[go: up one dir, main page]

WO2007055604A1 - Produit laitier et procédé associé - Google Patents

Produit laitier et procédé associé Download PDF

Info

Publication number
WO2007055604A1
WO2007055604A1 PCT/NZ2006/000294 NZ2006000294W WO2007055604A1 WO 2007055604 A1 WO2007055604 A1 WO 2007055604A1 NZ 2006000294 W NZ2006000294 W NZ 2006000294W WO 2007055604 A1 WO2007055604 A1 WO 2007055604A1
Authority
WO
WIPO (PCT)
Prior art keywords
yeast
yeasts
lactic acid
milk
product
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/NZ2006/000294
Other languages
English (en)
Inventor
Marlene Tsao
Shao Quan Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fonterra Cooperative Group Ltd
Original Assignee
Fonterra Cooperative Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fonterra Cooperative Group Ltd filed Critical Fonterra Cooperative Group Ltd
Publication of WO2007055604A1 publication Critical patent/WO2007055604A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0325—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using yeasts, alone or in combination with lactic acid bacteria or with fungi, without using other bacteria

Definitions

  • the invention relates to the preparation of dairy products and to extending the shelf life of dairy products.
  • 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.
  • 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.
  • yeasts can cause food spoilage
  • yeasts especially Saccharomyces cerevisiae
  • yeasts 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)
  • 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).
  • yeasts as biocontrol agents on fruits to control postharvest diseases
  • antagonistic yeasts have been used to control undesirable yeasts in brewing (Vaughan A, O'Sullivan T and van Sinderen D, 111: 355-371, 2005).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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
  • 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
  • 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
  • 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.
  • both processes entail an active role of yeasts (e.g. fermentation of lactose and/or galactose) during fermentation.
  • neither processes demonstrated the stabilising effect of yeasts on cell populations of lactic acid bacteria and probiotics 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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).
  • 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.
  • the yeast-containing mixture may be further processed.
  • 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.
  • 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.
  • 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.
  • 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.
  • lactose non-fermenting and galactose non-fermenting yeasts are preferred, or non-fermentative yeasts are preferred.
  • 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).
  • 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.
  • the yeast extends the viability of lactic acid bacteria and probiotics in food and beverages for longer periods of time under ambient conditions.
  • 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.
  • the starting material is milk or skim milk and the product is a yoghurt, or fermented milk drink or a cheese.
  • 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.
  • 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.
  • 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.
  • lactose non-fermenting and galactose non-fermenting yeasts are preferred, or non- fermentative yeasts are preferred.
  • 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 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.
  • 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.
  • 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).
  • 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).
  • yeast extracts see Examples 8 and 15.
  • dead yeasts or yeast extracts these need not necessarily be derived from yeasts that do not ferment lactose or galactose.
  • flavourings, colourings and texturisers commonly used in food and beverage manufacturing processes may be employed in the present invention.
  • 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.
  • 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.
  • 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.
  • a plurality of determinations of the numbers is made at different times.
  • 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,
  • 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).
  • B9010 Debaryomyces hasenii (Fonterra)
  • a B9014 Yarrowia lipolytica (Fonterra) o
  • B9052 Kluyveromyces marxianus (ATCC 8640) x
  • 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
  • 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.
  • 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.
  • B9043 Williopsis saturnus (CBS 254)
  • B9010 Debaryomyces hasenii (Fonterra)
  • B9042 Pichia membranifaciens
  • B9050 Kloekera apiculata
  • 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
  • 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
  • CBS 254 Williopsis saturnus
  • 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 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.
  • 10 ⁇ l 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 50 ⁇ l 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.
  • 10 ⁇ l 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 ⁇ m membrane filter. 300 ⁇ l 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.
  • Williopsis saturnus subsp. saturnus and Debaryomyces hansenii show 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.
  • Sac. bayanus B9033 (Lalvin Agglo 016) — —
  • Sac. cerevisiae B9034 (Lalvin 71B) — —
  • Sac. cerevisiae B9029 (Lalvin L2056) — —
  • Sac. cerevisiae B9037 (Uvaferm C52) — —
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • This method is designed to produce experimental cheese on a lab scale.
  • 1.2L of pasteurised milk pH about 6.6-6.7
  • 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.
  • 2.0% v/v cheese starter Stringeptococcus thermophilus, Fonterra
  • 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). 120 ⁇ l rennet is then added and mixed into the milk.
  • Curd 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.
  • 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.
  • 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.
  • 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. lOO ⁇ l 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
  • 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.
  • 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
  • Stabilising Lactic Acid Bacteria and Probiotics 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.
  • 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.
  • MRS Gibco
  • yoghurt bacteria and other lactic acid bacteria can be grown in 10% w/v reconstituted skim milk.
  • 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.
  • Oxoid oxytetracycline-glucose yeast extract
  • 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.
  • 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)
  • 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.
  • lactic acid bacteria in fermented milk with 20% w/v milk solids in the presence of added live yeast 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.
  • Yoghurt bacteria were added along with DR20.
  • the yeast added was Williopsis saturnus
  • 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 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).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Microbiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Dairy Products (AREA)

Abstract

L'invention concerne un produit laitier préparé par addition d'une levure ne fermentant pas le lactose et ne fermentant pas le galactose ou d'une levure non fermentaire à une matière de départ à base de lait ou à un produit laitier en quantité suffisante pour prolonger la durée de vie du mélange résultant ou des produits préparés à partir de celui-ci, et, si nécessaire, par traitement supplémentaire du mélange en vue de l'obtention d'un produit laitier supplémentaire, ladite levure étant faiblement protéolytique ou non protéolytique et faiblement lipolytique ou non lipolytique. L'invention concerne également un procédé destiné à préparer un produit laitier présentant un pH inférieur à 5,5 et une teneur en eau d'au moins 30 % (poids/volume), et consistant à additionner un ou plusieurs types de bactéries ou de probiotiques lactiques à une matière de départ à base de lait, une levure vivante stabilisatrice ne fermentant pas le lactose et ne fermentant pas le galactose ou une levure morte stabilisatrice ou un extrait de levure étant additionné(e) avant, pendant ou après l'étape d'addition de bactéries ou de probiotiques, ce qui permet de prolonger la survie des bactéries ou des probiotiques lactiques à des numérations supérieures ou égales à 105 par gramme ou millilitre, la levure ou l'extrait étant faiblement protéolytique ou non protéolytique et faiblement lipolytique ou non lipolytique.
PCT/NZ2006/000294 2005-11-11 2006-11-13 Produit laitier et procédé associé Ceased WO2007055604A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ54351105 2005-11-11
NZ543511 2005-11-11

Publications (1)

Publication Number Publication Date
WO2007055604A1 true WO2007055604A1 (fr) 2007-05-18

Family

ID=38023505

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ2006/000294 Ceased WO2007055604A1 (fr) 2005-11-11 2006-11-13 Produit laitier et procédé associé

Country Status (2)

Country Link
TW (1) TW200733887A (fr)
WO (1) WO2007055604A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173223B (zh) * 2007-10-24 2010-05-19 华中农业大学 一种作为微生物发酵剂的菌株,包含该菌株的复合发酵剂及应用
ITMI20122267A1 (it) * 2012-12-28 2014-06-29 Granarolo S P A Miscela aromatizzante per la produzione di mozzarelle.
WO2017108125A1 (fr) * 2015-12-23 2017-06-29 Compagnie Gervais Danone Procédé de désacidification, de neutralisation ou d'alcalinisation d'une composition laitière acide
JP2017143799A (ja) * 2016-02-18 2017-08-24 キユーピー株式会社 発酵乳飲食品
CN114097890A (zh) * 2021-11-26 2022-03-01 沈阳农业大学 一种酯香霉菌奶酪及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3995066A (en) * 1974-03-07 1976-11-30 Lever Brothers Company Yeast containing food emulsions
CA2086882A1 (fr) * 1992-01-23 1993-07-24 Rhodia Inc. Culture de levain a yogourt et methode

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3995066A (en) * 1974-03-07 1976-11-30 Lever Brothers Company Yeast containing food emulsions
CA2086882A1 (fr) * 1992-01-23 1993-07-24 Rhodia Inc. Culture de levain a yogourt et methode

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ADDIS E. ET AL.: "The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses", INT. J. FOOD MICROBIOL., vol. 69, 2001, pages 25 - 36, XP003013105 *
COSENTINO S. ET AL.: "Yeasts associated with Sardinian ewe's dairy products", INT. J. FOOD MICROBIOL., vol. 69, 2001, pages 53 - 58, XP003013106 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173223B (zh) * 2007-10-24 2010-05-19 华中农业大学 一种作为微生物发酵剂的菌株,包含该菌株的复合发酵剂及应用
ITMI20122267A1 (it) * 2012-12-28 2014-06-29 Granarolo S P A Miscela aromatizzante per la produzione di mozzarelle.
WO2014102711A1 (fr) 2012-12-28 2014-07-03 Granarolo S.P.A. Mélange d'aromatisants pour fabriquer de la mozzarella
WO2017108125A1 (fr) * 2015-12-23 2017-06-29 Compagnie Gervais Danone Procédé de désacidification, de neutralisation ou d'alcalinisation d'une composition laitière acide
WO2017108899A1 (fr) * 2015-12-23 2017-06-29 Compagnie Gervais Danone Procédé de désacidification, de neutralisation ou d'alcalinisation d'une composition laitière acide
JP2017143799A (ja) * 2016-02-18 2017-08-24 キユーピー株式会社 発酵乳飲食品
CN114097890A (zh) * 2021-11-26 2022-03-01 沈阳农业大学 一种酯香霉菌奶酪及其制备方法

Also Published As

Publication number Publication date
TW200733887A (en) 2007-09-16

Similar Documents

Publication Publication Date Title
EP2852691B1 (fr) Souches de lactobacillus aux propriétés antifongiques
Ferreira et al. Yeasts as adjunct starters in matured Cheddar cheese
Viljoen The interaction between yeasts and bacteria in dairy environments
Wszolek et al. Production of kefir, koumiss and other related products
Sarkar Biotechnological innovations in kefir production: a review
Liu et al. Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions
Narvhus et al. The role of interaction between yeasts and lactic acid bacteria in African fermented milks: a review
KR101947110B1 (ko) 락토바실러스 람노서스 균주를 이용한 바이오프로텍션
EP2693885B1 (fr) Effet antimicrobien synergetique
EP3279312B1 (fr) Bioprotection de produits laitiers
Awasti et al. The role of yeast and molds in dairy industry: An update
US7854950B2 (en) Pressure treating food to reduce spoilage
KR20180101636A (ko) 항진균 활성을 갖는 락토바실러스 퍼멘텀 박테리아
Fguiri et al. Isolation and characterization of lactic acid bacteria strains from raw camel milk for potential use in the production of yogurt
Noutsopoulos et al. Growth, nisA gene expression, and in situ activity of novel Lactococcus lactis subsp. cremoris costarter culture in commercial hard cheese production
Matejčeková et al. Evaluation of the potential of amaranth flour for lactic acid fermentation
Grattepanche et al. Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model
WO2007055604A1 (fr) Produit laitier et procédé associé
Vasiljevic et al. Cultured milk and yogurt
Liu et al. Enhancing stability of lactic acid bacteria and probiotics by Williopsis saturnus var. saturnus in fermented milks
Pappa et al. Chemical, microbiological and sensory characteristics of ‘Tsalafouti’traditional Greek dairy product
EP1224867A1 (fr) Aliments fermentes contenant bifidobacterium
Tharmaraj et al. Antimicrobial effects of probiotic bacteria against selected species of yeasts and moulds in cheese-based dips
WO2023073187A1 (fr) Bioprotection de produits laitiers
Abou Ayana et al. Research article using of dairy propionibacteria as bio-preservative in Kareish cheese

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06824385

Country of ref document: EP

Kind code of ref document: A1