate. Filtrate of a culture of protective yeast is used instead of whole cells. The filtrate is prepared by centrifuging the culture at 1500 g (SS-34 rotor) for 10 minutes at 4°C, followed by filtering the supernatant through a sterile 0.22 μm membrane filter. 300 μl of filtrate are inoculated into each well avoiding overflow. The inoculated plates are left on the bench to air-dry, followed by incubation for up to 7 days at 30°C and daily check for clear zones of inhibition. The inhibitory activity is recognized by the inhibition of growth (clear zones) around the protective yeast spots.

As shown in Table 1, two protective yeasts are identified, Williopsis saturnus subsp. saturnus and Debaryomyces hansenii. Williopsis saturnus subsp. saturnus shows inhibition against Schizosac. pombe, Can. krusei, Pic. membranaefaciens, Can. guillermondii, Han. subpelliculosa, Kloec. apiculata, Kluy. marxianus, Rhodo. glutinis and Pic. holstii. Debaryomyces hansenii shows inhibition against Schizosac. pombe, Schizosac. malidevorans, Brett, bruxellensis, Kloec. apiculata and Kluy. marxianus.

Filtrate of Williopsis saturnus subsp. saturnus shows inhibition against Han. subpelliculosa only. Filtrate of Debaryomyces hansenii does not show inhibition against any of the yeasts listed in Table 1.

Table 1 Inhibition of protective yeasts Williopsis saturnus subsp. saturnus

B9043 (CBS 254) and Debωyomyces hansenii B9010 (Fonterra) against potential spoilage yeasts based on agar diffusion assay ^a

Potential spoilage yeasts W. saturnus B9043 D. hasenii B9010

Sac. bayanUS B9039 (Red Star Premier Cuvee) — —

Sac. bayanus B9028 (La\ym ia) - -

Sac. bayanus B9032 (LaMn SBl) - -

Sac. bayanus B9033 (Lalvin Agglo 016) — —

Sac. cerevisiae B9038 (Red Star Montrachet) — —

Sac. cerevisiae B9026 (Lalvin OMl 3) - -

Sac. cerevisiae B9034 (Lalvin 71B) — —

Sac. cerevisiae B9029 (Lalvin L2056) — —

Sac. cerevisiae B9036 (Lalvin L2226) - -

Sac. cerevisiae B9027 (Lalvin ICV-D47) - -

Sac. cerevisiae B9037 (Uvaferm C52) — —

Sac. Cerevisiae B9030 (Laffort-Zymaflore VLl) — —

Sac. bayanus B9035 (Lalvin CVC-NF74) — —

Schizosac. pombe B9041 ++ ++

Schizosac. malidevorans B9045 — ++

Zygosac. bailli B9046 - -

Can. krusei B9040 44- -

Pic. membranaefaciens B9042 4- —

Brett, bruxellensis B9047 (CBS 74) - +

Can. guillermondii B9048 ++ -

Han. subpelliculosa B9049 +++ -

Kloec. apiculata B9050 ++ ++

Kluy. marxianus B9051 ++ ++

Kluy. marxianus B9052 (ATCC 8640) - -

Tor. delbrueckii B9053 - -

Rhodo. glutinis B9003 (NCYC 162) ++ -

Pic. holstii B9005 (NCYC 560) ^ ~

Can. kefyr B9006 (NCYC 143) _

Can. kefyr B9007 (NCYC 744) ~

Can. tropicalis B9009 (NCYC 997) __ _

Can. famata B9011 _ _

Yar. lipolytica B9013 ^~ _

Yar. lipolytica B9014 ^~ _

Can. kefyr B9015 ^~ Can. krusei B9016 ^a +, weak inhibition; -H- medium inhibition; +-H-, strong inhibition.

EXAMPLE 3

Inhibition of growth of potential spoilage yeasts in plain yoghurt

1 % v/v of yoghurt starter culture MY-900 (Danisco) pre-cultured in RSM is inoculated into each 100 ml of RSM (in duplicate) described in Example 1. This is followed by the addition of potential spoilage yeast {Sac. cerevisiae B9030, Sac. bayanus B9035, Kluy. marxianus B9052 or Can. kefyr B9006) at four concentrations 10 ¹ , 10 ² , 10 ³ , and 10 ⁴ cfu/ml. Two bottles of RSM are used for each yeast concentration, one with and one without the addition of protective yeast. 1 % v/v of protective yeast W. saturnus subsp. saturnus B9043 pre-cultured in YEPD broth (see Example 1) is inoculated into one of the RSMs. Inoculated RSMs are incubated at 30 °C for a period of 35 days. Gas formation serves as an indication of growth of potential spoilage yeast.

The protective yeast W. saturnus subsp. saturnus B9043 is neither lactose fermenting nor galactose fermenting, and thus, is not expected to grow in plain yoghurt. However, the four potential spoilage yeasts (Sac. cerevisiae B9030, Sac. bayanus B9035, Kluy. marxianus B9052 and Can. kefyr B9006) are either lactose fermenting and/or galactose fermenting and therefore, are expected to grow in plain yoghurt containing sufficient amounts of both lactose and galactose.

As indicated in Tables 2-5, there is no or delayed formation of gas (growth) during the incubation period in the plain yoghurt inoculated with the four potential spoilage yeasts (Sac. cerevisiae B9030, Sac. bayanus B9035, Kluy. marxianus B9052 and Can. kefyr B9006) and the protective yeast W. saturnus subsp. saturnus B9043, compared with the control (no added protective yeast), especially at the lower levels of yeast inoculum (10 ¹ and 10 ² cfu/ml). This example demonstrates that the initial yeast count is critical for the protective yeast to be effective.

It should be noted that the protective yeast W. saturnus subsp. saturnus B9043 does not show inhibition against the four potential spoilage yeasts (Sac. cerevisiae B9030, Sac. bayanus B9035, Kluy. marxianus B9052 and Can. kefyr B9006) according to the agar diffusion assay (see Table 1 in Example 2). This indicates that the protective yeast can inhibit a wide range of yeasts beyond those that show sensitivity on the agar diffusion assay. Further, it must be emphasised that protective culture is no substitute for good hygiene and that it is imperative to keep the initial yeast count as low as possible for the protective yeast to be effective.

Table 2 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 (inoculated at 10 ⁵ cfu/ml) against galactose fermenting yeast Saccharomyces cerevisiae B9030 in plain yoghurt incubated at 30°C ^a

Potential spoilage yeast Saccharomyces cerevisiae B9030

Added cell concentration (cfu/ml) 10 ² 10 ³ 10 ⁴

Day - B9043 + B9043 - B9043 + B9043 - B9043 + B9043

2 _

3 — — + — + +

4 +H- — +H- + +H- +H-

5 +H- — +H- +H- +H- +H-

6 +H- — +H- +H- +H- +H-

7 +H- —

8 +H- —

9 +H- —

10 +H- ^a Growth was indicated by gas formation; -, no gas; +, weak gas formation; ++, medium gas formation; t-, strong gas formation.

Table 3 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 (inoculated at 10 ⁵ cfu/ml) against lactose and galactose fermenting yeast Kluyveromyces marxianus B9052 in plain yoghurt incubated at 30°C ^a

Potential spoilage yeast Kluyveromyces marxianus B9052

Added cell concentration (cfu/ml) 10 ² 10 ³ 10 ⁴

Day - B9043 + B9043 - B9043 + B9043 - B9043 + B9043

1 1

2 __ __

3 + — + — + —

4 -H- — +H- — +H- +H-

5 +h — +H- +H- +H-

6 +h — +H- + +H- +H-

7 ^• H- + -H+ +

8 +r + +H- +h

9 +h + +H- +h

10 +h + +H- +H ^a Growth was indicated by gas formation; -, no gas; +, weak gas formation; ++, medium gas formation; +++, strong gas formation.

Table 4 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 (inoculated at 10 ⁵ cfu/ml) against lactose and galactose fermenting yeast Candida kefyr B9006 in plain yoghurt incubated at 30°C ^a

Potential spoilage yeast Candida kefyr B900(

Added cell concentration (cfu/ml) 10 ² 10 ³ 10 ⁴

Day - B9043 + B9043 - B9043 + B9043 - B9043 + B9043

2 — — - —

3 + — + —

4 + — +H- +H- +H- +H-

5 + — +H- +H- +H- +H-

6 + — +H- +H- +H- +H-

7

8 + —

9 4- ,

10 + — ^a Growth was indicated by gas formation; -, no gas; +, weak gas formation; ++, medium gas formation; 1-, strong gas formation.

Table 5 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 (inoculated at 10 ⁵ cfu/ml) against galactose fermenting yeast Saccharomyces cerevisiae B9030 (inoculated at 10 cfu/ml), galactose fermenting yeast Saccharomyces bay anus B9035 (inoculated at 10 cfu/ml) and lactose and galactose fermenting yeast Kluyveromyces marxianus B9052 (inoculated at 10 cfu/ml) in plain yoghurt incubated at 30°C ^a

^a Growth was indicated by gas formation; -, no gas; +, weak gas formation; -H-, medium gas formation; +++, strong gas formation.

EXAMPLE 4

Inhibition of growth of potential spoilage moulds on plain yoghurt

1 % v/v of yoghurt starter culture MY-900 (Danisco) pre-cultured in RSM is inoculated into each 100 ml of RSM (in duplicate) described in Example 1. This is followed by the addition of spore suspensions of potential spoilage moulds at three concentrations 10 ² , 10 ⁴ , and 10 ⁶ cfu/ml for each of the mould tested. Two bottles of RSM are used for each spore concentration, one with and one without the addition of protective yeast W. saturnus subsp. saturnus B9043 or D. hasenii B9010. 1 % v/v of protective yeast pre-cultured in YEPD broth (see Example 1) or cell-free filtrate (see Example 2) is inoculated into one of the RSMs. The content is mixed thoroughly after inoculation by gently swirling the bottles. Inoculated RSMs are incubated at 30 °C for up to 14 days. Appearance of mould colonies serves as an indication of growth of potential spoilage moulds.

As indicated in Tables 6-10, there is no or delayed (or reduced) mould growth during the incubation period in the plain yoghurt inoculated with potential spoilage moulds and the protective yeast W. saturnus subsp. saturnus B9043, compared with the control (no added protective yeast), especially at the lower level of yeast inoculum (10 ² cfu/ml). W. saturnus subsp. saturnus B9043 is effective against growth of mould species of Byssochlamys, Eurotium and Penicillium. Filtrate of whole cells of this protective yeast is also effective against growth of Penicillium roqueforti (Table 11).

As indicated in Tables 12-15, there is no or delayed (or reduced) mould growth during the incubation period in the plain yoghurt inoculated with potential spoilage moulds and the protective yeast D. hasenii B9010, compared with the control (no added protective yeast), especially at the lower level of yeast inoculum (10 ² cfu/ml). D. hasenii B9010 is effective against growth of mould species of Aspergillus, Byssochlamys, Eurotium and Penicillium. Filtrate of whole cells of this protective yeast is also effective against growth of Aspergillus (Table 16).

As with inhibition of yeasts, the initial mould spore count is critical for the protective yeast to be effective.

Table 6 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Byssochlamys fulva in plain yoghurt incubated at 30°C ^a

¹ -, no growth;+, weak growth; ++, medium growth; ++-+-, strong growth.

Table 7 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Byssochlamys nivea in plain yoghurt incubated at 30°C ^a

Potential spoilage

¹ -, no growth;+, weak growth; ++-, medium growth; +-++-, strong growth.

Table 8 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Penicillium sp in plain yoghurt incubated at 30°C ^a

Potential spoilage

-, no growth;+, weak growth; ++, medium growth; +++, strong growth.

Table 9 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Eurotium chevalieri in plain yoghurt incubated at 30°C ^a

Potential spoilage

Eurotium chevalieri mould

¹ -, no growth;+, weak growth; ++, medium growth; + ^■ ++-, strong growth.

Table 10 Inhibition of protective yeast Wilϊiopsis saturnus subsp. saturnus B9043 against mould Penicillium candidum in plain yoghurt incubated at 30°C ^a

Potential spoilage

Penicillium candidum mould

¹ -, no growth;+, weak growth; ++, medium growth; +++, strong growth.

Table 11 Inhibition of filtrate of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Penicillium roqueforti in plain yoghurt incubated at 30°C ^a

Potential spoilage

Penicillium roqueforti mould

^: -, no growth;+, weak growth; ++, medium growth; +++, strong growth.

Table 12 Inhibition of protective yeast Debaryomyces hasenii B9010 against mould Aspergillus sp. in plain yoghurt incubated at 30°C ^a

Potential spoilage

^a -, no growth;+, weak growth; ++, medium growth; +++. strong growth.

Table 13 Inhibition of protective yeast Debaryomyces hasenii B9010 against mould Byssochlamys fulva in plain yoghurt incubated at 30°C ^a

Potential spoilage

Byssochlamys fulva mould

¹ -, no growth;+, weak growth; ++, medium growth; +++, strong growth.

Table 14 . Inhibition of protective yeast Debaryomyces hasenii B9010 against mould Eurotium chevalieri in plain yoghurt incubated at 30°C ^a

Potential spoilage

¹ -, no growth;+, weak growth; -H-, medium growth; +-H-, strong growth.

Table 15 Inhibition of protective yeast Debaryomyces hasenii B9010 against mould Byssochlamys nivea in plain yoghurt incubated at 30°C ^a

Potential spoilage

Byssochlamys nivea mould

¹ -, no growth;+, weak growth; + ^• +, medium growth; +-H-, strong growth.

Table 16 Inhibition of Filtrate of protective yeast Debaryomyces hasenii B9010 against mould Aspergillus sp. in plain yoghurt incubated at 30°C ^a

Potential spoilage

Aspergillus sp. mould

Spore Concentration

10 ² 10 ⁴ 10 ⁶ (per ml)

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

1 — — _ _ + —

4 — — — — ^■ H- +

7 + — +H- M-

9 + — + +H- +H-

10 + — +H- +H- ^a -, no growth;+-, weak growth; + ^• +-, medium growth; +-H-, strong growth.

EXAMPLE 5 Inhibition of growth of potential spoilage yeasts in cheese

This method is designed to produce experimental cheese on a lab scale. 1.2L of pasteurised milk (pH about 6.6-6.7) is measured into a sterilised 2L beaker covered with tin foil. This milk is then warmed to 32 ⁰ C in pre-heated water bath, followed by the addition of 2.0% v/v cheese starter (Streptococcus thermophilus, Fonterra) to the milk. 1% v/v each of protective yeast and appropriately diluted potential spoilage yeast is then added (no protective yeast is added to the control milk). This is followed by incubation for 30 minutes and then measuring pH at 30°C (expected to be about pH 6.5). 120μl rennet is then added and mixed into the milk. Incubation is continued at 32°C until coagulum is firm (approx 30-45 minutes). The curd is cut using a sterilised miniature cheese knife. The temperature of the water bath is raised to 38 ⁰ C and maintained at 38°C for 2 minutes. The curd-whey mixture is stirred slowly for 10 min to break up larger curds. The pH of whey is measured regularly (every ¹ A hr) until pH reaches 6.2-6.3 (approx. 90 min.). This is followed by draining off approximately 80% of whey, breaking up curd and returning the curd to water bath. Curd is cut further; about 1/3 of the whey is drained

off and pH is measured every ¹ A hr until pH reaches 5.3. The beaker is then removed from water bath, and all whey is drained off. Curd is transferred into sterilised cheesecloth, and excess moisture is removed by squeezing. Squeezed curd is placed into sterilised centrifuge bottles and weighed. Food grade salt is then added to curd at rate of 1.8% curd salt and mixed in thoroughly manually. Curds are centrifuged at 22°C in swing bucket centrifuge for 60 minutes (1550 g). The cheese curd is then vacuum-packed in oxygen non-permeable plastic bags and stored at 2O ⁰ C for 3 weeks. Samples are taken at day zero and day 21 for microbiological analysis.

As mentioned in Example 3, the protective yeast W. saturnus subsp. saturnus B9043 is not expected to grow in the cheese, whereas the potential spoilage yeasts S. cerevisiae B9030 and K. marxianus B9052 are expected to grow in the cheese.

As shown in Figures 1 and 2, both spoilage yeasts S. cerevisiae B9030 and K. marxianus B9052 grow significantly in the cheese in the absence of the protective yeast W. saturnus subsp. saturnus B9043, compared with the control. The cell count declines when the protective yeast W. saturnus subsp. saturnus B9043 is used.

EXAMPLE 6

Inhibition of growth of potential spoilage moulds on cheese This example is designed to demonstrate the ability of protective yeasts to prevent or minimize mould growth on cheese blocks. A 10mm diameter well cutter is used to cut 36 wells on a cheese block (500 g size) with 5mm depth as indicated in Scheme I. 300μl of the protective yeast (Williopsis saturnus subsp. saturnus B9043 or Debaryomyces hansenii B9010) is inoculated into 18 wells on one half of the same cheese block (see the diagram). The cheese block covered with tinfoil is left to air-dry on the bench for around 2 hour. 200μl of protective yeast is then drawn off from each well. Mould spore suspension is diluted with sterile deionised water to give spore concentrations from 10 ⁸ -10 ² spore/ml, which is used to inoculate each well according to the experimental design. lOOμl of mould spore suspension is inoculated into wells on both sides of the same cheese block. The cheese block is then covered with ethanol soaked tissue and tinfoil to prevent contamination and drying out during incubation. The cheese block is incubated at 2O ⁰ C for 7 days. Observations are made daily for mould growth (colony formation).

Mould concentration

10 ⁸ 10 ⁵ 10 ² spore/ml 10 ⁸ 10 ⁵ 10 ² spore/ml

+ Protective yeast — Protective yeast

Scheme I - Wells cut in cheese block.

As indicated in Tables 17-18, there is delayed or reduced mould growth during the incubation period in the cheese inoculated with potential spoilage moulds and the protective yeast W. saturnus subsp. saturnus B9043, compared with the control (no added protective yeast). W. saturnus subsp. saturnus B9043 is effective against growth of mould species of Aspergillus and Cladosporium.

As indicated in Tables 19-23, there is no or delayed (or reduced) mould growth during the incubation period in the cheese inoculated with potential spoilage moulds and the protective yeast D. hasenii B9010, compared with the control (no added protective yeast). D. hasenii B9010 is effective against growth of mould species of Aspergillus, Byssochlamys, Cladosporium and Penicillium.

Table 17 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Aspergillus sp. on cheese incubated at 20°C ^a

Potential spoilage mould Aspergillus sp.

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9043 + B9043 - B9043 + B9043 - B9043 + B9043

5 0/6 0/6 0/6 0/6 2/6 0/6

7 0/6 0/6 0/6 0/6 2/6 0/6

9 0/6 0/6 3/6 0/6 3/6 0/6

12 . 4/6 2/6 3/6 2/6 6/6 4/6

14 4/6 2/6 4/6 2/6 6/6 4/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 18 Inhibition of protective yeast Williopsis saturnus subsp. saturnus B9043 against mould Cladosporium sp. on cheese incubated at 20°C ^a

Potential spoilage mould Cladosporium sp.

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9043 + B9043 - B9043 + B9043 - B9043 + B9043

2 0/6 0/6 0/6 0/6 0/6 0/6

5 0/6 0/6 0/6 0/6 4/6 0/6

6 0/6 0/6 0/6 0/6 4/6 0/6

9 0/6 0/6 0/6 0/6 4/6 0/6

12 0/6 0/6 0/6 0/6 4/6 0/6

13 0/6 0/6 0/6 0/6 4/6 0/6

14 0/6 0/6 0/6 0/6 4/6 1/6

^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 19 Inhibition of protective yeast Debaryomyces hansenii B9010 against mould Aspergillus sp. on cheese incubated at 20°C ^a

Potential spoilage mould Aspergillus

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

5 0/6 0/6 0/6 0/6 0/6 0/6

7 0/6 0/6 0/6 0/6 3/6 0/6

8 0/6 0/6 1/6 0/6 3/6 0/6

9 0/6 0/6 1/6 0/6 3/6 0/6

14 1/6 0/6 1/6 0/6 3/6 0/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 20 Inhibition of protective yeast Debaryomyces hansenii B9010 against mould Penicillium candidum on cheese incubated at 20°C ^a

Potential spoilage Mould Penicillium candidum

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

5 0/6 0/6 0/6 0/6 0/6 0/6

7 0/6 0/6 6/6 0/6 1/6 1/6

8 1/6 0/6 616 0/6 3/6 1/6

9 1/6 0/6 6/6 0/6 4/6 1/6

14 1/6 1/6 6/6 1/6 4/6 3/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 21 Inhibition of protective yeast Debaryomyces hansenii B9010 against mould Penicillium roqueforti on cheese incubated at 20°C ^a

Potential spoilage mould Penicillium roqueforti

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

5 0/6 0/6 0/6 0/6 0/6 0/6

7 0/6 0/6 2/6 0/6 2/6 0/6

8 0/6 0/6 5/6 0/6 2/6 0/6

9 1/6 0/6 5/6 0/6 3/6 0/6

14 1/6 0/6 5/6 0/6 3/6 0/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 22 Inhibition of protective yeast Debaryomyces hansenii B9010 against mould Byssochlamys nivea on cheese incubated at 20°C ^a

Potential spoilage Mould Byssochlamys nivea

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

5 0/6 0/6 0/6 0/6 0/6 0/6

7 0/6 0/6 0/6 0/6 1/6 0/6

8 0/6 0/6 0/6 0/6 3/6 1/6

9 0/6 0/6 0/6 0/6 3/6 1/6

14 0/6 0/6 0/6 0/6 3/6 1/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

Table 23 Inhibition of protective yeast Debaryomyces hansenii B9010 against mould Cladosporium on cheese incubated at 20°C ^a

Potential spoilage Mould Cladosporium sp.

Concentration (spores/per ml) 10 ² 10 ⁵ 10 ⁸

Day - B9010 + B9010 - B9010 + B9010 - B9010 + B9010

1 0/6 0/6 0/6 0/6 0/6 0/6

4 0/6 0/6 0/6 0/6 2/6 0/6

5 0/6 0/6 0/6 0/6 3/6 0/6

8 0/6 0/6 0/6 0/6 3/6 0/6

11 0/6 0/6 0/6 0/6 3/6 0/6

12 0/6 0/6 0/6 0/6 4/6 0/6

14 4/6 0/6 2/6 0/6 4/6 0/6 ^a The values given indicate the number of wells with mould colonies out of a total of 6 inoculated wells.

EXAMPLES 7-17

Stabilising Lactic Acid Bacteria and Probiotics The following examples serve to illustrate preferred practices of the present invention relating to stabilising lactic acid bacteria and probiotics and are again illustrative only and do not limit the present invention, hi all of these examples, the pH did not increase. The pH dropped to as low as about 3.4 in the case of 5% milk solids and to about 3.8 in the case of 20% milk solids.

EXAMPLE 7

Fermented milks (yoghurt and/or drinking yoghurt) were prepared as follows.

Whole milk powder (20% w/v) is constituted in water at 5O ⁰ C, followed by sterilisation at 9O ⁰ C for 10 minutes. After cooling to 3O ⁰ C, the reconstituted whole milk is inoculated with approximately 0.5-1 % v/v yoghurt starter culture MY-900 (Danisco,), with or without 0.5%- 1% v/v other lactic acid bacteria or probiotics, and 1-2 % v/v selected yeast culture. In some fermented milks, only lactic acid bacteria or probiotics were inoculated instead of yoghurt cultures. The inoculated milk is then dispensed in 50 mL aliquots into sterile plastic containers,

which are incubated at 3O ⁰ C for an extended period of time. Samples are taken at weekly intervals for microbiological testing (lactic acid bacteria, yeasts, moulds and pathogens).

Yeasts and lactic acid bacteria are cultured in standard microbiological media. Yeasts are pre- grown at 3O ⁰ C for 24 to 48 hours in a medium (pH 5.0) of 2% w/v glucose, 0.25% w/v each of yeast extract, malt extract and peptone). Streptococcus thermophilics (Fonterra) strains are pre- cultured in Ml 7 (Gibco) broth or plated on Ml 7 agar plates, and are incubated at 37 ⁰ C for 24 to 48 hours. Other lactic acid bacteria are cultured in MRS (Gibco) broth or plated on MRS agar plates, and are incubated at 3O ⁰ C for 24 to 48 hours. Alternatively, yoghurt bacteria and other lactic acid bacteria can be grown in 10% w/v reconstituted skim milk. In the case of plating lactic acid bacteria in fermented milks, an appropriate amount of natamycin as per manufacturer's instruction (Danisco) is added to M17 and MRS media to inhibit yeasts. Yeasts and moulds in fermented milks are plated on oxytetracycline-glucose yeast extract (Oxoid) agar with 0.1 g/L Chloramphenicol added and plates are incubated at 25 ⁰ C for 2-4 days.

Bifidobacteria are pre-cultured in MRS (Gibco) broth supplemented with 0.5 g/L of L- cysteine.HCl and are incubated at 37 ⁰ C for 24 to 48 hours. Bifidobacteria are enumerated by plating out on MRS agar supplemented with natamycin (see above), 0.3 g/L of L-cysteine.HCl and 0.5 mg /L of dicloxacillin. Plates are incubated at 37 ⁰ C for 5 days under anaerobic conditions. Yeasts and moulds are enumerated as described above.

EXAMPLE 8

Survival of yoghurt bacteria in set yoghurt with 20% w/v milk solids in the presence of added yeast extract (BBL) (1% w/v) was investigated during storage at 3O ⁰ C. The yoghurt bacteria were Streptococcus thermophiϊus and Lactobacillus bulgaricus (MY-900, Danisco). The results showed that the yeast extract increased survival of the yoghurt bacteria by Weeks 2-5 (Figure 3).

EXAMPLE 9

Survival of yoghurt bacteria in set yoghurt with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cfu/mL)) was investigated during storage at 3O ⁰ C.

The yoghurt bacteria were Streptococcus thermophilus and Lactobacillus bulgaricus (MY-900, Danisco).

Live yeasts added are:

B9030 = Saccharomyces cerevisiae (Laffort Zymaflore VLl) B9035 = Saccharomyces bayanus (Lalvin CVC-NF74, Lallemand, Canada) B9028 = Saccharomyces cerevisiae (Lalvin R2, Lallemand, Canada) B9043 - Williopsis saturnus (CBS 254, CBS Culture Collections, The Netherlands) B9010 = Debaryomyces hasenii (Fonterra, New Zealand) B9014 = Yarrowia lipolytica (Fonterra, New Zealand)

B9006 = Candida kejyr (NCYC 143, National Collection of Yeast Cultures, IFR, Norwich, UK) B9052 = Kluyveromyces marxianus (ATCC 8640)

At weeks 3-7, the yoghurt bacteria showed increased survival with all the live yeast additions relative to the control without added yeast. The effect was most pronounced for the first four yeasts in the above list where the number of colony forming units per gram did not fall below 10 ⁵ (Figure 4). The yeasts B9014, B9006 and B9052 were less effective than the other yeasts. The sample with the yeast B9014 gave an undesirable lipolytic off-odour relative to the control.

EXAMPLE 10

Survival of lactic acid bacteria in fermented milk with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cell counts/mL) was investigated during storage at 3O ⁰ C. The yeast added was Williopsis saturnus; Yoghurt bacteria were not added. The lactic acid bacteria used were Lactobacillus rhamnosus DR20 (deposited at AGAL on 18 August 1997 and given number NM97/09514), Lactobacillus reuteri (DSM 20016), Lactobacillus acidophilus (ATCC 4356), Streptococcus thermophilus (Fonterra), Lactobacillus bulgaricus (Fonterra), Lactobacillus johsonii. Improved survival was demonstrated over the period 1-9 weeks for the first three of these bacteria. Improved survival was not as significant for the final three bacteria over the period 1-3 weeks. The results are shown in Figure 5.

EXAMPLE 11

Survival of Lactobacillus rhamnosus DR20 in drinking yoghurt with 5% w/v milk solids in the presence of added live yeast (~10 cell count/mL) was investigated during storage at 3O ⁰ C.

Yoghurt bacteria were added along with DR20. The yeast added was Williopsis saturnus

(B9043). The presence of the live yeast allowed the DR20 strain to survive at numbers greater than 10 ⁵ cfu/g for 12 weeks (Figure 6). In contrast, without addition of the yeast the DR20 number had fallen well below 10 ⁵ by 6 weeks. DR20 was deposited at AGAL on 18 August 1997 and given number NM97/09514. This organism is commercially available.

EXAMPLE 12

Survival of Lactobacillus rhamnosus DR20 in fermented milk with 5% w/v milk solids in the presence of added live yeast (~10 ⁶ cell count/mL) was investigated during storage at 3O ⁰ C. Yoghurt bacteria were not added. The yeast added was Williopsis saturnus B9043. DR20 survival was greatly enhanced (see Figure 7). Strain DR20 was deposited at AGAL on 18 August 1997 and given number NM97/09514. This organism is commercially available.

EXAMPLE 13 Survival of Lactobacillus rhamnosus DR20 in fermented milk with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cfu/mL) was investigated during storage at 3O ⁰ C. Yoghurt bacteria were not added. Strain DR20 showed substantially enhanced survival with all the live yeast additions relative to the control without added yeast (Figure 8). The yeast B9014 was less effective than the other yeasts. The sample with the yeast B9014 gave an undesirable lipolytic off-odour relative to the control.

EXAMPLE 14

Survival of Lactobacillus rhamnosus DR20 in stirred yoghurt with 20% w/v milk solids in the presence of added live yeast (~10 ⁶ cell count/mL) was investigated during storage at different temperatures. Yoghurt bacteria were added along with DR20. B9043 = yeast added, Williopsis saturnus. Significantly enhanced survival of DR20 was observed at temperatures between 2O ⁰ C and 3O ⁰ C in the presence of added live yeast B9043 (Figure 9).

EXAMPLE 15 Survival of Lactobacillus rhamnosus DR20 in fermented milk with 20% w/v milk solids in the presence of added dead yeast (equivalent to ~10 ⁶ live cell counts/g) was investigated during storage at 3O ⁰ C. Yoghurt bacteria were not added. The yeast B9043 was autoclaved before being added to milk. Strain DR20 showed much higher survival rate with the addition of autoclaved yeast B9043 relative to the control without the added dead yeast (Figure 10).

EXAMPLE 16

Survival of Bifidobacterium lactis DRlO in set fermented milk with 20% w/v milk solids in the presence of added live yeast Williopsis saturnus B9043 was investigated during storage at 3O ⁰ C. Yoghurt bacteria were not added. Significantly enhanced survival of DRl 0 was shown from the

third week onwards in the presence of the added live yeast B 9043, compared with the control without the yeast B9043 (Figure 11).

EXAMPLE 17 Survival of Bifidobacterium lactis DRlO in set yoghurt with 20% w/v milk solids in the presence of added live yeast Williopsis saturnus B9043 was investigated during storage at different temperatures. Yoghurt bacteria were added along with DRlO. Improved survival of strain DRlO was observed at both 1O ⁰ C and ambient temperatures from around the 10 ^th week in the presence of added live yeast B9043, compared with the control without the yeast B9043 (Figure 12).

It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention. For example, the organisms used, the temperatures, and the milk solids contents may all be varied.