Strains of propionibacterium

The present invention relates to novel strains of Propionibacterium as well as preparations and compositions, such as fermentation broths, protective cultures, final food or feed product with Propionibacterium alone or in combination with bacteria of the genus Lactobacillus. The present invention further relates to methods for the controlling of growth of a contaminant, such as a bacteria, yeast or mold by using these novel strains of Propionibacterium.

FIELD OF THE INVENTION

The present invention relates to novel strains ofPropionibacteriumas well as preparations and compositions, such as fermentation broths, protective cultures, final food or feed products withPropionibacteriumalone or in combination with bacteria of the genusLactobacillus. The present invention further relates to methods for the controlling of growth of a contaminant, such as a bacteria, yeast or mould by using these novel strains ofPropionibacterium.

BACKGROUND OF THE INVENTION

Yeasts and moulds play a major role in spoilage of different types of dairy products, like yogurt, sweetened and sour cream and fresh and ripened cheese types and thus can lead to high economic losses. Chemical preservatives like organic acids and their salts (e.g. sorbate and propionate) are used to preserve dairy products and protect and prolong the shelf life. Drawbacks in using chemical preservatives are the labelling requirements (often as E numbers) and potential adverse effects on the sensory properties of the foodstuff.

In 1993 Valio, a finish dairy company described in EP 0576780 the use of a protective culture to inhibit fungal growth. The culture is a mixture of aLactobacillus rhamnosusstrain (deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) with the deposition number DSM 7061) and aPropionibacterium freudenreichiisubsp.shermaniistrain (DSM 7067). The specific characteristic of this co-culture was that the combination of theLactobacillusand thePropionibacteriumhad better antifungal activity than theLactobacillus rhamnosusalone. The culture was commercialized by Wiesby under the name “Bio Profit” and renamed into HOLDBAC™ YM-B by Danisco.

EP 0576780 relates to a novel microorganism strainLactobacillus caseissp.rhamnosusLC-705, DSM 7061, having a yeast and mould controlling effect, to bacterial preparation comprising this strain, alone or in combination with a bacterium of the genusPropionibacteriumor another strain of the bacteriumLactobacillus caseiand/or with conventional agents used for yeast and mould control.

EP1308506 (Swiss Federal Institute of Technology (ETH) in Zürich, Switzerland) also describes the combination oflactobacilliandpropionibacteria. ALactobacillus paracaseisubsp.paracaseistrain (DSM 14514) from the ETH was commercialized as a blend withPropionibacterium freudenreichiisubsp.shermaniiDSM 7067 from Valio under the name HOLDBAC™ YM-C.

Patent WO 03/040349 relates to a mixture of bacteria being a non starter culture which is free from metabolites and comprising the speciesPropionibacterium jenseniiand a second bacterium selected from the genusLactobacillus.

U.S. Pat. No. 4,728,516 relates to a method of inhibiting mould and psychrotrophic bacteria in creamed Cottage cheese using a mixture ofStreptococcus lactissubspeciesdiacetylactisandPropionibacterium shermaniiin the cream dressing or creamed Cottage cheese at refrigeration temperatures.

DE202006014937 relates to butterfat mixture with reduced fat content, comprises a butter and bio-protective cultures from lactic acid bacteria in the absence of preservatives.

U.S. Pat. No. 5,096,718A relates to metabolite material ofpropionibacteriaas well as uses thereof for inhibiting the growth of gram negative psychrotrophic bacteria, yeast, mould, gram positive bacteria, orListeriain food products.

WO9816124A1 relates to a food or beverage or animal feed composition which includes an antimycotic effective amount ofpropionibacteriametabolites.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide improved strains ofPropionibacteriumas well as preparations and compositions, such as fermentation broths, protective cultures, final food or feed product comprising such improvedPropionibacteriumalone or in combination with bacteria of the genusLactobacillus.

It is an object of embodiments of the invention to provide methods for the controlling of growth of a contaminant, such as a bacteria, yeast or mould by using these novel strains ofPropionibacterium.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that novel strains ofPropionibacteriumhave improved properties in terms of being able to control of growth of a contaminant, such as a bacteria, yeast or mould.

So, in a first aspect the present invention relates to a bacteria of the genusPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849; or functional equivalents thereof.

In a second aspect the present invention relates to a bacterial preparation, characterized in that it comprises aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, alone or in combination with a bacterium of the genusLactobacillus, with any other strain of the bacteriumPropionibacterium, or with both.

In a third aspect the present invention relates to the use of bacteria of the genusPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, in the preparation of a final food or feed product.

In a further aspect the present invention relates to a composition, such as a protective culture or a final food or feed product, comprising viable bacteria of the genusPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof.

In a further aspect the present invention relates to the use of a bacterial preparation comprising aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, to control the growth of a contaminant, such as a bacteria, yeast or mould.

In a further aspect the present invention relates to a method of controlling the growth of a contaminant, such as a bacteria, yeast or mould, characterized by using a bacterial preparation comprising aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, alone or in combination with a bacterium of the genusLactobacillus, with any other strain of the bacteriumPropionibacterium, or with both.

In a further aspect the present invention relates to a composition, such as a protective culture or a final food or feed product, comprising viable bacteria of the genusLactobacillusin combination with aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof.

In some embodiments the method further comprises as step of purifying and/or concentrating said bacteria of the genusLactobacillus.

In a further aspect the present invention relates to a method for controlling the growth of a contaminant, such as a bacteria, yeast or mould, in a composition, the method characterized by having in this composition the presence of a bacterial preparation according to the present invention.

In a further aspect the present invention relates to a method for storing a food or feed product, said food or feed product comprising viable bacteria of the genusLactobacillus, the method comprising a step of controlling the growth of a contaminant, such as a bacteria, yeast or mould, in this food or feed product by addition to said food or feed product of a bacteria according to the invention or a bacterial preparation according to the invention.

DETAILED DISCLOSURE OF THE INVENTION

The antifungal effects of the commercial available antifungal cultures HOLDBAC™ YM-B and HOLDBAC™ YM-C (DuPont, Denmark) has been demonstrated in various challenge studies in different types of fermented dairy products like yogurt, fresh cheese, sour cream and white brined cheese. Furthermore the application of HOLDBAC™ YM-B and HOLDBAC™ YM-C was evaluated in internal trials in Gouda-type cheese. The results demonstrated a delayed outgrowth of spoilage moulds on the cheeses prepared with either HOLDBAC™ YM-B or HOLDBAC™ YM-C compared to a reference without protective culture added. It is envisioned, that the mere presence of thePropionibacteriumstrain potentially may induce the formation of antifungal metabolites by theLactobacillusstrain. The inventors of the present invention have demonstrated that the major part of the antifungal activity is coming from theLactobacillusand combinations of livelactobacilliwithpropionibacteriathat express antifungal activity as well.

The present invention concerns improvedpropionibacteriahaving inhibition activity against yeast and moulds. The bacteria are suitable for the preservation of goods (e.g. food or feed).

As described in the examples, the strains were screened in an agar assay.

The strains may be used to develop antifungal cultures especially for dairy applications. Using antifungallactobacilli, antifungal cultures based on the co-culture concept of HOLDBAC™ YM would be possible, for use in, e.g., yoghurt (including drinking yoghurt), sour cream, semi-hard and hard cheese, cottage cheese, fresh cheese, cream cheese, and white-brined cheese.

Other food applications like e.g. sourdough may be a potential application as well as protection of feed products, such as e.g. silage.

Definitions

As used herein the term “viable” refers to bacterial cells with the potential to have an active metabolism, to survive, grow, or multiply.

As used herein the term “protective culture ofLactobacillus” refers to composition comprising liveLactobacillus(pure cultures or culture concentrates), which is not a final food product suitable for consumption, but which is added to food products with the aim of reducing risks by pathogenic or toxinogenic microorganisms. Included within this definition are compositions, such as fermentation media and concentrated preparations thereof, whereinLactobacillusis grown in a suitable medium.

As used herein the term “protective culture ofPropionibacterium” refers to composition comprising livePropionibacteria(pure cultures or culture concentrates), which is not a final food product suitable for consumption, but which is added to food products with the aim of reducing risks by pathogenic or toxinogenic microorganisms. Included within this definition are compositions, such as fermentation media and concentrated preparations thereof, whereinPropionibacteriumis grown in a suitable medium.

The European Food and Feed Cultures Association (EFFCA) has characterized protective cultures as follows: The term “Protective Cultures” has been applied to microbial food cultures (MFC) exhibiting a metabolic activity contributing to inhibit or control the growth of undesired microorganisms in food. These undesired microorganisms could be pathogenic or toxinogenic bacteria and fungi but spoilage causing species may also be included.

Protective cultures are considered as an integral part of starter cultures, which are the traditional tools of food technology used to produce fermented food such as cheese, yoghurt, certain sausages, wine etc. It is a general property of fermented foods that these possess a longer shelf life than the non-fermented raw materials (for instance cheese, has a much longer shelf-life than milk). This property is the result of the active metabolism of the fermenting culture, conducting its actions through a complex system of competition for nutrients and binding sites and by production of inhibitory metabolites like organic acids, hydrogen peroxide, diacetyl, reuterin and bacteriocins.

Depending on the specific cultures used the cultures commonly form numerous properties that are of sensory and nutritive value to the food product, too. In this way the same starter culture species used in fermentation processes have also been applied to food in order to make use of the “bioprotective” potential with or without sensory impact. For these starter cultures the term protective culture, has been applied.

Their usage is not limited to “classic” fermented foods but also plays an important role when their metabolic activities take place in food with a neutral pH and high water activity, which are subject to increased risk of growth of food pathogens. The application of “protective cultures” constitutes an additional measure to improve food hygiene and should not permit a neglecting of any measure of good manufacturing practice ensuring the high standard of food safety.

Protective cultures are an integral part of starter cultures rather than additives. It is clear that these cultures develop their protective and beneficial potential, like all starter cultures, as a result of their metabolic activity in or on the food. (EFFCA, December 2011).

The protective culture is added together with a starter culture to the food or feed matrix before the fermentation of the food or feed. Thus the protective culture undergoes the fermentation step and is able to grow and/or to be metabolic active. The starter culture is needed to produce fermented foods like yogurt, cheese and sour cream and contributes to the desired product changes in taste, texture and flavour development. In addition to this, protective cultures are added to the food to limit the growth of pathogenic or spoilage bacteria and thus reduce the risk of food poisonings and protect the shelf-life.

As used herein the term “final food or feed product” refers to a composition suitable for consumption, such as for human or animal consumption in the form of a food or feeding stuff.

The term “contaminant” as used herein refers to any unwanted and unintentional growth of any microorganism, such as bacteria, fungi, such as yeast or a mould. In some instances the contaminant may cause disease. However, often the contaminant just degrade and deteriorate the product wherein it is found and/or give an unpleasant and unwanted taste or mouth feel.

In some embodiments the contaminant is a bacteria different from the specificLactobacillusstrain used in the methods according to the invention, selected from the list consisting ofListeria monocytogenes, Pseudomonassp.,Staphylococcus aureus, Bacillussp.,Lactobacillussp.,Leuconostocsp.,Clostridiumsp.,EnterobacteriaceaelikeCitrobactersp.,Enterobactersp.,Escherichiasp.,Klebsiellasp.,Salmonellasp.

As used herein the term “any other strain of the bacteriumPropionibacterium” refers to any strain of the genusPropionibacteriumincluding the speciesPropionibacterium acidifaciens, Propionibacterium acidipropionici, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium cyclohexanicum, Propionibacterium freudenreichiisubsp.freudenreichii, Propionibacterium freudenreichiisubsp.shermanii, Propionibacterium granulosum, Propionibacterium jensenii, Propionibacterium microaerophilum, Propionibacterium propionicum, andPropionibacterium thoenii, as well as variants thereof. The term is intended to encompass specific strains described in the present disclosure and in any of EP0576780, EP1308506, WO03/040349, and US4728516, includingPropionibacterium freudenreichiisubsp.shermaniiDSM 7067.

Specific Embodiments of the Invention

In some embodiments the bacterial preparation according to the present invention is a cell suspension in fermentation broth, alone or in combination with a bacterium of the genusLactobacillusor another strain of thePropionibacterium.

In some embodiments the bacterial preparation according to the present invention is concentrated.

In some embodiments the bacterial preparation according to the present invention is freeze-dried or frozen.

In some embodiments the bacterial preparation according to the present invention is characterized in that it additionally comprises conventional agents used for yeast and mould control, such as propionate or phenylalanine.

In some embodiments the composition according to the present invention is a protective culture ofLactobacillusand said viable bacteria of the genusLactobacillusis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a protective culture ofPropionibacteriumand said viable bacteria of the genusPropionibacteriumis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×10 CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a final food or feed product and said viable bacteria of the genusLactobacillusis present in an amount of at least 105CFU/g or ml, such as at least 5×105CFU/g or ml, such as at least 106CFU/g or ml, such as at least 5×106CFU/g or ml, such as at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 5×108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 5×109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a final food or feed product and said viable bacteria of the genusPropionibacteriumis present in an amount of at least 105CFU/g or ml, such as at least 5×105CFU/g or ml, such as at least 106CFU/g or ml, such as at least 5×106CFU/g or ml, such as at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 5×108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 5×109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×10 CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition or preparation according to the present invention is a protective culture ofLactobacillus.

In some embodiments the composition or preparation according to the present invention is a protective culture ofPropionibacterium.

In some embodiments the composition or preparation according to the present invention is a final food or feed product.

In some embodiments the composition according to the present invention is with or without cell remnants of said bacteria of the genusPropionibacterium.

In some embodiments the composition according to the present invention is a protective culture ofLactobacillusand said viable bacteria of the genusLactobacillusis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a protective culture ofPropionibacteriumand said viable bacteria of the genusPropionibacteriumis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a final product and said viable bacteria of the genusLactobacillusis present in an amount of at least 105CFU/g or ml, such as at least 5×105CFU/g or ml, such as at least 106CFU/g or ml, such as at least 5×106CFU/g or ml, such as at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 5×108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 5×109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a protective culture ofLactobacillusand said viable bacteria of the genusLactobacillusis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a protective culture ofPropionibacteriumand said viable bacteria of the genusPropionibacteriumis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition according to the present invention is a final product and said viable bacteria of the genusLactobacillusis present in an amount of at least 105CFU/g or ml, such as at least 5×105CFU/g or ml, such as at least 106CFU/g or ml, such as at least 5×106CFU/g or ml, such as at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 5×108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 5×109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

In some embodiments the composition or preparation according to the present invention is used for the preparation of a food product of feed product, such as a milk product. In some embodiments the food product is selected from the group consisting of dairy products, yoghurt, drinking yogurt, cheese, such as fresh cheese, cream cheese, cottage cheese, semi-soft and soft cheese, semi-hard and hard cheese, white brine cheese, sour milk products, and sour cream, sauerkraut, pickles, beer, wine, cider, kimchi, cocoa, sour dough and other fermented foods, as well as animal feeds, such as silage.

In some embodiments according to the present invention, the bacteria of the genusLactobacillusis selected from the list consisting of

Numbered embodiments according to the invention:

2. A bacterial preparation, characterized in that it comprises aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, alone or in combination with a bacterium of the genusLactobacillus, with any other strain of the genusPropionibacterium, or with both.

3. A bacterial preparation according to embodiment 2, which is a cell suspension in fermentation broth, alone or in combination with a bacterium of the genusLactobacillus, with another strain of thePropionibacterium, or with both.

5. A bacterial preparation according to any one of embodiments 2-4, which preparation is concentrated.

6. A bacterial preparation according to any one of embodiments 2-4, which preparation is freeze-dried.

7. A bacterial preparation according to any one of embodiments 2-6, characterized in that it additionally comprises conventional agents used for yeast and mould control, such as propionate or phenylalanine.

8. Use of bacteria of the genusPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, in the preparation of a final food or feed product.

9. Use of a bacterial preparation comprising the aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, to control the growth of a contaminant, such as a bacteria, yeast or mould.

10. A method of controlling the growth of a contaminant, such as a bacteria, yeast or mould, characterized by using a bacterial preparation comprising aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof, alone or in combination with a bacterium of the genusLactobacillus, with another strain of the genusPropionibacterium, or with both.

11. A composition, such as a protective culture or a final food or feed product, comprising viable bacteria of the genusLactobacillusin combination with aPropionibacteriumselected from the list consisting of:a.Propionibacterium acidipropioniciDSM 25845;b.Propionibacterium freudenreichiisubsp.shermaniiDSM 25846;c.Propionibacterium freudenreichiiDSM 25847;d.Propionibacterium thoeniiDSM 25848; ande.Propionibacterium thoeniiDSM 25849;
or functional equivalents thereof.

12. The composition according to embodiment 11, wherein said composition is a protective culture ofLactobacillusand said viable bacteria of the genusLactobacillusis present in an amount of at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

13. The composition according to embodiment 11, wherein said composition is a final food or feed product and said viable bacteria of the genusLactobacillusis present in an amount of at least 105CFU/g or ml, such as at least 5×105CFU/g or ml, such as at least 106CFU/g or ml, such as at least 5×106CFU/g or ml, such as at least 107CFU/g or ml, such as at least 5×107CFU/g or ml, such as at least 108CFU/g or ml, such as at least 5×108CFU/g or ml, such as at least 109CFU/g or ml, such as at least 5×109CFU/g or ml, such as at least 1010CFU/g or ml, such as at least 5×1010CFU/g or ml, such as at least 1011CFU/g or ml, such as at least 5×1011CFU/g or ml, such as at least 1012CFU/g or ml, such as at least 5×1012CFU/g or ml, such as at least 1013CFU/g or ml, such as at least 5×1013CFU/g or ml of composition.

14. The composition according to any one of embodiments 11-13 or preparation according to embodiments 2-7, which is or is used for the preparation of a food product of feed product, such as a milk product.

15. The composition according to embodiment 14, wherein said food product is selected from the group consisting of dairy products, yoghurt, drinking yogurt, cheese, such as fresh cheese, cream cheese, cottage cheese, semi-soft and soft cheese, semi-hard and hard cheese, white brine cheese, sour milk products, and sour cream, sauerkraut, pickles, beer, wine, cider, kimchi, cocoa, sour dough and other fermented foods, as well as animal feeds, such as silage.

16. The composition according to any one of embodiments 11-13 or preparation according to embodiments 2-7, wherein said bacteria of the genusLactobacillusis selected from the list consisting of

Lactobacillus rhamnosusDSM 7061, or a combination of any thereof.

17. Use of the composition according to any one of embodiments 11-16 to control the growth of a contaminant, such as a bacteria, yeast or mould.

Thepropionibacteriawere propagated in sodium lactate broth that consisted of 2% peptone from casein (Merck KGaA, Darmstadt, Germany), 1% yeast extract (Merck) and 1.6% sodium lactate solution 50% (Merck) at 30° C. in anaerobic jars. A passage was done by adding 0.1 ml from the pre-cultures to 10 ml sodium lactate broth, cultivation as described above. The cultures were kept at 4-6° C. prior to use.

Yeast and Mould Indicator Strains

In the following the yeast and mould strains are listed in table 1 and 2.

Prior to use, the yeasts were cultivated in a broth consisted of 2% glucose (vwr, Herlev, Denmark), 0.5% yeast extract (Oxoid Limited, Basingstoke, United Kingdom) and 0.01% peptone from casein (Oxoid) dissolved in a potassium di-hydrogen phosphate solution (312 μmol/l, pH 7.20±0.10). Sterile glycerol was added in a concentration of 33% v/v to the medium with grown yeast before storage in cryo tubes at −80° C. After freezing the yeast cultures were enumerated on malt extract agar (Oxoid).

The mould strains were cultivated on malt extract agar slants (Oxoid) until sporulation was visible. The spores were harvested by adding twice 5 ml sterile tap water supplemented with 0.01% Tween 80 (Merck). Sterile glycerol was added in a concentration of 33% v/v to the spore solutions before storage in cryo tubes at −80° C. A spore count was made after freezing on malt extract agar (Oxoid).

Thepropionibacteriatest strains were spot inoculated (three spots per plate) on sodium lactate agar that consisted of 2% tryptone (Oxoid), 1% yeast extract (Oxoid), 1.6% sodium lactate solution 50% (Merck) and 1.5% agar agar (Merck). The plates were incubated at 30° C. in anaerobic jars.

The plates with grown colonies of thepropionibacteriawere overlaid with malt extract soft agar consisted of 2% malt extract broth (Oxoid) and 0.8% agar agar (Merck) tempered to 47° C. containing either 104yeast cells/ml or 104mould spores/ml. The overlaid plates were incubated for 3-5 days at 25° C. and inspected for inhibition zones around the test colonies.

As a growth control agar plates without spotted test strains were prepared for each indicator organisms. The area of the zones was graded as follows:

No inhibition, plate fully overgrown−No fungal growth on the spot(+)No fungal growth for 5 mm around the spot+
Results

Fivepropionibacteriawere tested for antifungal activities. Therefore, the strains were spot inoculated on sodium lactate agar plates on which the fungi listed in table 1 and 2 were applied incorporated in malt extract soft agar. The plates were stored at 25° C. and inspected for inhibition zones around thelactobacillicolonies.

All five testedpropionibacteriashowed antifungal activity in the applied overlayer assay as summarised in table 3.

Thepropionibacteriashowed only slightly different inhibition spectra. The majority of the moulds were more sensitive and inhibited to a higher extent than the tested yeast strains.

Antifungal Activity in Yogurt

The antifungal activity ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 was evaluated in experimental blends with eitherLactobacillus paracaseisubsp.paracaseiDSM 14514 orLactobacillus rhamnosusDSM 7061. A reference sample without protective cultures added was included for comparison. Two individual trials were carried out. In the first trial the experimental protective culture blends were prepared from freeze-dried strains. In the second trial the experimental protective culture blends were prepared from both freeze-dried and frozen strains.

The yogurt samples were prepared using whole milk with 3.5% fat. The milk was heat-treated for 360 seconds at 95° C. and afterwards cooled down to the fermentation temperature of 43° C. The milk was inoculated with a commercial available thermophilic yogurt starter culture. The yogurt starter culture was consisting of strains ofStreptococcus thermophilusandLactobacillus delbrückiisubsp.bulgaricus(DuPont, Denmark). Test samples were additional inoculated with the experimental protective culture blends to give a level of either 5.0·106CFU/ml for the blend ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 andLactobacillus rhamnosusDSM 7061 or 5.35·106CFU/ml for the blend ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 andLactobacillus paracaseisubsp.paracaseiDSM 14514. The fermentation was done for about 6-7 hours at 43° C. until the pH reached 4.60. The yogurt was dispatched in sterile beakers and inoculated with a pool of yeasts. The yeast pool contained the strainsRhodotorula mucilaginosaDCS 1087 (CFSQE 63 (Magnusson, 3. et al 2003. FEMS Microbiology Letters 219: 129-135) andDebaryomyces hanseniiDCS 605 (DSM 70238, Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany)) and was added at a final level of 2.0E00 CFU/g of yogurt. The yogurt samples were stored for 36 days at 5° C. and yeasts were enumerated on YGC agar.

The yogurt samples were prepared using whole milk with 3.5% fat. The milk was heat-treated for 360 seconds at 95° C. and afterwards cooled down to the fermentation temperature of 43° C. The milk was inoculated with a commercial available thermophilic yogurt starter culture. The yogurt starter culture was consisting of strains ofStreptococcus thermophilusandLactobacillus delbrüickiisubsp.bulgaricus(DuPont, Denmark). Test samples were additional inoculated with the experimental protective culture blends to give the following cell levels: 5.0·106CFU/ml for the freeze-dried blend ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 andLactobacillus rhamnosusDSM 7061, 1.0·107CFU/ml for the frozen blend ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 andLactobacillus rhamnosusDSM 7061, and 5.35·106CFU/ml both for the freeze-dried and frozen blend ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 andLactobacillus paracaseisubsp.paracaseiDSM 14514. The fermentation was done for about 6-7 hours at 43° C. until the pH reached 4.60. The yogurt was dispatched in sterile beakers and inoculated with a pool of yeasts. The yeast pool contained the strainsRhodotorula mucilaginosaDCS 1087 andDebaryomyces hanseniiDCS 605 and was added at a final level of 8.0E00 CFU per 10 g of yogurt. The yogurt samples were stored for 33 days at 5° C. and yeasts were enumerated on YGC agar.

Results

The freeze-dried experimental culture blends ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 with eitherLactobacillus paracaseisubsp.paracaseiDSM 14514 orLactobacillus rhamnosusDSM 7061 were evaluated for inhibitory activity against yeasts in yogurt. A reference sample without antifungal cultures was included for comparison. Portions of whole milk with 3.5% fat were fermented with a commercial yogurt culture. Test samples were inoculated additionally with the protective strains. After fermentation the yogurt samples were dispatched in beakers and inoculated with either a pool of yeasts. The samples were stored at 6° C. and the outgrowth of the added yeast strains was monitored.FIG. 1displays the development of the yeast pool in yogurt samples with and without antifungal cultures. An increase in yeast cell counts was observed in all samples but the reference sample without antifungal cultures showed higher yeast counts than the samples prepared with the experimental antifungal cultures after approx. seven days of storage.

The freeze-dried and frozen experimental culture blends ofPropionibacterium freudenreichiisubsp.shermaniiDSM 25846 with eitherLactobacillus paracaseisubsp.paracaseiDSM 14514 orLactobacillus rhamnosusDSM 7061 were evaluated for inhibitory activity against yeasts in yogurt. A reference sample without antifungal cultures was included for comparison. Portions of whole milk with 3.5% fat were fermented with a commercial yogurt culture. Test samples were inoculated additionally with the protective strains. After fermentation the yogurt samples were dispatched in beakers and inoculated with a pool of yeasts. The samples were stored at 6° C. and the outgrowth of the added yeast strains was monitored.FIG. 2displays the development of the yeast pool in yogurt samples with and without antifungal cultures. An increase in yeast cell counts was observed in all samples but the reference sample without antifungal cultures showed higher yeast counts than the samples prepared with the experimental antifungal cultures after approx. seven days of storage.