WO2017050980A1

WO2017050980A1 - Lactobacillus rhamnosus and supernatants thereof for inhibition of pathogens

Info

Publication number: WO2017050980A1
Application number: PCT/EP2016/072715
Authority: WO
Inventors: Corina Diana CEAPA; Jan Knol; Kees VAN LIMPT
Original assignee: N.V. Nutricia
Priority date: 2015-09-25
Filing date: 2016-09-23
Publication date: 2017-03-30

Abstract

The present invention, in embodiments, provides the use of Lactobacillus rhamnosus and/or supernatants thereof for the inhibition of pathogens. The present invention, in embodiments, further provides the use of Lactobacillus rhamnosus and/or supernatants thereof 5 in the treatment, prevention or amelioration of a disease caused by a pathogen or symptoms thereof as well as compositions comprising Lactobacillus rhamnosus and/or supernatants thereof for use according to embodiments of the present invention.

Description

Lactobacillus rhamnosus and supernatants thereof for inhibition of pathogens.

FIELD OF THE INVENTION

The present invention relates to the field of probiotics. In particular, the invention pertains to the use of Lactobacillus rhamnosus and/or supernatants thereof for inhibition of pathogens.

BACKGROUND

According to a definition approved by a joint Food and Agriculture Organization of the United Nations/World HealthOrganization (FAO/WHO) expert Consultation on Health and Nutritional properties of powder milk with live lactic acid bacteria in 2001 , probiotics are "live microorganisms which when administered in adequateamounts confer a health benefit on the host". Probiotic bacteria have been described among species belonging to the genera Lactobacillus, Bifidobacterium, Streptococcus and Lactococcus, which are commonly used in the dairy industry. Probiotics are thought to intervene at the level of the gut microbiota by impeding the development of pathogenic microorganisms and/or by acting more directly on the immune system.

Opportunistic bacterial infections responsible for healthcare associated infections (HAIs) contribute significantly to patient mortality and morbidity, as well as healthcare costs both in developed and developing countries (WHO, 2008). The gastrointestinal tract (GIT) is a reservoir for opportunistic pathogens, which benefit from the disruption of the intestinal microbiota balance, or dysbiosis, to invade and infect susceptible patients. In particular, antibiotic treatments have deleterious effects on the diversity of the intestinal microbiota and they promote expansion of bacterial human opportunistic pathogens such as Enterococcus faecalis, and Clostridium difficile.

It is clear from the above that there is a need for alternatives or complements to antibiotics for the treatment or for the prevention of gastrointestinal infection.

SUMMARY OF THE INVENTION

A subject of embodiments of the present invention is the use of

Lactobacillus rhamnosus and/or supernatants thereof for the inhibition of pathogens. In a further aspect of embodiments of the present invention provides the use of Lactobacillus rhamnosus and/or supernatants thereof in the treatment, prevention or amelioration of a disease caused by a pathogen. In embodiments, the present invention also provides compositions comprising Lactobacillus rhamnosus for use according to embodiments of the present invention.

DETAILED DESCRIPTION

As used herein the term "CNCM I-" followed by a 4 digit number shall be taken to refer to a strain deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) 25 rue du Docteur Roux, Paris, France under the Budapest Treaty with an accession number corresponding to said 4 digit number, e.g. CNCM 1-4316.

As used herein the term "LMG" followed by a 5 digit number shall be taken to mean a strain available from the Belgian Coordinated Collection of Microorganisms Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium with an accession number corresponding to said 5 digit number, e.g. LMG 18020, LMG 18025 or LMG 18030.

As used herein the term "supernatant" shall be taken to mean the culture medium in which bacteria has been grown under conditions suitable for growth. The culture media may be separated from the bacterial cells and fragments thereof by means of centrifugation.

As used herein the term "stable composition" shall be taken to mean a composition that does not present sedimentation and/or serum separation.

As used herein the term "x% (w/w)" is equivalent to "x g per 100 g".

As used herein the term "fermented milk" shall be taken to mean a product or composition derived from milk by the acidifying action of at least one lactic acid bacterium.

As used herein the term "spoonable" shall be taken to mean a solid or semi-solid that may be consumed by means of a spoon or other utensil.

As used herein the term "fermentation" shall be taken to mean the metabolism of a substance by bacteria, yeasts, or other microorganisms.

As used herein the term "cfu" or "CFU" shall be taken to be an abbreviation of the term "colony forming unit".

As used herein the term "in the free form" shall be taken to mean that which is not bound to other amino acids by a peptide bond within peptides, polypeptides or proteins.

As used herein reference to a bacterial strain or species shall be taken to include functionally equivalent bacteria derived therefrom such as but not limited to mutants, variants or genetically transformed bacteria. These mutants or genetically transformed strains can be strains wherein one or more endogenous gene(s) of the parent strain has (have) been mutated, for instance to modify some of their metabolic properties (e.g., their ability to ferment sugars, their resistance to acidity, their survival to transport in the gastrointestinal tract, their post- acidification properties or their metabolite production). They can also be strains resulting from the genetic transformation of the parent strain to add one or more gene(s) of interest, for instance in order to give to said genetically transformed strains additional physiological features, or to allow them to express proteins of therapeutic or prophylactic interest that one wishes to administer through said strains. These mutants or genetically transformed strains can be obtained from the parent strain by means of conventional techniques for random or site-directed mutagenesis and genetic transformation of bacteria, or by means of the technique known as "genome shuffling". In the present text, strains, mutants and variants derived from a parent species or strain will be considered as being encompassed by reference to said parent species or strain, e.g. the phrases " L Rhamnosus" and "strain CNCM 1-4316" shall be taken to include strains, mutants and variants derived therefrom.

Accordingly, as used herein reference to a bacterial strain specified by an accession or deposit number shall be taken to encompass variants thereof having at least 80 % identity with the 16S rRNA sequence of said specified strain, preferably at least 85 % identity, more preferably at least 90 % identity, most preferably at least 95 % identity (see: Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). In a preferred embodiment, said variant has at least 97 % identity with the 16S rRNA sequence of said specified strain, more preferably at least 98 % identity, more preferably at least 99 % identity.

As used herein the term "yeast extract" shall be taken to mean concentrates of soluble compounds of yeast cells. In this regard reference may be made in particular to the article "Yeast extracts: production, properties and components" by Rolf Sommer (9th International Symposium on Yeasts), from which the information below is extracted.

Yeast extracts are mainly produced by autolysis, i.e. cell hydrolysis is carried out without the addition of other enzymes. Yeast extract or yeast autolysate are used mainly in the fermentation industry and in the agri-food industry. The main raw material used in order to produce the yeast extract is constituted by yeasts with a high concentration of proteins (strains of Saccharomyces cerevisiae) cultured on media based on molasses or is constituted by yeasts from debittered beer (strains of Saccharomyces cerevisiae or Saccharomyces uvarum). Other raw materials used are yeasts such as Kluyveromyces fragilis (fermented on lactoserum) or Candida utilis (cultured on carbohydrate-rich waste originating from of the timber industry or on ethanol) or also special strains of baker's yeasts, in order to produce yeast extract containing 5'-nucleotides. Methods for the preparation of yeast extract are known in the art, autolysis is the dissociation process most frequently used production process. During this process, the yeasts are degraded by their endogenous enzymes. The autolysis process can be initiated by osmotic shock or controlled temperature, causing cell death without inactivating the endogenous enzymes (in particular the proteases). The temperature, controlled pH and the duration of the autolysis are decisive factors in a standardized autolysis process. By adding salts or enzymes (for example proteases or mixtures of proteases and peptidases) relative to the "standard" autolysis, the protein degradation of the yeast cells can be controlled.

As an alternative to autolysis, the yeast extract can be produced by thermolysis (for example by boiling the yeasts in water at 100° C), plasmolysis (treatment with strong saline solutions at a temperature below 100° C.) and mechanical degradation (high-pressure homogenization or grinding). Soluble compounds are separated from the insoluble cell walls and concentrated with an evaporator with stirring or falling film evaporator, followed by optional stages of filtration, concentration under partial vacuum and rapid sterilization.

Uses and methods of embodiments of the invention

The present inventors have found that the bacterial species Lactobacillus rhamnosus and its supernatants are capable of inhibiting pathogen growth or replication. Accordingly, in

embodiments, the present invention provides the use of Lactobacillus rhamnosus and/or supernatants thereof for the inhibition of a pathogen, or the growth or replication thereof.

Preferably said pathogen is a microorganism, most preferably a bacterial pathogen. In one embodiment the pathogen is an opportunistic pathogen. In an alternative embodiment the pathogen is a gastrointestinal pathogen. Accordingly in one embodiment the present invention provides the use of Lactobacillus rhamnosus and/or supernatants thereof in the treatment, prevention or amelioration of a disease or infection caused by a pathogen or symptoms thereof. Preferably said disease or infection is a gastrointestinal disease or infection. In an alternative embodiment the present invention provides the non-therapeutic use of Lactobacillus rhamnosus and/or supernatants thereof for the inhibition of a pathogen, or the growth or replication thereof.

In a further embodiment the present invention provides a method for inhibiting a pathogen or the growth or replication thereof comprising providing or administering to a subject a composition comprising Lactobacillus rhamnosus and/or supernatants thereof, such that the pathogen, or the growth or replication thereof, is inhibited. In a further aspect of embodiments of the present invention provides a method for the treatment, prevention or amelioration of a pathogen infection or symptoms thereof in a subject, said method comprising administering to said subject in need thereof a therapeutically effective amount of Lactobacillus rhamnosus and/or supernatants thereof, or a composition as provided herein. Determination of a therapeutically effective amount is well known from the person skilled in the art, especially in view of the detailed disclosure provided herein.

A further subject of embodiments of the present invention is also a method for the manufacture of a medicament for treating, preventing or ameliorating pathogen infection or symptoms thereof, said method comprising incorporating Lactobacillus rhamnosus and/or supernatants thereof, with at least one pharmaceutically acceptable diluent, carrier or excipient.

Symptoms associated with or caused by pathogens or infections thereof according to

embodiments of the present invention are preferably gastrointestinal symptoms typically vomiting, nausea, diarrhea, fever and/or gastrointestinal inflammation. Preferably the Lactobacillus rhamnosus according to the uses and methods of embodiments of the present invention is selected from the group consisting of CNCM 1-4316, LMG 18020, LMG 18025 and LMG 18030.

Lactobacillus rhamnosus strain CNCM 1-4316 has been deposited by Compagnie Gervais Danone, 17 Boulevard Haussman, 75009 Paris, France in accordance with the Budapest

Treaty, at the Collection Nationale de Cultures de Microorganismes (CNCM) 25 rue du Docteur Roux, Paris, France on May 19 2010.

Lactobacillus rhamnosus strains LMG 18020, LMG 18025 & LMG 18030 are available from the Belgian Coordinated Collection of Microorganisms Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium.

Preferably the gastrointestinal pathogens according to the uses and methods of embodiments of the present invention are selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia, coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis. In one embodiment it is preferred that the pathogen is Clostridium difficile. In a further embodiment it is preferred that the pathogen is Escherichia, coli. In one embodiment of the invention said Lactobacillus rhamnosus is strain CNCM 1-4316 and the pathogen is selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia, coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis; it is particularly preferred that the pathogen is Salmonella Enteritidis or Enterococcus faecalis.

In an alternative embodiment of the invention said Lactobacillus rhamnosus is strain LMG 18020 and the pathogen is selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia, coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis; it is particularly preferred that the pathogen is Enterococcus faecalis.

In a further alternative embodiment of the invention said Lactobacillus rhamnosus is strain LMG 18025 and the pathogen is selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis; it is particularly preferred that the pathogen is Salmonella Enteritidis or Enterococcus faecalis.

In an additional alternative embodiment of the invention said Lactobacillus rhamnosus is strain LMG 18030 and the pathogen is selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis; it is particularly preferred that the pathogen is Salmonella Enteritidis or Enterococcus faecalis.

Compositions of embodiments of the invention and methods for the preparation thereof

It is particularly preferred that the Lactobacillus rhamnosus and/or supernatants thereof are provided in a suitable composition, accordingly in a further aspect of embodiments of the present invention provides compositions suitable for the uses and methods of embodiments of the invention.

The composition for use according to embodiments of the invention is suitable for consumption or ingestion, preferably by oral means. Accordingly the composition comprises or consists of comestible matter. It is particularly preferred that the compositions of embodiments of the invention are substantially free of pathogenic or toxicogenic matter. The composition according to embodiments of the invention may be a medicament or pharmaceutical

composition. In an alternative embodiment the composition according to the invention may be a non-therapeutic composition, preferably a nutraceutical composition, a nutritional composition and/or a food composition.

Nutritional compositions which can be used according to embodiments of the invention also include baby foods, infant milk formulas and infant follow-on formulas. Preferably, the composition comprises at least 10⁶, more preferably at least 1 0⁷ and most preferably at least 10⁸ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. Preferably also the composition according to embodiments of the invention comprises at least about 10¹¹ , more preferably at least 10¹⁰ and most preferably at least 10⁹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In a further embodiment the Lactobacillus rhamnosus is selected from the group consisting of CNCM 1-4316, LMG 18020, LMG 18025 and LMG 18030. In embodiments, the composition comprises 10⁶ to 10¹¹ colony forming unit (CFU) of

Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁷ to 10¹¹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁸ to 10¹¹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁹ to 10¹¹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10¹⁰ to 10¹¹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁶ to 10¹⁰ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁶ to 10⁹ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁶ to 10⁸ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention. In embodiments, the composition comprises 10⁶ to 10⁷ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention.

Preferably the composition suitable for the uses and methods of embodiments of the invention further comprises milk, more preferably fermented milk. Preferably the composition comprises at least about 30 % (w/w) milk, more preferably at least about 50% (w/w) milk and even more preferably at least about 70% (w/w) milk. In embodiments, the composition comprises at 30 % to 100% (w/w) milk. In embodiments, the composition comprises 50% to 100% (w/w) milk. In embodiments, the composition comprises 70% to 100% (w/w) milk. Preferably said milk is vegetal and/or animal milk, more preferably soya, almond, oat, hemp, coconut, rice, goat, ewe, camel, mare or cow milk, and most preferably to cow milk. Preferably said milk(s) are heat-treated, typically pasteurized, to ensure sterility. Preferably said heat treatment is carried out prior to the preparation of the fermented milk composition.

Preferably said mixture comprises one or more of skimmed, partially-skimmed or non- skimmed milk. Preferably said milk or milks may be in liquid, powdered and/or concentrated form. In one embodiment said mixture further comprises cream. In one embodiment said mixture further comprises plant and/or fruit juices. In one embodiment said milk or milks may be enriched or fortified with further milk components or other nutrients such as but not limited to vitamins, minerals, trace elements or other micronutrients.

Preferably said milk has been subjected to heat treatment at least equivalent to pasteurization, preferably said heat treatment is carried out prior to the preparation of the composition.

Typically, milk is pasteurized by means of the following successive steps: 1 ) a stage of standardization of fatty substances of the raw material so as to obtain a standardized substance,

2) a stage of enrichment with dried matter of the standardized substance obtained in the preceding stage, so as to obtain an enriched substance,

3) a stage of preheating of the enriched substance obtained in the preceding stage, so as to obtain a starting substance,

4) a stage of pasteurization and holding of the starting substance obtained in the preceding stage, so as to obtain a pasteurized and held substance,

5) an optional stage of homogenization of the pasteurized and held substance obtained in the preceding stage, so as to obtain a pasteurized, held and optionally homogenized substance,

6) a stage of initial cooling of the pasteurized, held and optionally homogenized substance obtained in the preceding stage, so as to obtain a pasteurized starting substance that has been held, optionally homogenized, and cooled down. As used herein "standardization of fatty substances" is taken to mean a stage of bringing the quantity of fats present in the starting substance to a pre-determined level. Enrichment with dried matter involves the addition of proteins and fatty substance in order to modify curd firmness.

As used herein "Holding" is taken to mean a rapid thermization of the milk and makes it possible to destroy the vegetative microbial flora, including pathogenic forms. Its typical duration is from 4 to 10 minutes, in particular from 5 to 8 minutes, and in particular approximately 6 minutes.

As used herein "homogenization" is taken to mean the dispersion of the fatty substances in the milk-type substance into small fat globules. The homogenization is carried out for example at a pressure of 100 to 280 bars, in particular 100 to 250 bars, in particular 100 to 200 bars, in particular approximately 200 bars. This homogenization stage is purely optional. It is in particular absent from the production process of products with 0% fatty substances.

Preferably the composition comprises above about 0.3 g per 100 g by weight free lactic acid, more preferably above about 0.7 g or 0.6 g per 100 g by weight free lactic acid. In embodiments, the composition comprises 0.3 g to 0.7 grams per 100 g by weight free lactic acid.

Preferably the composition comprises a protein content at least equivalent to that of the milk or milks from which it is derived, preferably at least about 2.5%, more preferably at least about 3% or 3.5% (w/w). Preferably the composition has a pH equal to or lower than 5, preferably between about 3 and about 4.5 and more preferably between about 3.5 and about 4.5. Preferably the composition has a viscosity lower than 200 mPa.s, more preferably lower than 100 mPa.s and most preferably lower that 60 mPa.s, at 10°C, at a shear rate of 64 s^" . In embodiments, the composition has a viscosity range of 1 to 200 mPa.s, 1 to 100 mPa.s, or 1 to 60 mPa.s, at 10°C, at a shear rate of 64 s^" . In embodiments, the composition has a viscosity range of 10 to 200 mPa.s, 10 to 100 mPa.s, or 10 to 60 mPa.s, at 10°C, at a shear rate of 64 s^"1. In embodiments, the composition has a viscosity range of 30 to 200 mPa.s, 30 to 100 mPa.s, or 30 to 60 mPa.s, at 10°C, at a shear rate of 64 s^"1.

The composition according to embodiments of the invention is preferably a product selected from the group comprising yogurt, set yogurt, stirred yogurt, pourable yogurt, yogurt drink, frozen yogurt, kefir, buttermilk, quark, sour cream, fresh cheese and cheese. In one embodiment the composition according to embodiments of the invention is a drinkable composition, more preferably a fermented milk drink such as but not limited to a yogurt drink, kefir etc.. In an alternative embodiment the composition according to embodiments of the invention is a composition that is spoonable such as a set or stirred yogurt or equivalent thereof.

Preferably the composition, according to embodiments of the invention, may be stored, transported and/or distributed at a temperature of from 1 °C to 10°C for at least about 30 days, at least about 60 days or at least about 90 days from packaging and remain suitable for consumption.

Preferably, the composition is a packaged product that comprises at least 10⁶, more preferably at least 10⁷ and most preferably at least 10⁸ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention subsequent to storage, transport and/or distribution at a temperature of from 1 °C to 10°C for at least about 30 days, at least about 60 days or at least about 90 days from packaging.

In embodiments, the composition is a packaged product that comprises 10⁶ to 10⁸ or 10⁶ to 10⁷ colony forming unit (CFU) of Lactobacillus rhamnosus bacteria according to embodiments of the invention per gram (g) of composition according to embodiments of the invention subsequent to storage, transport and/or distribution at a temperature of from 1 °C to 10°C for at least about 30 days, at least about 60 days or at least about 90 days from packaging. According to a further embodiment, the composition further comprises an intermediate preparation comprising a preparation of fruits and/or cereals and/or additives such as flavorings and/or colorings. Said intermediate preparation can in particular contain thickeners (soluble and insoluble fibres, alginates, carragheenans, xanthan gum, pectin, starch, in particular gelatinized, gelan gum, cellulose and its derivatives, guar and carob gum, inulin) or sweeteners (aspartame, acesulphame K, saccharine, sucralose, cyclamate) or preservatives. Examples of flavorings are: apple, orange, strawberry, kiwi fruit, cocoa flavoring etc. Examples of colorings are: beta- carotene, carmine, cochineal red. Moreover, the preparation of the abovementioned fruits can comprise fruits which are whole or in pieces or in jelly or in jam, making it possible for example to obtain fruit yogurts.

Preferably the composition according to embodiments of the invention comprises up to about 30% (w/w) of said intermediate preparation, e.g. up to about 10%, 15%, 20%, 25% (w/w). In embodiments, the composition according to embodiments of the invention comprise 1 % to 30% (w/w) of said intermediate preparation. In embodiments, the composition according to embodiments of the invention comprise 1% to 30% (w/w) of said intermediate preparation. In embodiments, the composition according to embodiments of the invention comprise 1 % to 25% (w/w) of said intermediate preparation. In embodiments, the composition according to embodiments of the invention comprise 1% to 20% (w/w) of said intermediate preparation. In embodiments, the composition according to embodiments of the invention comprise 1 % to 15% (w/w) of said intermediate preparation. In embodiments, the composition according to embodiments of the invention comprise 1% to 10% (w/w) of said intermediate preparation.

Preferably the composition, according to embodiments of the invention is provided in a sealed or sealable container containing about 50 g, 60 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, 100 g, 105 g, 1 10 g, 1 15 g, 120 g, 125 g, 130 g, 135 g, 140 g, 145 g, 150 g, 200 g, 300 g, 320 g or 500 g or about 1 oz, 2 oz, 3 oz, 4 oz, 5 oz, 6 oz or 12 oz product by weight.

In embodiments, the composition, according to embodiments of the invention is provided in a sealed or sealable container containing about 50 g to 500 g, 60 g to 500 g, 70 g to 500 g, 75 g to 500 g, 80 g to 500 g , 85 g to 500 g, 90 g to 500 g, 95 g to 500 g, 100 g to 500 g, 105 g to 500 g, 1 10 g to 500 g, 1 15 g to 500 g, 120 g to 500 g, 125 g to 500 g, 130 g to 500 g, 135 g to 500 g, 140 g to 500 g, 145 g to 500 g, 150 g to 500 g, 200 g to 500 g, 300 g to 500 g, 320 g to 500 g or 500 g product by weight. In embodiments, the composition, according to embodiments of the invention is provided in a sealed or sealable container containing about 1 oz to 12 oz, 2 oz to 12 oz, 3 oz to 12 oz, 4 oz to 12 oz, 5 oz to 12 oz, 6 oz to 12 oz or 12 oz product by weight.

It is particularly preferred that the composition comprises fermented milk. It is particularly preferred that the fermented milk composition is obtained by the acidifying action of at least one, two, three, four, five, six, seven or more lactic acid bacteria strains. Accordingly in one embodiment the composition further comprises at least one, two, three, four, five, six, seven or more lactic acid bacteria strains.

The selection of suitable lactic acid bacteria strains is within the scope of the skilled person and is typically a thermophillic lactic acid bacteria. Examples of lactic acid bacteria that can be used include but are not limited to Lactobacilli (for example Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbruckei, in particular L. delbruckei supsb. bulgaricus or lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus rhamnosus ); Streptococci (for example Streptococcus thermophilus); Lactococci (for example Lactococcus lactis, typically Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris). Typically a mixture or association of a plurality of species of lactic acid bacteria may be used, typically a mixture or association of Lactobacillus and Streptococcus. For the preparation of yogurt this typically includes Lactobacillus bulgaricus (also referred to as Lactobacillus delbruckei subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms such as but not limited to probiotic species or other species that may provide desirable organoleptic or other qualities to the composition, e.g. Lactococcus lactis.

Accordingly in one embodiment, the composition further comprises a plurality of species of lactic acid bacteria comprising either, or more preferably both, Streptococcus thermophilus and/or Lactobacillus delbrueckii subsp. bulgaricus. In a further embodiment, the composition further comprises a plurality of species of lactic acid bacteria comprising Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis. Preferably the total amount of lactic acid bacteria in said composition is about 10⁶ -10⁸ cfu/g, more preferably about 10⁶ -10⁷ cfu/g. Preferably the amount of Lactobacillus bulgaricus in said composition is about 10⁶ -10⁸ cfu/g, more preferably about 10⁶ -10⁷ cfu/g. Preferably the amount of Streptococcus thermophilus in said composition is about 10⁶ -10⁸ cfu/g, more preferably about 10⁶ -10⁷ cfu/g.

Methods for the preparation of fermented milk products, such as yogurts or equivalents thereof, are well-known in the art. Typically a fermented milk product is prepared by culture of milks at a suitable temperature with suitable microorganisms to provide a reduction in pH, preferably to a pH equal to or lower than 5, preferably between about 3 and 4.5; more preferably between about 3.5 and about 4.5. The pH can be adjusted by controlling the fermentation by the microorganism and stopping it when appropriate, for example by cooling.

Suitable temperatures for milk fermentation are typically about 36°C to about 44°C and the temperature is maintained for an incubation time sufficient to provide the desired reduction in pH.

For the preparation of a fermented milk product the temperature at the start of fermentation is typically about 36°C to about 43°C, in particular about 37°C to about 40°C, the temperature at the end of fermentation is typically about 37°C to about 44°C, in particular about 38°C to about 41 °C. The fermentation time is typically about 6 to about 1 1 hours.

Subsequent to the fermentation the fermented milk is cooled. Optionally a stage of intermediate cooling of the fermented milk may be performed to provide a pre-cooled fermented milk having a temperature of between about 22°C and about 4°C. Typically the intermediate cooling time is about 1 hour to about 4 hours, in particular about 1 hour 30 minutes to about 2 hours. The pre-cooled fermented milk is typically stored for up to 40 hours or less.

Preferably a stage of final cooling of the fermented milk is performed such that the temperature at the start of the final cooling is less than about 22°C and the temperature at the end of the final cooling is about 4°C to about 10°C. The cooled product may then be stored, transported and/or distributed at a temperature from about 1 °C to about 10°C for at least about 30 days, at least about 60 days or at least about 90 days. According to a further embodiment, the process for the preparation of a fermented milk composition as defined above optionally comprises a stage of stirring at a pressure of at least 20 bars, or performing a dynamic smoothing, to obtain a composition having the desired viscosity, typically a viscosity of up to 20 mPa.s. Stirring or dynamic smoothing operations provide some shear to composition that typically allow a viscosity drop. Such operations are known by the one skilled in the art, and can be operated with conventional appropriate equipment. This stage is typically performed at cold temperature, for example at a temperature of form 1 °C to 20°C. Without intending to be bound to any theory, it is believed that applying some shear at cold temperature, typically by stirring at high pressure or by performing a dynamic smoothing, can lead to a fluid gel formation within the composition, that provides improved stability even at a low viscosity of up to 20 mPa.s.

According to a further embodiment, the process for the preparation of a fermented milk composition as defined above optionally comprises a stage of addition of an intermediate preparation prior or subsequent to fermentation, said intermediate preparation comprising a preparation of fruits and/or cereals and/or additives such as flavorings and/or colorings.

EXAMPLES

Example 1 : Pathogen growth inhibition assay Growth of L rhamnosus strains:

L rhamnosus strains were grown in 40ml_ milk based milk medium in 5% C02 atmosphere at 37°C for 16 hours (overnight). Overnight cultures of the L rhamnosus strains were washed once with 3m L of fermentation medium and resuspended in 40m L of fermentation medium after centrifugation for 10 minutes at 2000rpm. The number of viable L. rhamnosus cells used in the experiment was determined by plating serial dilutions in duplicate onto deMan, Rogosa and Sharpe (MRS) agar (Biotrading 1017414970). The plates were incubated in a 5% C02 atmosphere at 37°C for 48 hours.

Growth of pathogenic bacteria:

Pathogenic bacteria (S. enteritidis, P. aeruginosa, E. coli, E. feacalis and C. difficile) were aerobically cultured in Brain Heart Infusion (BHI) broth (Oxiod CM0225) at 37°C for 16 hours (overnight). For the assay, cells were washed once with 3ml_ of fermentation medium and resuspended in 40ml_ of fermentation medium after centrifugation for 10 minutes at 2000rpm. For counting, serial dilutions of bacteria were plated in duplicate onto Xylose lysine deoxycholate (XLD) agar (Oxoid CM0469) and incubated under aerobic conditions at 37°C for 24 hours.

Fecal suspension preparation:

l OOgram feces, from a single individual, was collected and diluted 2.5 times with fermentation medium containing 2,6gram/l K2HP04.3H20. The suspension was stored in aliquots at -80°C. The initial pathogen colony forming formation present in the fecal suspension was verified by plating serial dilutions onto selective medium for Enterobacter/ Salmonella, S. aureus and Pseudomonas and incubated in 5% C02 atmosphere at 37°C for 24 hours.

Assay:

The assay was performed in an anaerobic environment. One aliquot of fecal suspension was thawed on ice. 120μΙ pathogenic bacteria culture, 480μΙ L. rhamnosus culture and 600μΙ fecal suspension were mixed in a 15ml_ tube. 1 ml_ of the prepared mixtures was added in a transwell insert. The transwell inserts were wrapped in an additional membrane with a pore size of 500 Dalton. Each insert was placed in a designated well of a 12-well plate containing 2ml_ of dialysis medium diagram in Figure 1 . The pathogen growth inhibition was monitored over a period of 6 hours. Samples of 100μΙ were taken at the start (tO) followed by a measurement after 3 and 6hours (t1 and t2). The samples were serial diluted in PBS+1% BSA ranging from 1 :10- 1 :1.8 x 10⁶. 5μΙ of each dilution was spot plated in duplicate onto XLD-agar, selective medium for the pathogen of interest, and incubated at 37°C for 24 hours. The number of CFU of the quantifiable dilutions was counted and the average of the duplicates was calculated. The average of the viable cells was used to determine the CFU of pathogenic bacteria per ml_ experimental sample.

Assay using bacterial supernatants L rhamnosus bacterial cells were inoculated in 40ml_ milk based medium (pH 6.5), and incubated under anaerobic conditions (5% C02) for 16 hours at 37°C. The supernatants were harvested by centrifuging overnight cultures of various L rhamnosus strains for 15 minutes at 3750rpm. The pH of the cell free supernatants was adjusted at 6.5 with NaOH and HCI and filter- sterilized through 0.2μηη filter. Aliquots of filter-sterilized supernatants were stored at -20°C until used for the assay.

Pathogenic bacteria (S. enteritidis, P. aeruginosa, E. coli, E. feacalis and C. difficile) were grown overnight in milk based medium. The antimicrobial activity of the supernatants was determined in a 96 wells plate by combining 100μΙ of filtered milk based medium, 100μΙ supernatant and 20μΙ of 1 :100 dilution of pathogen overnight culture. The positive control contained 200μΙ of filtered milk based medium and 20μΙ 1 :100 diluted overnight cultured pathogen and the negative control consisted exclusively of 200μΙ filtered milk based medium. The 96-well plate was incubated in a spectrophotometer for 24 hours at 37°C. The growth of the pathogen was determined measuring absorbance at 600nm, preceded by shaking.

Statistical analysis Data are presented as the means of duplicated or triplicates. The error bars represent the standard deviation. One-way analysis of variance (ANOVA) test was used for statistical analysis. A P-value less than 0.05 was considered statistically significant.

Results: Bacterial cells

The fecal sample contained 1 .7 x 10⁴ Enterobacteriaceae CFU/mL of fecal suspension, S. aureus 300 CFU/mL feacal suspension and undetectable levels of Salmonella and Pseudomonas. Plate counting showed an initial inoculation of 10^Λ8 for the Lactobacilli.

The results show an average reduction of 3 log CFU S. enteritidis over a time period of 6 hours in the experimental samples incubated with L rhamnosus cells.

Bacterial supernatants

An experiment was performed to assess the pathogen growth inhibitory effect of compounds secreted by L. rhamnosus bacterial cells. There are three types of effects that were observed:

(A) an identical growth as the control group;

(B) a lower stationary phase than the control group and

(C) a longer lag phase than the control group.

The level of activation was further classified in three groups namely a low, an intermediate and a strong effect. The groups were annotated with the following symbols: the low effect: +, the intermediate effect: ++ and the strong effect: +++ as described in table 4. The classification was based on the difference in growth between the experimental sample and the control in OD (600nm) or time for respectively a longer lag phase or later stationary phase. The results are provided in table 2.

Table 1 : Classification level of effect in pathogen growth due to the presence of supernatant of L. rhamnosus strains

Effect Classification of level Level of inhibition compared to In table 2 indicated as control group

Lower stationary Low 0.05-1 OD600 + phase Intermediate 1 -1 .9 OD600 ++

Strong <2 OD600 +++

Later exponential Low 2-4 hours + phase Intermediate 5-7 hours ++

Strong >7 hours +++

Table 2: Evaluation of bactericidal effect of L. rhamnosus supernatant (pH 6.5) activity on S. enteritidis, P. aeruginosa, E. coli, E. feacalis and C. difficile. The A represents identical growth as the control group. B represents a lower exponential phase in which + is 2-4h, ++ is 5-7h and +++ is >7h difference with the control group. C is a longer stationary phase in which + is 0.05-1 , ++ is 1 -1.9 and +++ is <2 difference in OD600 compared to the control.

Scoring:

A=0

B+=1

B++=2

B+++=3

C+=3

C++=4

C+++=5 Table 3: Phenotypic traits in L. rhamnosus (heatmap). Data is processed by mean averaging, in order to make it comparable between different methods. While surface properties and metabolic traits show very little variation, strains have diverse pathogen inhibition effects that can be used in a gene trait matching analysis.

the International Bureau later

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Peschier-Van den Berg, Y.

FOR INTERNATIONAL BUREAU USE ONLY

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Claims

1. The use of Lactobacillus rhamnosus and/or supernatants thereof for the inhibition of a pathogen.

2. The use of Lactobacillus rhamnosus and/or supernatants thereof for the treatment, prevention or amelioration of a disease or infection caused by a pathogen or symptoms thereof.

3. The use according to any preceding claim wherein said pathogen is selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes, Clostridium difficile and Enterococcus faecalis.

4. The use according to any preceding claim wherein said pathogen is Escherichia coli or Clostridium difficile.

5. The use according to any preceding claim wherein said Lactobacillus rhamnosus is selected from the group consisting of CNCM 1-4316, LMG 18020, LMG 18025 and LMG 18030.

6. The use according to any preceding claim wherein said Lactobacillus rhamnosus is selected from the group consisting of CNCM 1-4316, LMG 18025 and LMG 18030 and the pathogen is Salmonella Enteritidis or Enterococcus faecalis.

7. The use according to any preceding claim wherein said Lactobacillus rhamnosus is LMG 18020 and the pathogen is Enterococcus faecalis.