PROBIOTIC PET FOOD COMPOSITION WITH NON-VIABLE PROBIOTICS

FIELD OF THE INVENTION

The present invention relates to a method for the production of a pet food. The invention further relates to pet food obtainable using the methods herein and uses of such pet foods.

BACKGROUND OF THE INVENTION

Probiotic microorganisms (hereinafter referred to as probiotics) are currently used in many applications including animal feed. Increasing knowledge about the health benefits of probiotics has increased the use of probiotics in animal feeds. For example in Lebeer et al., Microbiol Mol Biol Rev 72, 728-764, 2008, probiotic micro-organisms are shown to adhere to the epithelial cells and mucosal surfaces.

Probiotics has recently been introduced into pet food. However, modern methods of manufacturing, storing and applying pet foods are often harmful to living biologic organisms. Probiotics are sensitive to the physico-chemical conditions in their environment and for example moisture, heat, salts, nutrients and other factors plays a significant role for their biological functions.

To reduce harm to probiotics in the pet food manufacture processes. It is known from US 5,968,569 to apply probiotics as part of a lipid coating on a gelatinized starch matrix after extrusion.

SUMMARY OF THE INVENTION

The present invention is based on the seminal finding that at least Lactobacillus farciminis can be used in the manufacture of an extruded pet food and is able to still provide probiotic effects even after the extrusion manufacturing process is completed. Advantageously, Lactobacillus farciminis is able to still provide a probiotic effect when administered in an effective amount to a pet following manufacturing processes comprising extrusion. This is highly surprising, especially as many extrusion processes require the use of high

temperatures and/or pressure.

Therefore in a first aspect the invention provides a method for producing a pet food comprising admixing at least Lactobacillus farciminis with at least one pet food ingredient wherein said admixing is carried out either before extrusion or during extrusion and wherein during extrusion the admixture is heated to a temperature of at least 71 °C. The Lactobacillus farciminis (also referred to herein as L. farciminis) and/or Lactobacillus rhamnosus (also referred to herein as L. rhamnosus) for use in the methods and/or uses and/or pet food of the present invention may be present in a number of suitable forms. In one embodiment the Lactobacillus farciminis and/or L. rhamnosus may be intact. Suitably the L. farciminis and/or L. rhamnosus cells may be intact but non-viable and referred to herein as "intact cells of a non-viable probiotic microorganism". In another embodiment intact cells of L. farciminis and/or L. rhamnosus may be non-cultivatable and referred to herein as "intact cells of a non- cultivatable probiotic microorganisms".

In a second aspect there is provided an extruded pet food obtainable (e.g. obtained) by the methods of the present invention.

In a third aspect of the present invention there is provided the use of an extruded pet food comprising at least Lactobacillus farciminis and at least one pet food ingredient in an effective amount for inducing a probiotic effect in a pet.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the level of pathogen adherence (% of maximum) on intestinal mucus in vitro at the presence of Sample D soluble fraction, tested at three different concentrations. The test concentrations correspond to 0.1 %, 1 % or 4% (w/v) of product in reaction.

Figure 2 shows the level of pathogen adherence (% of maximum) on intestinal mucus in vitro at the presence of Sample E soluble fraction, tested at three different concentrations. The test concentrations correspond to 0.02%, 0.2% or 2% (w/v) of product in reaction.

Figure 3 shows a schematic of sample preparation, PMA cross-linking and DNA extraction as per the "Intact Cell Assay" as taught herein.

Figure 4 shows a continuation of the schematic for sample preparation begun in Figure 3. Figure 5 shows calculations for calibration curves and active substance as per the "Intact Cell Assay" as taught herein.

DEFINITIONS

The term "intact cells of a non-viable probiotic microorganism" (used synonymously with "intact non-viable cells") as used herein means cells originating or derived from a probiotic microorganism, which cells are non-viable but has retained one or more intact plasma membrane. The cell may include but is not limited to cells that are inactivated by heat. "Nonviable" in this context is to be understood that the cell no longer have biological functions required for categorizing the cell as being alive.

The term "intact cells of a non-cultivatable probiotic microorganisms" (used synonymously with "intact non-cultivatable cells") as used herein, means cells, that still have an active metabolism but are incapable of division (that is the living microorganism lacks one or more biological functions necessary for dividing cells) or growth on a medium that would normally support growth, such as a suitable medium, temperature and pH.

The term "pet" or "companion animal" as used herein means an animal kept for a person's company, as opposed to livestock, laboratory animals, working animals and sport animals which are kept for economic reasons. Pets include dogs, cats, rabbits, rodents, avian pets and reptiles.

The terms "pet food", or "pet food composition" as used herein means nutritional compositions intended for ingestion by a pet.

In one embodiment a nutritional composition may refer to a dietary supplement intended for ingestion by a pet. A dietary supplement is intended to refer to a composition that provides nutrients that may otherwise not be consumed in sufficient quantities by the pet.

The term "pet treat" as used herein refers to a food for consumption by a pet that is intended as an occasional reward or indulgence and not as the sole source of a pet's nutrition. As such, there are no specific nutritional requirements for treats. Pet treats can be produced via numerous methods, one example of such a production method is via extrusion, which may be at a lower temperature than the temperatures taught herein for the extrusion of pet food. The term "extrudate" as used herein means a product that is made by a process by which a set of mixed ingredients are forced through an opening in a perforated plate or die, usually under elevated pressure and temperature and then cut to a specified size by blades. Any type of extruder can be used, non-limiting examples of which include single screw extruders and twin-screw extruders.

The term "admixing" as used herein means mixing one or more substances. The term encompasses mixing the substances in any order, thus substances can be mixed sequentially, or simultaneously.

DETAILED DESCRIPTION OF THE INVENTION

In the first embodiment there is provided a method for producing a pet food comprising admixing at least Lactobacillus farciminis with at least one pet food ingredient wherein said admixing is carried out either before extrusion or during extrusion and wherein during extrusion the admixture is heated to a temperature of at least 71 °C.

Suitably, the L. farciminis for use in the methods and/or uses of the present invention may be Lactobacillus farciminis CNCM I-3699.

In another embodiment the method of the present invention may further comprise admixing Lactobacillus rhamnosus.

Suitably, the L. rhamnosus for use in the methods and/or uses of the present invention may be L. rhamnosus CNCM I-3698. Suitably L. rhamnosus CNCM 1-3698 may be used in combination with L. farciminis CNCM I- 3699 in the methods and/or uses of the present invention.

These strains were deposited at the Collection Nationale de Cultures de Microorganims (CNCM) 25, Rue due Docteur Roux, F75724 Paris Cedex 15, France on 8 December 2006 by Sorbial, Route de Spay 72700 Allonnes, France and all right, title and interest in the deposits were subsequently transferred to Danisco France SAS of 20, Rue de Brunei, 75017 Paris, France.

Danisco France SAS has authorised DuPont Nutrition Biosciences ApS of Langebrogade 1 , PO Box 17, DK-1001 , Copenhagen K, Denmark to refer to these deposited biological materials in this patent application and have given unreserved and irrevocable consent to the deposited material being made available to the public.

In a preferred embodiment the at least L. farciminis (optionally in combination with L.

rhamnosus) is a fermentate composition. Preferably the bacteria are provided as a fermentate composition.

When present in a fermentate the at least L. farciminis (optionally in combination with L. rhamnosus) may be comprised in the fermentate at more than about 1 x10 ⁵ cells/g of fermentate.

In one embodiment the at least L. farciminis (optionally in combination with L. rhamnosus) may be comprised in the fermentate at between about 1 x10 ⁵ to about 1 x10 ¹³ cells/g of fermentate.

Suitably the at least L. farciminis (optionally in combination with L. rhamnosus) may be comprised in the fermentate at between about 1x10 ⁶ to about 1 x10 ¹² cells/g of fermentate. More suitably, the at least L. farciminis optionally in combination with L. rhamnosus) may be comprised in the fermentate at between about 3x10 ⁷ to about 1 x10 ¹¹ cells/g of fermentate. Suitably the fermentate composition is a composition obtained from the co-culture of at least L. farciminis and L. rhamnosus as taught herein.

Suitably any appropriate ratio of L. rhamnosus to L. farciminis may be used. In one embodiment the ratio of L. rhamnosus to L. farciminis may be from about 1 :100 to about 100:1 , suitably about 1 :50 to 50:1 .

In one embodiment the ratio of L. rhamnosus to L. farciminis may be from about 1 :9 to about 9:1 .

Suitably the ratio may be from 1 :8 to 8:1 . Suitably the ratio may be from 1 :7 to 7:1 . Suitably the ratio may be from 1 :6 to 6:1 . Suitably the ratio may be from 1 :5 to 5:1 . Suitably the ratio may be from 1 :4 to 4:1. Suitably the ratio may be from 1 :3 to 3:1 . Suitably the ratio may be from 1 :2 to 2:1.

Suitably the ratio may be about 1 :1. In one embodiment the ratio of L. rhamnosus to L. farciminis may be presented in terms of the total number of cells. In one embodiment there may be between at least about 1x10 ³ L. farciminis cells to at least about 1 x10 ³ L. rhamnosus cells/g of fermentate.

Suitably there may be about 1 x10 ⁵ L. farciminis cells to about 1 x10 ⁵ L. rhamnosus cells/g of fermentate.

The L. farciminis and/or L. rhamnosus may be present as viable cells, non-viable cells or combinations thereof.

The at least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) for use in the methods and/or uses may be dosed at any effective amount. In one embodiment the at least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) may be dosed at more than about 1x10 ³ cells/g of pet food and/or pet food admixture.

In another embodiment, the at least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) may be dosed at between about 1 x10 ³ to about 1x10 ⁹ cells/g of pet food and/or pet food admixture.

Suitably, the at least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) may be dosed at between about 1 x10 ⁴ to about 1x10 ⁸ cells/g of pet food and/or pet food admixture.

Suitably, the at least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) may be dosed at between about 7.5x10 ⁴ to about 1 x10 ⁷ cells/g of pet food and/or pet food admixture.

Preferably, the least L. farciminis (optionally in combination with L. rhamnosus or further probiotic microorganism) may be dosed at about 1 x10 ⁶ cells/g of pet food and/or pet food admixture.

In embodiments where the pet food is a pet treat the number of cells/g dosed may be between about 2 times to about 20 times, suitably between about 4 times to about 15 times the number of cells/g dosed in a pet food and/or pet food admixture. Preferably the number of cells/g dosed may be about 10 times the number of cells/g dosed in a pet food and/or pet food admixture.

The term "viable cells" as used herein means cells that have an active metabolism. The term "viable cells" encompasses cells that are unable to propagate (e.g. replicate) and/or those that are able to propagate (e.g. replicate).

The term "non-viable cells" as used herein means that a cell has an inactive metabolism. The term encompasses cells that have been fragmented (such as those where the cell wall and/or plasma membrane and/or other important cellular structures have been broken down) as well as cells that are intact. In one embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may comprise viable cells.

In another embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may consist of viable cells.

In one embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may comprise more than about 50% viable cells. Suitably the bacteria may comprise more than about 60% or 70% viable cells.

Suitably the bacteria may comprise more than about 75% viable cells. Preferably the bacteria may comprise about 75% viable cells.

Suitably the viable cells may be intact non-cultivatable cells.

In a further embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may comprise non-viable cells.

In a yet further embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may consist of non-viable cells.

In one embodiment the bacteria (e.g. L. farciminis and/or L. rhamnosus) for use in the methods and/or uses of the present invention may comprise more than about 50% nonviable cells. Suitably the bacteria may comprise more than about 60% or 70% non-viable cells. Suitably the bacteria may comprise more than about 75% non-viable cells.

Preferably the bacteria may comprise about 75% non-viable cells.

Suitably the non-viable cells may be intact non-viable cells.

In one embodiment L. farciminis tot use in the methods and/or uses of the present invention comprises viable L. farciminis cells.

In another embodiment the L. farciminis for use in the methods and/or uses of the present invention may consist essentially of, or consist of, viable L. farciminis cells.

In a further embodiment the L. farciminis for use in the methods and/or uses of the present invention may comprise non-viable L. farciminis cells.

In a yet further embodiment the L. farciminis for use in the methods and/or uses of the present invention may consist essentially of, or consist of, non-viable L. farciminis cells.

In one embodiment L. rhamnosus for use in the methods and/or uses of the present invention comprises viable L. rhamnosus cells.

In another embodiment the L. rhamnosus for use in the methods and/or uses of the present invention may consist essentially of, or consist of, viable L. rhamnosus cells.

In a further embodiment the L. rhamnosus for use in the methods and/or uses of the present invention may comprise non-viable L. rhamnosus cells.

In a yet further embodiment the L. rhamnosus for use in the methods and/or uses of the present invention may consist essentially of, or consist of, non-viable L. rhamnosus cells. Suitably the L. farciminis and/or L. rhamnosus for use in the methods and/or uses of the present invention is/are present in the composition as intact viable cells, intact non- cultivatable cells or combinations thereof.

In one embodiment the L. farciminus may comprise intact non-viable L. farciminus cells. In another embodiment the L. farciminis may consist essentially of, or consist of intact nonviable L. farciminus cells.

In one embodiment the L. rhamnosus may comprise intact non-viable L. rhamnosus cells. In another embodiment the L. rhamnosus may consist essentially of, or consist of intact nonviable L. rhamnosus cells.

In one embodiment the L. farciminus may comprise intact non-cultivatable L. farciminus cells. In another embodiment the L. farciminis may consist essentially of, or consist of intact non- cultivatable L. farciminus cells.

In one embodiment the L. rhamnosus may comprise intact non-cultivatable L. rhamnosus cells.

In another embodiment the L. rhamnosus may consist essentially of, or consist of intact non- cultivatable L. rhamnosus cells.

The person skilled in the art will appreciate that where two or more strains are used (e.g. L. farciminis and L. rhamnosus) then there may be a mixture of viable and non-viable strains. Suitably a mixture of intact non-viable and intact non-cultivatable strains.

In some embodiments where two or more strains are used, one strain may comprise viable cells and the other strain may comprise non-viable cells (e.g. intact non-viable cells).

Suitably one strain may consist essentially of (or consist of) viable cells and the other strain may consist essentially of (or consist of) non-viable cells.

In one embodiment the L. farciminis for use in the methods and/or uses of the present invention may be a mixture of viable and non-viable cells (e.g. intact non-viable cells). In one embodiment the ratio of non-viable to viable cells may be about 1 :1 , suitably about 1 :20. In another embodiment the ratio of non-viable to viable cells may be about 1 :50, suitably about 1 :100 or about 1 :150. Preferably the non-viable cells may be intact non-viable cells and/or preferably the viable cells may be intact non-cultivatable cells.

In one embodiment the ratio of viable to non-viable cells may be about 1 :1 , suitably about 1 :20. In another embodiment the ratio of viable to non-viable cells may be about 1 :50, suitably about 1 :100 or about 1 :150. Preferably the non-viable cells may be intact non-viable cells and/or preferably the viable cells may be intact non-cultivatable cells.

In one embodiment the at least L. farciminis may be dosed at more than about 1 x10 ³ intact viable cells/g of pet food and/or pet food admixture. In another embodiment, the at least L. farciminis may be dosed at between about 1 x10 ³ to about 1 x10 ⁹ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Suitably, the at least L. farciminis may be dosed at between about 1 x10 ⁴ to about 1 x10 ⁸ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Suitably, the at least L. farciminis may be dosed at between about 7.5x10 ⁴ to about 1 x10 ⁷ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Preferably, the least L. farciminis may be dosed at about 1 x10 ⁶ viable cells/g (e.g. intact non- cultivatable cells) of pet food and/or pet food admixture.

In embodiments where the pet food is a pet treat the number of viable (e.g. intact non- cultivatable cells) cells/g dosed may be between about 2 times to about 20 times, suitably between about 4 times to about 15 times the number of cells/g dosed in a pet food and/or pet food admixture.

Preferably the number of (e.g. intact non-cultivatable cells) cells/g dosed may be about 10 times the number of cells/g dosed in a pet food and/or pet food admixture.

In another embodiment the at least L. farciminis may be dosed at more than about 1 x10 ³ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

In another embodiment, the at least L. farciminis may be dosed at between about 1 x10 ³ to about 1 x10 ⁹ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

Suitably, the at least L. farciminis may be dosed at between about 1 x10 ⁴ to about 1x10 ⁸ nonviable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

Suitably, the at least L. farciminis may be dosed at between about 7.5x10 ⁴ to about 1 x10 ⁷ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

Preferably, the least L. farciminis may be dosed at about 1 x10 ⁶ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

In embodiments where the pet food is a pet treat the number of non-viable (e.g. intact nonviable cells) cells/g dosed may be between about 2 times to about 20 times, suitably between about 4 times to about 15 times the number of cells/g dosed in a pet food and/or pet food admixture.

Preferably the number of non-viable (e.g. intact non-viable cells) cells/g dosed may be about 10 times the number of cells/g dosed in a pet food and/or pet food admixture.

When present in a fermentate the at least L. farciminis may be comprised in the fermentate at more than about 1 x10 ⁵ viable (e.g. intact non-cultivatable) cells/g of fermentate.

In one embodiment the at least L. farciminis may be comprised in the fermentate at between about 1x10 ⁵ to about 1 x10 ¹³ viable (e.g. intact non-cultivatable) cells/g of fermentate. Suitably the at least L. farciminis may be comprised in the fermentate at between about 1 x10 ⁶ to about 1 x10 ¹² viable (e.g. intact non-cultivatable) cells/g of fermentate.

More suitably, the at least L. farciminis may be comprised in the fermentate at between about 3x10 ⁷ to about 1 x10 ¹¹ viable (e.g. intact non-cultivatable) cells/g of fermentate.

In another embodiment the at least L. farciminis may be comprised in the fermentate at between about 1 x10 ⁵ to about 1 x10 ¹³ non-viable (e.g. intact non-viable) cells/g of fermentate. Suitably the at least L. farciminis may be comprised in the fermentate at between about 1 x10 ⁶ to about 1 x10 ¹² non-viable (e.g. intact non-viable) cells/g of fermentate.

More suitably, the at least L. farciminis may be comprised in the fermentate at between about 1 x10 ⁷ to about 1 x10 ¹¹ non-viable (e.g. intact non-viable) cells/g of fermentate.

In one embodiment there may be about 1x10 ⁷ to about 8x10 ⁸ non-viable cells to about 7.5x10 ⁷ to about 1 x10 ⁹ viable cells per gram of fermentate.

Suitably there may be about 1 x10 ⁷ to about 5x10 ⁸ non-viable cells to about 1 x10 ⁸ to about 8x10 ⁸ viable cells per gram of fermenate.

In one embodiment the L. rhamnosus for use in the methods and/or uses of the present invention may be a mixture of viable and non-viable cells. In one embodiment the ratio of non-viable to viable cells may be about 1 :1 , suitably about 1 :20. In another embodiment the ratio of non-viable to viable cells may be about 1 :50, suitably about 1 :100. Preferably the non-viable cells may be intact non-viable cells and/or preferably the viable cells may be intact non-cultivatable cells.

In another embodiment the ratio of viable to non-viable cells may be about 1 :1 , suitably about 1 :20. In another embodiment the ratio of viable to non-viable cells may be about 1 :50, suitably about 1 :100 or about 1 :150. Preferably the non-viable cells may be intact non-viable cells and/or preferably the viable cells may be intact non-cultivatable cells.

In one embodiment the L. rhamnosus may be dosed at more than about 1 x10 ³ intact viable cells/g of pet food and/or pet food admixture.

In another embodiment, the at least L. farciminis may be dosed at between about 1 x10 ³ to about 1 x10 ⁹ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Suitably, the L. rhamnosus may be dosed at between about 1 x10 ⁴ to about 1 x10 ⁸ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Suitably, the L. rhamnosus may be dosed at between about 7.5x10 ⁴ to about 1x10 ⁷ viable cells/g (e.g. intact non-cultivatable cells) of pet food and/or pet food admixture.

Preferably, the L. rhamnosus may be dosed at about 1 x10 ⁶ viable cells/g (e.g. intact non- cultivatable cells) of pet food and/or pet food admixture. In embodiments where the pet food is a pet treat the number of viable (e.g. intact non- cultivatable cells) cells/g dosed may be between about 2 times to about 20 times, suitably between about 4 times to about 15 times the number of cells/g dosed in a pet food and/or pet food admixture.

Preferably the number of (e.g. intact non-cultivatable cells) cells/g dosed may be about 10 times the number of cells/g dosed in a pet food and/or pet food admixture.

In another embodiment the L. rhamnosus may be dosed at more than about 1 x10 ³ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

In another embodiment, the L. rhamnosus may be dosed at between about 1 x10 ³ to about 1 x10 ⁹ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture. Suitably, the L. rhamnosus may be dosed at between about 1 x10 ⁴ to about 1 x10 ⁸ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

Suitably, the L. rhamnosus may be dosed at between about 7.5x10 ⁴ to about 1 x10 ⁷ nonviable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

Preferably, the L. rhamnosus may be dosed at about 1 x10 ⁶ non-viable cells/g (e.g. intact non-viable cells) of pet food and/or pet food admixture.

In embodiments where the pet food is a pet treat the number of non-viable (e.g. intact nonviable cells) cells/g dosed may be between about 2 times to about 20 times, suitably between about 4 times to about 15 times the number of cells/g dosed in a pet food and/or pet food admixture.

Preferably the number of non-viable (e.g. intact non-viable cells) cells/g dosed may be about

10 times the number of cells/g dosed in a pet food and/or pet food admixture.

When present in a fermentate the L. rhamnosus may be comprised in the fermentate at more than about 1x10 ⁵ viable (e.g. intact non-cultivatable) cells/g of fermentate.

In one embodiment the L. rhamnosus may be comprised in the fermentate at between about

1 x10 ⁵ to about 1 x10 ¹³ viable (e.g. intact non-cultivatable) cells/g of fermentate.

Suitably the L. rhamnosus may be comprised in the fermentate at between about 1 x10 ⁶ to about 1x10 ¹² viable (e.g. intact non-cultivatable) cells/g of fermentate.

More suitably, the L. rhamnosus may be comprised in the fermentate at between about 3x10 ⁷ to about 1x10 ¹¹ viable (e.g. intact non-cultivatable) cells/g of fermentate.

In another embodiment the at least L. farciminis may be comprised in the fermentate at between about 1 x10 ⁵ to about 1 x10 ¹³ non-viable (e.g. intact non-viable) cells/g of fermentate.

Suitably the L. rhamnosus may be comprised in the fermentate at between about 1 x10 ⁶ to about 1x10 ¹² non-viable (e.g. intact non-viable) cells/g of fermentate.

More suitably, the L. rhamnosus may be comprised in the fermentate at between about 1 x10 ⁷ to about 1x10 ¹¹ non-viable (e.g. intact non-viable) cells/g of fermentate. In one embodiment there may be about 1 x10 ⁷ to about 8x10 ⁸ non-viable cells to about 7.5x10 ⁷ to about 1 x10 ⁹ viable cells per gram of fermentate.

Suitably there may be about 1 x10 ⁷ to about 5x10 ⁸ non-viable cells to about 1 x10 ⁸ to about 8x10 ⁸ viable cells per gram of fermenate.

In one embodiment a composition comprising viable cells (e.g. viable L. farciminis cells and/or L. rhamnosus cells) comprises at least 25% intact non-cultivatable cells.

In one embodiment at least 50% of the Lactobacillus farciminis cells may be intact non- cultivatable cells. Suitably at least 70% of the L. farciminis cells may be intact non- cultivatable cells. More suitably at least 90% of the L. farciminis cells may be intact non- cultivatable cells.

In one embodiment at least 50% of the Lactobacillus farciminis cells may be intact non-viable cells. Suitably at least 70% of the L. farciminis cells may be intact non-viable cells. More suitably at least 90% of the L. farciminis cells may be intact non-viable cells.

In one embodiment at least about 50% to about 90% (suitably about 60% to about 80%) of the Lactobacillus farciminis cells may be intact non-viable cells.

In one embodiment at least 50% of the Lactobacillus rhamnosus cells may be intact non- cultivatable cells. Suitably at least 70% of the L. rhamnosus cells may be intact non- cultivatable cells. More suitably at least 90% of the L. rhamnosus cells may be intact non- cultivatable cells.

In one embodiment at least 50% of the Lactobacillus rhamnosus cells may be intact nonviable cells. Suitably at least 70% of the L. rhamnosus cells may be intact non-viable cells. More suitably at least 90% of the L. rhamnosus cells may be intact non-viable cells.

In one embodiment at least about 50% to about 90% (suitably about 60% to about 80%) of the Lactobacillus rhamnosus cells may be intact non-viable cells.

In another embodiment the method of the present invention may comprise the admixing of a further probiotic microorganism. The further probiotic may be one or more genus, species or strain or a mixture of genera, species or strains.

In one embodiment the further probiotic microorganism may be selected from but not limited to the group consisting of: Lactobacillus, Bifidobacterium, Bacillus, Lacotococcus,

Saccharomyces, Streptococcus, Propionibacterium and Streptomyces.

Suitably the further probiotic microorganism may be selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Lacotococcus, Saccharomyces, Streptococcus and Streptomyces or a combinations thereof.

Suitably, the further probiotic may be a strain of_Lactobacillus or a combination of

Lactobacillus strains. In one embodiment the Lactobacillus is selected from the group consisting of cremoris, lactis, acidophilus, casei, kefiri, bifidus, brevis, helveticus, paracasei, rhamnosus, salivarius, curvatus, bulgaricus, sakei, reuteri, fermentum, farciminis, delbreuckii, plantarum, paraplantarum, crispatus, gasseri, iohnsonii, jensenii and kimchi or combinations thereof. In one embodiment the Bifodobacterium is selected from the group consisting of lactis, bifidium, longum , animalis , breve , infantis , catenulatum , pseudocatenulatum ,

adolescentis and angulatum or combinations thereof.

In one embodiment the Saccharomyces is selected from the group consisting of boulardii and cerevisiae or combinations thereof.

In one embodiment the Streptococcus is selected from the group consisting of oralis, uberis and rattus or combinations thereof.

In one embodiment the Bacillus is selected from the group consisting of coagulans, subtilis, cereus, clausii, pumilus or combinations thereof.

In one embodiment the Propionibacterium is selected from the group consisting of freundenreichii, acidipropionici, jensenii or combinations thereof.

FERMENTATES

The at least L. farciminis (optionally in combination with L. rhamnosus) may be used in any suitable form. In some embodiments the at least L. farciminis (optionally in combination with L. rhamnosus) for admixing with a pet food ingredient may be present in a fermentate.

In a particularly preferred embodiment the fermentate may be a co-culture of L. farciminis and L. rhamnosus.

Without wishing to be bound by theory it is believed that L. farciminis and L. rhamnosus have a co-operative interaction with one another when present in a fermentate and that the cells do not compete with one another, making them suitable bacterial species to grow in a co- culture.

A "fermentate" in this context is to be understood as a composition for which one or more living microbial strains have been propagated in a nutrient medium to produce one or more metabolites.

In one embodiment a fermentate comprising L. farciminis (optionally in combination with L. rhamnosus) may be used in the methods and/or uses of the present invention.

In some embodiments L. farciminis (optionally in combination with L. rhamnosus) may be added separately or in combination with other additives to a fermentate.

The nutrient medium used for preparing the fermentate is any medium comprising necessary nutrients suitable for propagating selected microorganisms. Suitable nutrients include but are not limited to amino peptides, peptides, yeast extract and/or vitamins. The medium can be based on dairy products, such as milk, cereals, fruits and/or vegetables. Milk based media may include and milk derivatives from mammals, specifically cows, horses, donkeys, goats, sheep, camels or lamas. Cereal based media may include grains - such as corn, rice, wheat, oats, sorghum, barley, rye and/or derivatives thereof. Beer is a cereal based medium.

Vegetable extract based media may include starch or cocoa bean components. Fruit based media may include fruit or vegetables juices.

In one embodiment the nutrient medium used for preparing a fermentate comprising L.

farciminis and/or L. rhamnosus may be a dairy medium.

Suitably the nutrient medium used for preparing a fermentate comprising farciminis and/or L. rhamnosus may be a milk medium.

In one embodiment the L. farciminis (optionally in combination with the L. rhamnosus) may be present as non-viable cells (e.g. intact cells) and/or viable cells (e.g. intact non- cultivatable cells) comprised within a fermentate, suitably a milk-based fermentate. Suitably such a fermentate may be a soluble fermented skimmed milk powder comprising the non- viable cells (e.g. intact cells) and/or viable cells (e.g. intact non-cultivatable cells) of L.

farciminis (optionally in combination with the L. rhamnosus).

In one embodiment the fermentate (suitably the milk-based fermentate) may comprise L. farciminis (optionally in combination with the L. rhamnosus) consisting essentially of nonviable cells (e.g. intact cells). Suitably such a fermentate may be a soluble fermented skimmed milk powder comprising L. farciminis (optionally in combination with the L.

rhamnosus) consisting essentially of non-viable (e.g. intact cells).

In another embodiment the fermentate (suitably the milk-based fermentate) may comprise L. farciminis (optionally in combination with the L. rhamnosus) consisting essentially of viable cells (e.g. intact non-cultivatable cells). Suitably such a fermentate may be a soluble fermented skimmed milk powder comprising L. farciminis (optionally in combination with the L. rhamnosus) consisting essentially of viable cells (e.g. intact non-cultivatable cells).

The term "consisting essentially of" in the context of the fermentate means that at least 90% of the L. farciminis (optionally in combination with the L. rhamnosus) have the indicated property (e.g. intact non-viable cells) or viable cells (e.g. intact non-cultivatable cells).

Suitably at least 95% have the indicated property. Suitably at least 97% have the indicated property. Suitably at least 99% have the indicated property. In some embodiments at least 100% have the indicated property.

The L. farciminis, L rhamnosus or combinations thereof, or fermentates thereof for use in the present invention may be prepared using a method having: a first stage wherein bacteria is reproduced for 12 to 48 hours; a second plugging phase wherein the bacteria following reproduction are contacted with a medium that absorbs water; and a third stage where the product from the second phase is put in contact with a medium low in water.

Suitably, the following method may be used:

(a) mix about 0.5 to about 2 L (suitably 1 L) of bacteria with about 18L to about 22L (suitably 20L) of water;

(b) add about 3-7kg (suitably 5kg) of dried milk enriched in biocatalyst (such as a yeast extract or a live yeast) and minerals (such as mineral salts, for example but not limited to magnesium or calcium salts);

(c) grow for about 20-30 hours (suitably 24 hours) at about 30-42 °C (suitably at about 37°C); (d) add about 22-27 kg (suitably about 25 kg) of dried milk enriched in biocatalyst and minerals;

(e) incubate for about 20-30 hours (suitably 24 hours) at 30-42°C (suitably at about 37°C);

(f) mix about 550-650kg (suitably 600 kg) of dry product (e.g. a carrier such as corn raids) per 100 kg of the product obtained from (e) or if the dry product is a milk powder mix at approximately a 1 :1 ratio of milk powder to the product obtained from (e) or if the dry product is a cereal carrier at approximately a 10:1 ratio of cereal carrier to the product obtained from (e);

(g) leave the mixture to rest for about 35-50 minutes (suitably for about 45 minutes) at 30-42 °C (suitably at 37 °C).

Suitably the method may also comprise drying the fermentate obtainable (e.g. obtained) after step (g). Alternatively or additionally the method may comprise admixing a carrier with the fermentate.

The fermentate may be formulated with a suitable carrier.

In one embodiment the carrier may be a milk powder carrier. Suitably when the carrier is a milk powder carrier the fermentate may be admixed at about 35-65% w/w with milk powder, more suitably about 50% w/w with milk powder.

When the fermentate is formulated with a milk powder carrier the admixture may be dosed at about 150 g per ton to about 300 g per ton of pet food. Suitably, the admixture may be dosed at about 250 g per ton of pet food.

In another embodiment when the fermentate is formulated with a milk powder carrier and wherein the pet food is a pet treat, the admixture may be dosed at about 1.5 kg per ton to about 3 kg per ton of pet treat. Suitably, the admixture may be dosed at about 2.5 kg per ton of pet treat.

In another embodiment the carrier may be a cereal carrier. Suitably when the carrier is a cereal carrier the fermentate may be admixed at about 5%-15% w/w with cereal carrier, more suitably about 10% w/w with cereal carrier. When the fermentate is formulated with a cereal carrier the admixture may be dosed at about 0.5 kg per ton to about 2 kg per ton of pet food. Suitably, the admixture may be dosed at about 1 kg per ton of pet food.

In another embodiment when the fermentate is formulated with a cereal carrier and wherein the pet food is a pet treat, the admixture may be dosed at about 5 kg per ton to about 20 kg per ton of pet treat. Suitably, the admixture may be dosed at about 10 kg per ton of pet treat. In one embodiment the method for preparing a fermentate may be that taught in FR2649719 the teaching of which is incorporated herein by reference.

In a preferred embodiment the method for preparing a fermentate as taught herein is carried out using L. rhamnosus CNCM 1-3698 in combination with L. farciminis CNCM 1-3699.

Advantageously use of such methods for preparing a fermentate may result in cells which are more stable and more likely to remain intact following subsequent procedures in the pet food manufacturing processes, such as after extrusion.

In one embodiment of the invention the fermentate is prepared by fermenting

microorganisms at a temperature between about 15°C to about 49°C, suitably between 20°C to about 45°C. Suitably the microorganisms may be fermented at a temperature between about 35°C to about 40°C.

The duration of the fermentation may be adjusted depending on the organism used. In one embodiment the duration of the fermentation may be up to about 24 hours, suitably between about 10 -24 hours. Suitably the duration of the fermentation may be between about 12-17 hours.

In one embodiment the fermentate may be prepared at a temperature of between about 15°C to about 49°C and at a temperature of up to about 24 hours.

In one embodiment the L. farciminis and L. rhamnosus for use in the present invention may be a co-culture fermentate of L. farciminis and L. rhamnosus produced by the method taught in FR2649719 and comprising intact non-viable cells.

Suitably the fermentate may be a milk fermentate.

In one embodiment the L. farciminis and L. rhamnosus for use in the present invention may be a co-culture fermentate of L. farciminis and L. rhamnosus produced by the method taught in FR2649719 and comprising intact non-cultivatable cells.

Suitably the fermentate may be a milk fermentate.

It is not intended that the at least L. farciminis (optionally in combination with L. rhamnosus) for admixing with the at least one pet food ingredient is used as a cell-free fermentate.

A "cell-free fermentate" (synonymous to the term "fermentation product") as used herein means that the fermentate is substantially free of viable bacterial cells, typically containing less than about 10 ⁵ viable bacterial cells/mL fermentate, less than about 10 ⁴ viable bacterial cells/mL fermentate, less than about 10 ³ viable bacterial cells/mL fermentate, less than about 10 ² viable bacterial cells/mL fermentate, or less than about 10 viable bacterial cells/mL fermentate. Preferably, the fermentate is substantially free of cells, typically containing less than about 10 ⁵ cells/mL fermentate, less than about 10 ⁴ cells/mL fermentate, less than about 10 ³ cells/mL fermentate, less than about 10 ² cells/mL fermentate, or less than about 10 cells/mL fermentate.

Suitably, the fermentate may be substantially free of viable bacterial cells, typically containing less than about 10 ² viable cells/mL fermentate.

Suitably, the fermentate may be substantially free of viable bacterial cells, typically containing less than about 10 viable cells/mL fermentate.

The fermentate for use in the methods and/or uses of the present invention may be substantially free of viable bacterial cells, typically containing zero (or substantially) viable cells/mL fermentate.

In some embodiments, the term "cell-free" means that the fermentate is substantially free of viable spores in addition to viable cells, typically containing less than about 10 ⁵ viable spores/mL fermentate, less than about 10 ⁴ viable spores/mL fermentate, less than about 10 ³ viable spores/mL fermentate, less than about 10 ² viable spores/mL fermentate, or less than about 10 viable spores/mL fermentate. Preferably, the fermentate is substantially free of spores, typically containing less than about 10 ⁵ spores/mL fermentate, less than about 10 ⁴ spores/mL fermentate, less than about 10 ³ spores/mL fermentate, less than about 10 ² spores/mL fermentate, or less than about 10 spores/mL fermentate.

Suitably, the fermentate may be substantially free of viable spores, typically containing less than about 10 ² viable spores/mL fermentate.

Suitably, the fermentate may be substantially free of viable spores, typically containing less than about 10 viable spores/mL fermentate.

Suitably, the fermentate may be substantially free of viable spores, typically containing zero (or substantially zero) viable spores/mL fermentate.

In one aspect, the term "cell-free" as used herein means that the fermentate is substantially free of viable bacterial cells and viable spores, typically containing less than about 10 ⁵ viable bacterial cells and viable spores/mL fermentation product, less than about 10 ⁴ viable bacterial cells and viable spores/mL fermentate, less than about 10 ³ viable bacterial cells and viable spores/mL fermentate, less than about 10 ² viable bacterial cells and viable spores/mL fermentate, or less than about 10 viable bacterial cells and viable spores/mL fermentate. Preferably, the fermentate is substantially free of cells and/or spores, typically containing less than about 10 ⁵ cells and/or spores/mL fermentate, less than about 10 ⁴ cells and/or spores/mL fermentate, less than about 10 ³ cells and/or spores/mL fermentate, less than about 10 ² cells and/or spores/mL fermentate, or less than about 10 cells and/or spores/mL fermentate.

Suitably, the fermentate may be substantially free of viable bacterial cells and viable spores, typically containing less than about 10 ² viable cells and/or viable spores/mL fermentate. Suitably, the fermentate may be substantially free of viable bacterial cells and viable spores, typically containing less than about 10 viable cells and/or viable spores/mL f fermentate. Suitably, the fermentate may be substantially free of viable bacterial cells and viable spores, typically containing zero (or substantially zero) viable cells and/or viable spores/mL fermentate.

In some aspects, the fermentate for use in the methods and/or uses of the present invention may be treated (e.g. heat treated or irradiated) so that no cells, or spores, or combinations thereof, remain viable.

Spores are "viable" when they are dormant and capable of germinating. PREPARATION OF INTACT NON-VIABLE CELLS

Preparation of intact non-viable cells may be achieved by subjecting at least one probiotic microorganism(s) (e.g. L. farciminis (optionally in combination with L. rhamnosus)) comprised in a suitable (suitably liquid) composition to any treatment allowing deterioration of biological function but preservation of one or more plasma membranes.

Intact non-viable cells may be prepared using a method comprising:

a) providing at least one probiotic microorganism(s) in a suitable, preferably liquid, composition; and

b) subjecting the composition to a treatment deteriorating biological function of the microorganism, while one or more plasma membranes remain intact. In one embodiment the treatment is a heat treatment and the heat is provided by steam, electric resistance, exposure to infra red light, microwaves or other sources of heat. In one embodiment the heat treatment includes heating a composition containing a selected probiotic microorganism to a temperature of at least about 100°C to about 1 10°C.

The at least one probiotic microorganism(s) (e.g. L. farciminis (optionally in combination with L. rhamnosus)) may be heated to a temperature of about 100°C to about 1 10°C and maintained at this temperature for about 30 seconds to about 300 seconds.

Suitably, the at least one probiotic microorganism(s) (e.g. L. farciminis (optionally in combination with L. rhamnosus)) may be heated to a temperature of about 102°C to about 103°C and maintained at this temperature for about 30 seconds to about 300 seconds, (suitably for about 60 seconds to about 100 seconds). The probiotic microorganism is subjected to the aforesaid temperatures only for enough time to deteriorate biological function while maintaining one or more plasma membranes intact. The presence of an intact plasma membrane can be determined using the "Intact Cell Assay" taught herein.

In one embodiment the at least one probiotic microorganism is at least Lactobacillus farciminis.

In another the at least one probiotic microorganism is at least Lactobacillus rhamnosus. Suitably, the at least one probiotic microorganism(s) is a combination of L. farciminis and L. rhamnosus.

PREPARATION OF INTACT NON-CULTIVATABLE CELLS

Preparation of intact non-cultivatable cells may be achieved by subjecting at least one probiotic microorganism(s) to a treatment causing stress to the at least one probiotic microorganism(s) (e.g. a stressful condition). Stressful conditions in this context are to be understood as conditions that cause the living microorganism to become incapable of division or growth on a medium that would normally support growth. Stressful conditions include but are not limited to metabolic stress such as excess metabolites (for example organic acids and peptides) or insufficient amounts of one or more nutrients (starvation), thermal stress such as heating or freezing, acid/base stress, such as shifts in pH, shifts in ionic strength and mechanical stress such as high shear mixing, shifts or fluctuating levels of oxygen and/or light intensity or drying.

The skilled person will appreciate that the combinations of stressful conditions may also be used.

In order to determine if a cell is an intact cell the "Intact Cell Assay" taught herein can be used.

Intact non-cultivatable cells may be prepared using a method comprising:

a) providing at least one probiotic microorganism(s) in a suitable, preferably liquid, composition; and

b) subjecting said at least one probiotic microorganism(s) to a treatment selected from the group consisting of metabolic stress, thermal stress and mechanical stress making the probiotic microorganisms incapable of division or growth on a medium that would normally support growth.

In one embodiment the at least one probiotic microorganism(s) is subjected to the treatment during fermentation in a suitable medium.

In a further embodiment the fermentation is carried out at a temperature of between about 15°C to about 49°C and for a duration of up to about 24 hours. In one embodiment the at least one probiotic microorganism is at least Lactobacillus farciminis.

In one embodiment the at least one probiotic microorganism is at least L. rhamnosus CNCM 1-3698

In another the at least one probiotic microorganism is at least Lactobacillus rhamnosus. Suitably, the at least one probiotic microorganism(s) may be a combination of L. farciminis and L. rhamnosus.

Suitably the at least one probiotic microorganism(s) may be at least L. rhamnosus CNCM I- 3698 used in combination with L. farciminis CNCM 1-3699.

Methods for determining between intact non-cultivatable cells and intact cultivatable cells can be determined by a suitable assay such as a classical plating method. Classical plating methods are summarised in the microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N.Y. 790 pm, which is incorporated herein by reference.

Typically, the absence of viable cells can be shown as follows: no visible colony on agar plates or no increasing turbidity in liquid growth medium (e.g. suitably MRS standard medium) after inoculation with different concentrations of bacterial preparations and incubation under appropriate conditions, such as for incubation at a suitable temperature for at least 24 hours.

A culture contains intact non-cultivatable cells if it does not increase the turbidity of a MRS liquid growth medium when the optical density (OD) of the test sample is compared to a control sample containing only MRS liquid growth medium after incubation at 35-37°C for about 24 hours. "INTACT CELL ASSAY"

A cell having an intact cell membrane can be measured using the "Intact Cell Assay" as taught herein. The assay is carried out according to the method detailed below.

1 Unit: CFU of Lactobacillus strain. g-1 of pet food

Pet food is supplemented with a known concentration of CFU of targeted Lactobacillus determined by numeration on MRS agar Media and referred to an equivalent CT value after DNA extraction and qPCR amplification with and without PMA (Propidium Mono Azide). The established standard curve without PMA (CT = f(log starting quantity)) is used to calculate total bacteria. The standard curve with PMA is used to calculate only viable bacteria. The difference between total bacteria and intact bacteria represents the quantity of non-intact bacterial cells. The method is based on the use of DNA-modifying dyes like Propidium Mono Azide

(abbreviated as PMA) coupled with real time PCR overcome this problem as they can only enter the non-intact cells. Pre-treating a sample with PMA prior to PCR analysis permits to selectively detect only viable bacteria in a highly accurate and reliable manner. PMA is a photo-reactive dye with a high affinity for DNA. The dye intercalates in DNA and forms a covalent linkage upon exposure to intense visible light, resulting in chemically modified DNA, which cannot be amplified by PCR. Intact membranes of cells are impermeable to PMA. Thus, when a sample comprising of both intact and non-intact bacterial cells are treated with PMA, only bacteria with compromised cell membrane permeability are susceptible to DNA modification.

Quantitative PCR is performed with TaqMan® probes. TaqMan® probes are dual labelled hydrolysis probes and are a registered trademark of the Roche Molecular Systems, Inc. TaqMan® probes utilize the 5' exonuclease activity of the enzyme Taq Polymerase for measuring the amount of target sequences in the samples. TaqMan® probes consist of an 18-22 bp oligonucleotide probe which is labelled with a reporter fluorophore at the 5' end and a quencher fluorophore at the 3' end. Each strain specific probe developed for this purpose was labelled at the 5' end with the reporter dyes FAM or Texas Red and at the 3' end with the quencher BHQ1 or BHQ2. The cycle threshold (CT) is defined as the cycle number at which the reaction begins the exponential phase (calculated by the Biorad CFX Manager 2.0) and was used to create standard curve (CT values against the logarithm of serial 10-fold diluted standard concentration give a linear relationship).

2. Reagents and materials

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or ultra-pure distilled water.

2.1 MRS broth (Merck KGaA, Darmstadt, Germany)

2.2 MRS Agar Media (Merck KGaA, Darmstadt, Germany)

2.3 Sodium carbonate, (Na2C03) (#1 .06392.0500) (Merck)

2.4 Sodium bicarbonate (NaHC03) (#S8875) (Sigma-Aldrich, lllkirch, France)

2.5 Tween 20 (0.1 %) (#P-9416) (Sigma-Aldrich, lllkirch, France)

2.6 Sodium thiosulphate (Na2S203-5H20) (20 g/l) (#217247) (Sigma-Aldrich, lllkirch, France)

2.7 Albumin from Bovine Serum (BSA) (#A9418) (Sigma-Aldrich, lllkirch, France)

2.8 Sodium hydroxide (NaOH) (10%) (Sigma-Aldrich, lllkirch, France)

2.9 Polyvinyl polypyrrolidone (PVPP) (#77627) (Sigma-Aldrich, lllkirch, France) 2.10 Propidium Mono Azide, PMATM dye, 20mm in H20 (#BTIU40019) (Biotium, Hayward, Canada)

2.1 1 Strain specific probes (Sigma-Aldrich, lllkirch, France), for L. farciminis and L.

rhamnosus are shown as below

2.12 Strain specific primers (Invitrogen) for L. farciminis and L. rhamnosus the probes are shown as below

SsoADV Univer Probes Supermix (#172-5280) (Bio-Rad)

2.14 Sterile molecular biology grade water (Sigma-Aldrich, lllkirch, France)

2.15 Extraction buffer without PVPP: Weigh 3.18 g Sodium carbonate (2.3) and 5.86 g Sodium bicarbonate (2.4) into a 1 L volumetric flask. Add demineralised water. Adjust pH to 9.6 with 10% Sodium hydroxide (2.8). Adjust volume to 1 L with demineralised water (2.1 1 ). Add 20 g Sodium thiosulphate (2.6). Autoclave at 120oC for 15 min. Allow the solution to cool down to below 50oC. Add 1 ml Tween 20 (2.5, sterilized by filtration 0.2μη"ΐ) and 4g BSA (2.7) (Note: this solution can be stored at 4oC for up to 15 days).

2.16 Extraction buffer with PVPP: Weigh 3.18 g Sodium carbonate (2.3) and 5.86 g Sodium bicarbonate (2.4) into a 1 L volumetric flask. Add demineralised water. Adjust pH to 9.6 with 10% Sodium hydroxide (2.8). Adjust volume to 1 L with demineralised water (2.1 1 ). Add 20 g Sodium thiosulphate (2.6). Autoclave at 120oC for 15 min. Allow the solution to cool down to below 50oC. Add 1 ml Tween 20 (2.5, sterilized by filtrationO^m), 40 g Polyvinyl pyrrolidone (2.9) and 4g BSA (2.7). (Note: this solution can be stored at 4oC for up to 15 days).

2.17 PBS: phosphate buffer saline (Sigma-Aldrich, lllkirch, France) 5.18 GENbox anaerobic generators (Biomerieux)

3. Apparatus

Usual laboratory apparatus, in particular, the following:

3.1 Plastic Petri Dishes 90 mm diameter (Starsted, Nijmbrecht)

3.2 BagPage® microperforated bag: This is a sterile bag with a full surface non-woven filter. Flora is extracted from the sample and goes through the filter during blending. The flora can be pipetted from behind the filter with no residue or particles of the sample.

3.3 Whatman FTA Elute cards (#512-0527) (Whatman, UK)

3.4 Sterile Syringe filter 0.2μηι cellulose acetate (VWR)

3.5 Sterile Syringe without needle 10ml (Terumo, Leuven, Belgium)

3.6 Blender type Stomacher® set at 120 seconds, fast power (AES Laboratoire)

3.7 Blender (Warring Commercial, Blender 8010, Model 32BL79)

3.8 Sterile plastic disposable tubes (50 ml and 10 ml)

3.9 Ultrasonic Liquid Processor Sonicator Model 400 W (Bioblock Scientific #72408) 3.10 Centrifuge for 10 ml and 50 ml plastic tubes, max X 10,000g.

3.1 1 Vortex shaker

3.12 Light-transparent plastic 1 .5 ml microcentrifugation tubes

3.13 A 650-W Tungsten halogen light source

3.14 Heater block for test tubes: digital hot dry Incubator 100°C

3.15 pH-meter, with two decimal digital readout

3.16 Analytical balance, sensitivity 0.0001 g

3.17 Technical balance, sensitivity 0.01 g

3.18 Pipettes in the range of 0.5 μΙ to 1000μΙ

3.19 Glass test tubes, 15.5 x 100 mm (15 ml)

3.20 Harris Micro-Punch® 2 mm and cutting mat

3.21 Alarm clock 3.22 Magnetic stirrers

3.23 Magnetic bars

3.24 Plastic 96 well plate

3.25 qPCR adhesive clear seals

3.26 Thermocycler Bio-Rad CFX-96 for qPCR

3.27 Centrifuge for 96 well plates, max rpm 1000

3.28 Pipette Boy Acu

3.29 Plastic pipettes 10 ml

3.30 Microplate shaker, max rpm 1350

3.31 Ice

3.32 Anaerobic chambers

3.33 Benchtop centrifuge

4. Sample preparation

4.1 . Pet food admixture

Mix pet food admixture manually with a spoon. Weigh 1 1 .0 g of pet food admixture into a BagPage® and add 99 ml of extraction solution with PVPP (2.16). Incubate for 15 minutes at 20°C and stirr simultaneously (rpm 70). Homogenize the sample with Stomacher® shaker (3.6) for 120 seconds, fast power and leave for 4 minutes. Then remove 15 ml of the suspension from the filtered side of the BagPage® (3.2) and place into a sterile plastic disposable 50ml plastic tube.

Sonicate the suspension for 10 seconds, 40 W, pulse 2s/1 s at 4°C. Seal with a plastic cork and then centrifuge for 2min at 100 g. Remove 10 ml of supernatant and transfer into a sterile plastic tube of 15 ml. Centrifuge for 10 minutes at 10,000 g and discard the

supernatant.

Resuspend the pellet in 1 ml of extraction solution without PVPP (2.15). Vortex until the pellet is completely resuspended. Transfer 500 μΙ of this suspension in a 1.5 ml microcentrifuge tube to perform the DNA extraction (4.4) and transfer another 500 μΙ of this suspension in light-transparent 1.5 ml microcentrifuge tube for PMA (2.10) treatment.

4.2. Extruded pet food

Stir extruded pet food samples manually with a spoon. Weigh 100.0 g of extruded pet food in the blender and blend for 5 minutes. Weigh 1 1.0 g of extruded pet food and proceed as described above (4.1 ) for pet food admixture.

4.3. PMA cross-linking

Add 1 .25 μΙ of PMA (2.10) to the 500 μΙ of the suspension of pet food admixture or extruded pet food, obtained as detailed in 4.1 and 4.2 (final concentration of PMA is 50 μΜ) in a light- transparent 1.5 ml microcentrifuge tube. Following an incubation period of 5 minutes in the dark with occasional mixing, sample tubes are placed on ice (to avoid excessive heating) and exposed to light for 15 min at a distance of 20 cm from the light source (a 650-W halogen light -3.13) to activate and photolyse the PMA.

4.4. DNA extraction with FTA® Elute Cards

40 μΙ of sample suspension treated with PMA (2.10) (or not treated with PMA) are deposited on two different collection areas of the FTA® Elute Cards (3.3) and dry overnight at room temperature or for 30 min at 50°C. These FTA Elute cards can be stored at room

temperature for one year. DNA is recovered from the FTA® Elute matrix through a simple hot water elution procedure. Inhibitory components are retained on the FTA® Elute matrix or disposed of during a short washing step.

Using a 2 mm Harris Micro-Punch® (3.20) and a cutting mat, punch 5 discs from the centre of the sample area and place into a 1 .5 ml microcentrifuge tube. Wash with 500 μΙ of sterile distilled ultra-pure H20 (2.14) by pulse vortexing (3.1 1 ) 3 times for a total of 5 s. Discard wash solution. Centrifuge 30 s (3.33) and remove traces of wash solution. Add 1 10 μ I of sterile distilled H20 ultra-pure (2.14). Centrifuge 30 s (3.33) and transfer to a heating block (3.14) at 100°C for 30 min. Remove the sample from the heating block and pulse vortex (3.1 1 ) the sample 60 pulses. Centrifuge for 30 s (3.33) to pellet the discs and collect 90 μΙ_ of upper suspension in a 1.5 ml microcentrifuge tube. The suspension contains the purified DNA and can be stored at -20°C until required.

Schema of the sample preparation from weight to DNA extraction is presented in Figures 3 and 4. Sample preparation leads to two DNA suspensions from the same pet food sample and treated with PMA (labelled PMA) and without (labelled NoPMA). 5. Procedure for duplex real time PCR analysis

5.1 Standards

5.1 .1. DNA from artificial supplemented pet food for standard curves

Bacterial strains for analysis (e.g. Lb. farciminis CNCM I-3699 and Lb. rhamnosus CNCM I- 3698) are inoculated separately in 9 ml MRS broth (2.1 ) and incubated for 24 hours at 37°C. Weigh 8.8 g of blank pet food admixture into a BagPage® and add 1 .1 mL of 24h culture of each strain and 99 ml of extraction solution with PVPP (2.16). Sample for RT-qPCR is prepared according to Figures 3 and 4.

In parallel, bacterial (e.g. Lb. farciminis CNCM I-3699 and Lb. rhamnosus CNCM I-3698) counts are determined by plating onto MRS agar (2.2) and incubating at 37°C for 48 hours in anaerobic conditions using GENbox generators and chambers (2.18 and 3.32). 5.1 .2 Preparation of standard curves Two dilutions series of the suspension PMA and NoPMA (see Figures 3 and 4) are realised in sterile distilled ultra-pure H20 (2.14).

5.1.3 RT-qPCR reaction mixture A master mix is prepared by adding the following

components for each 15 μΙ reaction to a tube at room temperature. All solutions were gently vortexed and briefly centrifuged after thawing. Negative controls are obtained by mixing 5 μΙ of sterile distilled ultra-pure H20 (2.14) to 15 μΙ of reaction mixture.

The table below shows the reaction mixture used for qPCR run:

5.1.4 RT-qPCR run

The reaction mixture is thoroughly mixed and dispensed in wells of the PCR plate (3.24). Template DNA is added to a volume of δμΙ/reaction to one well containing the reaction mixture. The reactions are mixed by gently centrifuging taking care not to create any bubbles. Reactions are run in an Biorad CFX96TM detection system (Biorad) using the following program: 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 min. Data acquisition was performed using the Biorad CFX Manager version 2.0 (Biorad). 5.2. Pet food samples containing pet food additive (e.g. based on Lb. farciminis CNCM I- 3699 and Lb. rhamnosus CNCM 1-3698).

5 μΙ of DNA extracted from pet food as described in 4.4 is added to 15 μΙ of reaction mixture (table 1 ) in one well of the 96-well plate (3.24). Control and test samples are assayed at the same time as the standards. The assay procedure used is the same as for standards (5.1 .4). Samples are deposited in triplicate in a 96-well plate (3.24). Bacteria (e.g. Lb. farciminis CNCM I-3699 and Lb. rhamnosus CNCM I-3698) estimated counts are calculated by interpolation of the CT values to the standard curve constructed with a serial dilution of a DNA extract pet food model.

6. Calculations 6.1 . Calibration curves Two calibration curves are made for each strain by plotting a serial dilution of extracted DNA of a known concentration of CFU of the targeted bacteria in pet food, after PMA treatment or not, against their CT values, forming two linear relationships. The slope of the curves are used to determine the amplification efficiency from the equation E=10-1/slope-1. The established standard curve without PMA (CT = f(log starting quantity)) is used to calculate total bacteria. Standard curve with PMA is used to calculate only viable bacteria. The difference between total bacteria and intact bacteria represents the proportion of non-intact bacteria.

Baceria (e.g Lb. farciminis CNCM I-3699 and Lb. rhamnosus CNCM I-3698) estimated counts are calculated by interpolation of the CT values to the standard curves constructed as explained in Figure 5.

6.2. Calculation of active substances in pet food

Bacteria (e.g. Lb. farciminis CNCM 1-3699 and Lb. rhamnosus CNCM 1-3698) estimated counts in pet food samples were calculated by interpolation of the CT values to a standard curve constructed with serial dilutions of a DNA extract of a known concentration of CFU of the targeted Lactobacillus in pet food, after PMA treatment (See 6.1 ).

In some embodiments the primers specific for the bacteria may be primers developed based on the sequence of a CRISPR locus present in the bacterium. When the bacterium for use in the assay is a L. farciminis bacterium then the specific primer used may be a primer which amplifies a region of the CRISPR locus of the L. farciminis bacterium. When the bacterium for use in the assay is a L. rhamnosus bacterium then the specific primer used may be a primer which amplifies a region of the CRISPR locus of the L. rhamnosus bacterium.

The term "CRISPR locus" as used herein has its typical meaning in the art, teaching of CRISPR loci can be found at WO2006/073445, WO2007/025097, WO2007/136815, WO2008/108989, WO2010/054154 and/or WO2012/054726, the contents of which are incorporated herein by reference.

"VIABLE/NON-VIABLE CELL ASSAY"

Cells can be categorised as viable or non-viable cells in accordance with the assay presented below. In the assay cells which are blue under the microscope are live cells, whilst those that are green are dead cells. Suitably this assay can be used to distinguish between intact non-viable cells and intact non-replicating cells. "Intact non-viable cells" as used herein are cells that are green in the following assay. "Intact non-cultivatable cells" as used herein are cells that are blue in the following assay.

1 . Weigh 10 g of dried bacterial sample in a sterile Stomacher® bag (available from Seward Ltd.); 2. Add 90 mL of thinner which is a surface-active agent made up using Tween 80 at 2ml per litre in sterile tryptone salt;

3. Mix the Stomacher for 2 min rapid speed;

4. Sonicate for 10s 40W at pulse 2s/1 s using a Vibracell™ sonicator (BIOBLOCK Scientific); 5. , Centrifuge for 1 minute at 1000 revolutions per minute (rpm) in a Chemfiltre™

(CHEMUNEX) where the cellulose paper has a pore of 20 micrometers;

6. Recover the supernatant (1 mL) in an eppendorf tube;

7. Centrifuge the tube for 1 min at 10 000 rpm in a Sigma 3K10 centrifuge (available from BIOBLOCK Scientific) to recover cells;

8. Discard the supernatant;

9. Resuspend the pellet in PBS (1 mL);

10 Sonicate for 5s 25W, pulse 2s/1 s;

1 1. Enumeration of lactobacilli contained in the precipitate is evaluated by epifluorescence and by standard cell count on petri dishes. Carry out the plate count by performing 10-fold serial dilutions from the suspension obtained from the precipitate and inoculating thereof on specific agar medium using spiral plates (available from major laboratory equipment suppliers, for example from Don Whitley Scientific Ltd.);

12. Epifluorescence of cells is measured using DAPI and Viability Substrate (from

CHEMUNEX);

13. 200ul of DAPI at a concentration of 30ug/ml is added to 2ml of the bacterial solution;

14. Incubate for 15 minutes in darkness then filter through a Isopore™ membrane (available from Merck Millipore);

15. Add the Viability Substrate and incubate for 15 minutes in darkness;

16. Air dry the membrane for a few minutes before depositing on a glass slide and the counting the cells under a microscope (x 1000) using epifluorescence;

17. Make the count by counting 30 visual surfaces (or fields) of the membrane.

An example of results is presented in the table below:

Green =

Number Fields Blue = Live Dead

1 21 10

2 18 5

3 20 8

4 34 8

5 15 8

6 14 2

7 18 8 8 18 6

9 12 6

10 13 6

1 1 26 6

12 14 6

13 19 9

14 20 2

15 22 5

16 19 4

17 22 8

18 19 6

19 24 6

20 19 8

21 36 2

22 20 1

23 26 2

24 18 0

25 26 8

26 17 0

27 20 6

28 27 4

29 24 4

30 22 1

Number fields 30 30

Sum 653 185

Average cells/field 21 ,7666667 6,16666667

Dilution factor 100 100

Deposited volume in

ml 0,1 0,1

Number of cells/g 559010880 158372148

Expressed in Log 8,75 8,20

A similar assay is also taught in FR2789398 (which is incorporated herein by reference) which may be used in addition to or alternatively to the assay stated above.

Another assay based on epifluorescence taught in Bernardeau et al. Journal of Applied Microbiology (2001 ), Vol. 91 , pp.1 103-1 109 (which is incorporated herein by reference) may be also additionally or alternatively used. PET FOOD

The term "pet food" as used herein means a food suitable for consumption by a domesticated animal such as a dog, cat, horse, pig, bird, hamster, gerbil, guinea pig, rodent e.g. rat, mouse, rabbit and chinchilla.

In one aspect, the term "pet food" as used herein means a food suitable for consumption by a domesticated dog or cat.

The L. farciminis (optionally in combination with L. rhamnosus) may be applied on, or in, the pet food itself and/or constituent(s) (e.g. ingredients) of the pet food. For example, the anti- contaminant composition may be applied on, or in, a palatant.

Examples of typical constituents found in dog and cat food include palatants, Whole Grain Corn, Soybean Mill Run, Chicken By-Product Meal, Powdered Cellulose, Corn Gluten Meal, Soybean Meal, Chicken Liver Flavor, Soybean Oil, Flaxseed, Caramel Color, Iodized Salt, L- Lysine, Choline Chloride, Potassium Chloride, vitamins (L-Ascorbyl-2- Polyphosphate (source of vitamin C), Vitamin E Supplement, Niacin, Thiamine Mononitrate, Vitamin A Supplement, Calcium Pantothenate, Biotin, Vitamin B12 Supplement, Pyridoxine

Hydrochloride, Riboflavin, Folic Acid, Vitamin D3 Supplement), Vitamin E Supplement, minerals (e.g., Ferrous Sulfate, Zinc Oxide, Copper Sulfate, Manganous Oxide, Calcium lodate, Sodium Selenite), Taurine, L-Carnitine, Glucosamine, Mixed Tocopherols, Beta- Carotene, Rosemary Extract.

In one embodiment, the pet food may be a wet or dry pet food, which may be in the form of a moist pet food (e.g. comprising 18-35% moisture), semi-moist pet food (e.g. 14 to 18% moisture), dry pet food, pet food supplement or a pet treat. Some pet food forms (e.g. moist and semi-moist pet food) are particularly susceptible to contamination due to the fact that the processing conditions for preparing the pet food are not sufficient to kill all microorganisms on, or in, the pet food.

Suitably, the pet food may be in kibble form.

In one embodiment the pet food may be suitable for a dog or a cat.

In another embodiment the pet food may be a bird food. Bird food may mean food that is used to feed pet birds. Typically bird food comprises of a variety of seeds, but may also encompass suet (beef or mutton fat).

The L. farciminis (optionally in combination with L. rhamnosus) may be incorporated within the pet food or on the surface of the pet food, such as, by spraying or precipitation thereon. In one embodiment, the L. farciminis (optionally in combination with L. rhamnosus) is formulated for use in pet food. In this embodiment, the L. farciminis (optionally in

combination with L. rhamnosus) may be present in a composition comprising additional anti- contaminant agents such as phosphoric acid, propionic acid and propionates, sulfites, benzoic acid and benzoates, nitrites, nitrates and parabens. Alternatively, the L. farciminis (optionally in combination with L. rhamnosus) may not comprise any chemicals.

The L. farciminis (optionally in combination with L. rhamnosus) is admixed with the at least one pet food ingredient in amounts sufficient to provide a probiotic effect in a pet fed with the pet food composition. In one embodiment L. farciminis (optionally in combination with L. rhamnosus) is present in the pet food in amounts corresponding to at least 10 ⁵ CFU per gram pet food.

Suitably the at least L. farciminis (optionally in combination with L. rhamnosus) is present at at least 10 ⁶ CFU per gram pet food.

Suitably at at least 10 ⁷ CFU per gram pet food.

Suitably at at least 10 ⁸ CFU per gram pet food.

Suitably at at least 10 ⁹ CFU per gram pet food.

Suitably at at least 10 ¹⁰ CFU per gram pet food.

Suitably at at least 10 ¹¹ CFU per gram pet food.

Suitably at at least 10 ¹² CFU per gram pet food.

In one aspect, the pet food may be a kibble (e.g. dog kibble). An illustrative method of preparing a kibble comprises the following steps:

a. preconditioning by mixing wet and dry ingredients at elevated temperature to form a kibble dough;

b. extruding the kibble dough at a high temperature and pressure;

c. drying the extruded kibble; and

d. enrobing or coating the dried kibble with topical liquid and/or dry ingredients.

Suitably, the L. farciminis (optionally in combination with L. rhamnosus) can be applied to the kibble at any stage in the process, such as at step a and/or step b.

The method of the present invention involves subjecting the pet food ingredient to extrusion. In one embodiment the L. farciminis (optionally in combination with L. rhamnosus) may be admixed with the at least one pet food ingredient prior to extrusion.

In another embodiment, the L. farciminis (optionally in combination with L. rhamnosus) may be added to the at least one pet food ingredient (e.g. kibble) during extrusion.

In one embodiment additional L. farciminis (optionally in combination with L. rhamnosus) may be added after extrusion. Suitably such addition may be via a vacuum coating step.

In another embodiment the additional L. farciminis (optionally in combination with L.

rhamnosus) may be added by a dusting step.

In the method of the present invention the L. farciminis (optionally in combination with L. rhamnosus) may be added following pre-conditioning of the at least one food ingredient. Pre-conditioning may be where the cooking process is initiated (e.g. via steam injection into the pre-conditioning apparatus) and takes place prior to extrusion.

In the method of the present invention it is intended that the admixture comprising L.

farciminis (optionally in combination with L. rhamnosus) is heated to a temperature of at least 71 °C. Suitably the admixture may be heated to a temperature of at least about 75°C, more suitably of at least about 80°C.

In one embodiment, the admixture may be heated at a temperature between about 71 °C to about 85°C.

In another embodiment the admixture may be heated to a temperature of at least about 85°C.

Suitably, the admixture may be heated to a temperature of at least about 100°C.

Suitably, the admixture may be heated to a temperature of at least about 120°C.

Preferably, the admixture may be heated to a temperature of up to about 130°C.

In some embodiments, preferably the admixture is not heated to a temperature of more than about 130°C.

The temperature to which the admixture may be heated may be measured at the die outlet of an extruder.

The temperature during extrusion may be calculated by measuring the temperature of the die plate.

In one embodiment extrusion in the present invention may be carried out at at least 70°C. Suitably the extrusion may be carried out at at least 75°C, more suitably at at least 80°C. Suitably the extrusion may be carried out at at least 85°C.

In one embodiment extrusion may be carried out at about 100°C to about 200°C, suitably at about 1 10°C to about 190°C. Suitably at about 120°C to about 190°C. Suitably at about 130°C to about 190°C. Suitably at about 140°C to about 190°C. Suitably at about 150°C to about 190°C. Suitably at about 150°C to about 180°C. Suitably at about 160°C to about 180°C.

In one embodiment the extrusion may be carried out at about 170°C.

In one embodiment the method may include heating the one or more pet food ingredient or admixture comprising at least one pet food ingredient and at least L. farciminis (optionally in combination with L. rhamnosus) to at least 100°C in an extrusion process. Suitably to at least about 105°C in the extrusion process. Suitably to at least 1 10°C in the extrusion process.

Suitably to at least 1 15°C before extrusion. Suitably to at least 120°C before extrusion.

Suitably to at least 125°C before extrusion. Suitably to at least 130°C before extrusion.

In one embodiment the method may include a cooling step wherein the heated pet food ingredient is subsequently cooled to below 100°C before extrusion. Suitably to below 95°C in the extrusion process. Suitably to below 90°C in the extrusion process, Suitably to below 85°C in the extrusion process. Suitably to below 80°C in the extrusion process.

In one embodiment L. farciminis (optionally in combination with L. rhamnosus) may be added during the cooling step.

The skilled person will appreciate that the pressure during extrusion will be dependent upon both the temperature as well as the mechanical forces applied to the pet food or admixture. In one embodiment the extrusion may be carried out at a pressure of about 10 to about 35 Bar. In another embodiment extrusion may be carried out at a pressure of about 20 to about 35. Suitably the extrusion may be carried out at a pressure of about 25 to about 33 Bar. In one embodiment the method of the invention further comprises one of more of the following steps

a) a drying step and/or

b) a coating step and/or

c) a sifting step and/or

d) a dusting step and/or

e) a quality control step, such as qPCR

In some embodiments, the extrudate may have a moisture content less than 12%, specifically less than 10%, more specifically less than 8%.

Suitably the term "pet food" as used herein does not encompass feed for livestock animals. The term "livestock", as used herein refers to any farmed animal. Preferably, livestock is one or more of ruminants such as cattle (e.g. cows or bulls (including calves)), mono-gastric animals such as poultry (including broilers, chickens and turkeys), pigs (including piglets), birds, or sheep (including lambs).

In one embodiment the pet food of the present invention may be a pet food comprising L farciminis (optionally in combination with L. rhamnosus). Suitably the pet food may comprise farciminis and L. rhamnosus.

In one embodiment the pet food may be an extruded pet food comprising farciminis and L. rhamnosus. MEAT BASED PET FOOD PRODUCT

A "meat based pet food product" as used herein means any pet food product based on meat.

The meat based pet food product is suitable for animal consumption.

In one embodiment the pet food of the invention may be a meat based extruded pet food product. A meat based pet food product may comprise non-meat ingredients such as for example water, salt, flour, milk protein, vegetable protein, starch, hydrolysed protein, phosphate, acid, spices, colouring agents and/or texturising agents.

A meat based pet food product in accordance with the present invention preferably comprises between 5-90% (weight/weight) meat. In some embodiments the meat based pet food product may comprise at least 30% (weight/weight) meat, such as at least 50%, at least 60% or at least 70% meat.

In some embodiments the meat based pet food product may comprise a cooked meat, such as ham, loin, picnic shoulder, bacon and/or pork belly for example.

The meat based pet food product may be one or more of the following:

Dry or semi-dry cured meats - such as fermented products, dry-cured and fermented with starter cultures;

Emulsified meat products (e.g. for cold or hot consumption), such as mortadella, frankfurter, luncheon meat and pate;

Fresh meat muscle, such as whole injected meat muscle, for example loin, shoulder ham, marinated meat;

Ground and/or restructured fresh meat - or reformulated meat, such as upgraded cut-away meat by cold setting gel or binding, for example raw, uncooked loin chops, steaks, roasts, fresh sausages, beef burgers, meat balls, pelmeni;

Poultry products - such as chicken or turkey breasts or reformulated poultry; and

Retorted products - autoclaved meat products, for example picnic ham, luncheon meat, emulsified products.

In one embodiment of the present invention the meat based pet food product is a processed meat product, such as for example a sausage, bologna, meat loaf, comminuted meat product, ground meat, bacon, polony, salami or pate.

A processed meat product may be for example an emulsified meat product, manufactured from a meat based emulsion, such as for example mortadella, bologna, pepperoni, liver sausage, chicken sausage, wiener, frankfurter, luncheon meat, meat pate.

The meat based emulsion may be cooked, sterilised or baked, e.g. in a baking form or after being filled into a casing of for example plastic, collagen, cellulose or a natural casing. A processed meat product may also be a restructured meat product, such as for example restructured ham. A meat product of the invention may undergo processing steps such as for example salting, e.g. dry salting; curing, e.g. brine curing; drying; smoking; fermentation; cooking; canning; retorting; slicing and/or shredding.

In one embodiment the meat to be contacted with the L. farciminis (optionally in combination with L. rhamnosus) may be minced meat. In another embodiment the pet food may comprise an emulsified meat product. MEAT

The term "meat" as used herein means any kind of tissue derived from any kind of animal. The term meat as used herein may be tissue comprising muscle fibres derived from an animal. The meat may be an animal muscle, for example a whole animal muscle or pieces cut from an animal muscle.

In another embodiment the meat may comprise inner organs of an animal, such as heart, liver, kidney, spleen, thymus and brain for example.

The term meat encompasses meat which is ground, minced or cut into smaller pieces by any other appropriate method known in the art.

The meat may be derived from any kind of animal, such as from cow, pig, lamb, sheep, goat, chicken, turkey, ostrich, pheasant, deer, elk, reindeer, buffalo, bison, antelope, camel, kangaroo; horse, rodent, chinchilla.

In one embodiment the meat is beef, pork, chicken, lamb and/or turkey.

METABOLITES

In some embodiments the method for producing a pet food may comprise admixing metabolites of a fermented microorganism. Alternatively, or additionally, the metabolites may be produced in the pet food obtainable or obtained by the method of the present invention by at least L. farciminis (optionally by at least L. farciminis in combination with L. rhamnosus) in situ in the pet food.

The metabolites may be added to the pet food composition separately from the at least L. farciminis (optionally the at least L. farciminis in combination with L. rhamnosus) or in the form of a mixture with said at least L. farciminis (optionally at least L. farciminis in

combination with L. rhamnosus).

Metabolites may be obtained by using any standard techniques known to the person skilled in the art to which the present invention pertains. The metabolites may suitably be produced by fermenting a microorganism, in particular a probiotic microorganism in a viable form in a suitable medium and at suitable conditions. Suitable media are as described supra.

In some embodiments the metabolites may be produced or already be present in a fermentate comprising the at least L. farciminis (optionally in combination with L. rhamnosus) for use in the methods herein.

In one embodiment the metabolites may be made by fermenting microorganisms in a dairy product, such as milk based medium. In some embodiments the metabolites may be used in the method and/or uses of the present invention in the form of a cell-free fermentate.

Metabolites include but are not limited to metabolites selected from the group consisting of organic acid, peptide, peroxides (e.g. hydrogen peroxide), polysaccharides, amino acids, enzymes, vitamins, sugars, lipids and lipoproteins or combinations thereof.

In one embodiment the metabolites include one or more organic acids selected from the group consisting of lactic acid, acetic acid, propionic acid, succinic acid and orotic acid or combinations thereof.

In another embodiment the metabolites include one or more peptides (e.g. a bacteriocin). The person skilled in the art will appreciate that the metabolites may be admixed with the at least L. farciminis (optionally in combination with L. rhamnosus) and at least one pet food ingredient in any suitable manner. For example, the metabolites may be applied onto the exterior surface of the pet food (e.g. the extrudate) or homogeneously distributed throughout the pet food (e.g. extrudate).

Suitably, the metabolites may be homogeneously distributed through the pet food.

In one embodiment metabolites taught herein may be admixed with the at least L. farciminis

(optionally in combination with L. rhamnosus) and at least one pet food ingredient before extrusion, during extrusion or after extrusion.

Suitably the metabolites may be admixed before extrusion.

Suitably the metabolites may be admixed during extrusion.

Suitably the metabolites may be admixed after extrusion.

In one embodiment the at least L. farciminis (optionally in combination with L. rhamnosus) and metabolite may be admixed before extrusion.

In one embodiment the at least L. farciminis (optionally in combination with L. rhamnosus) and metabolite may be admixed during extrusion.

PROBIOTIC EFFECT

The present invention also concerns the use of a pet food (e.g. the pet food obtainable by the methods of the present invention) comprising at least Lactobacillus farciminis and at least one pet food ingredient in an effective amount for inducing a probiotic effect in a pet.

Suitably the pet food for the use stated above may be a pet food that comprises

Lactobacillus farciminis, Lactobacillus rhamnosus or a combination thereof in an effective amount and at least one pet food ingredient.

Suitably, the ratio between Lactobacillus rhamnosus and Lactobacillus farciminis may be from 1 :9 to 9:1 . The L. farciminis (optionally in combination with L. rhamnosus) or fermentate for use in the methods and/or uses of the present invention may be comprised in a probiotic culture.

The term "probiotic culture" as used herein defines microorganisms (including bacteria or yeasts for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism. Probiotics may improve the microbial balance in one or more mucosal surfaces. For example, the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin. Whilst there are no lower or upper limits for probiotic intake, it has been suggested that at least 10 ⁶ -10 ¹² , preferably at least 10 ⁶ -10 ¹⁰ , preferably 10 ⁸ -10 ⁹ , cfu as a daily dose will be effective to achieve the beneficial health effects in a subject.

The term "effective amount" or "effective amount for inducing a probiotic effect" as used herein means that the L. farciminis (optionally in combination with L. rhamnosus) or the admixture comprising the L. farciminis (optionally in combination with L. rhamnosus) and at least one food ingredient is administered to a pet (e.g. by feeding) in an amount that produces a probiotic effect in the pet. Suitable dosages of L farciminis (optionally in combination with L. rhamnosus) are taught in the preceding embodiments and it is intended that such dosages encompass effective amounts for inducing a probiotic effect in a pet.

The probiotic effect may be any probiotic effect including but not limited to one or more of the following: a reduction in the growth of pathogenic microorganisms (e.g. an antipathogenic or antimicrobial effect) in the pet or alterations in the immunity of the pet, improvements in gastrointestinal health, improvements in diabetic conditions, improvements in dental health, a reduction in the activity of endotoxins in the pet, oxidative effects, or a reduction in the incidence of cancer.

It is intended that the probiotic effect is determined by comparing a pet administered with an effective amount of the pet food of the present invention to a pet that has not been administered with the pet food, this is particularly true where terms such as "improvement" or "alteration" are used with regard to the probiotic effect.

Without wishing to be bound by theory it is believed that the L. farciminis and/or L.

rhamnosus for use in the methods and/or uses of the present invention may exert its probiotic effect by a number of different methods, such as the production of metabolites, adhesion to cell membranes (e.g. mucosal membranes) in the gut, aggregation and/or co- aggregation of pathogenic microorganisms, competition with pathogenic microorganisms for nutrients, production of biosurfactants, production of lactic acid or combinations thereof. The exopolysaccharides composition of the L. farciminis and/or L. rhamnosus may contribute to these effects and/or facilitate adhesion to cell membranes in the pet. Without wishing to be bound by theory it is believed that the L. farciminis and/or L. rhamnosus for use in the present invention may exert its effect at the intestinal lumen (e.g. via immune stimulation or effecting enterocytes) or in the intestinal mucosa (e.g. via signalling or competition with pathogens for nutrients).

When the probiotic effect is a reduction in the growth of pathogenic microorganisms in the pet this may be a reduction in the growth of pathogenic organisms in the gut.

Suitably the pet food comprising L. farciminis (optionally in combination with L. rhamnosus) present as intact non-cultivatable cells may be able to reduce growth of Clostridium, Salmonella and Escherichia.

The pathogenic microorganism that is reduced in growth may be selected from one or more of the genera Clostridium, Helicobacter, Escherichia, Staphylococcus, Salmonella, Listeria, Brachyspira or Pasteurella.

Suitably the pet food comprising L. farciminis (optionally in combination with L . rhamnosus) present as intact non-cultivatable cells is able to reduce growth of Clostridium, Salmonella and Escherichia .

Suitably the pet food comprising L. farciminis (optionally in combination with L . rhamnosus) present as intact non-viable cells may be able to reduce growth of Clostridium.

Suitably the pathogenic microorganism that is reduced in growth may be Clostridium perfingens.

Suitably the pathogenic microorganism that is reduced in growth may be Helicobacter pylori. Suitably the pathogenic microorganism that is reduced in growth may be Escherichia coli. Suitably, E. coli selected from one or more of the strains: K88, 02K1 , 078K80, 0138K81 or 01 K1 .

Suitably the pathogenic microorganism that is reduced in growth may be a Staphylococcus aureus.

Suitably the pathogenic mircroorganism that is reduced in growth may be a Salmonella typhimurium, Salmonella enteritidis or combinations thereof. More suitably S. typhimurium PS1 , VS2 or combinations thereof.

Suitably the pathogenic microorganism that is reduced in growth may be Listeria

monocytogenes.

Suitably the pathogenic microorganism that is reduced in growth may be Brachyspira pilosicoli.

Suitably the pathogenic microorganism that is reduced in growth may be a Pasteurella multocida. When the probiotic effect is the alteration of the immunity of a pet this may lead to an immune stimulation and/or another effect such as the reduction in allergies of a pet and/or the prevention or treatment of arthritis.

In embodiments where the probiotic effect is the immune stimulation of a pet then this stimulation may be characterised by an increase in the levels of macrophages and/or lymphocytes (e.g. B-lymphocytes, T-lymphocytes or combinations thereof). Alternatively or additionally the immune stimulation may lead to an increase in the level of cytokines in the pet. In some embodiments immune stimulation may result in an increase in the level of interferons in the pet. In other embodiments the immune stimulation may lead to or be mediated by the following: inactivation of microbial endotoxins, neutralisation of viruses, stimulation of interferons or combinations thereof.

When the probiotic effect is an improvement in gastrointestinal health this may result in a reduction in the incidence of diarrhoea, a reduction in symptoms associated with irritable bowel disease and/or Crohn's disease, alterations in intestinal mucosal permeability, a reduction in the colonisation of the gastrointestinal tract by pathogenic bacteria or a restoration of the microecology (e.g. after administration of medicaments, for example antibiotics), or combinations thereof.

FORMS

The product and/or the L. farciminis (optionally in combination with L. rhamnosus) of the present invention may be used in any suitable form - whether when alone or when present in a composition.

The L. farciminis (optionally in combination with L. rhamnosus) may be formulated as a composition.

The composition may be formulated in any suitable way to ensure that the composition has the desired characteristics such as that the cells remain viable, non-viable or combinations.

Alternatively or additionally the fermentate and/or metabolite for use in the methods and/or uses of the present invention may be formulated as a composition.

Suitably the composition may be formulated to ensure that the cells comprised in the composition remain intact, suitably intact non-viable or intact non-cultivatable, or

combinations thereof.

The L. farciminis (optionally in combination with L. rhamnosus) may be formulated as a liquid, a dry powder or a granule. Alternatively or additionally the fermentate and/or metabolite may be formulated as a liquid, a dry powder or a granule. The dry powder or granules may be prepared by means known to those skilled in the art, such as, in top-spray fluid bed coater, in a buttom spray Wurster or by drum granulation (e.g. High sheer granulation), extrusion, pan coating or in a microingredients mixer.

Suitably, any of the compositions may be provided as a spray-dried or freeze-dried powder. In one aspect, the composition is in a liquid formulation. Such liquid consumption may contain one or more of the following: a buffer, salt, sorbitol and/or glycerol.

In one embodiment the composition may be formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na ₂ S0 ₄ , Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol, 1 ,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.

In some embodiments the composition may be admixed with another component.

The components may be prebiotics. Here, a prebiotic is:

"a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of beneficial bacteria".

Prebiotics are typically non-digestible carbohydrate (oligo- or polysaccharides) or a sugar alcohol which is not degraded or absorbed in the upper digestive tract. Known prebiotics used in commercial products and useful in accordance with the present invention include inulin (fructo-oligosaccharide, or FOS) and transgalacto-oligosaccharides (GOS or TOS). Suitable prebiotics include palatinoseoligosaccharide, soybean oligosaccharide, alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), non-degradable starch, lactosaccharose, lactulose, lactitol, maltitol, maltodextrin, polydextrose (i.e. Litesse®), lactitol, lactosucrose, soybean

oligosaccharides, palatinose, isomalto-oligosaccharides, gluco-oligosaccharides and xylo- oligosaccharides, pectin fragments, dietary fibres, mannan-oligosaccharides.

Dietary fibres may include non-starch polysaccharides, such as arabinoxylans, cellulose and many other plant components, such as resistant dextrins, inulin, lignin, waxes, chitins, pectins, beta-glucans and oligosaccharides.

In one embodiment the composition according to the present invention may be admixed with a prebiotic. In another embodiment the present invention may relate to a pet food comprising at least L. farciminis (optionally in combination with L. rhamnosus) and a prebiotic. Suitably the present invention may relate to a pet food comprising at least L. farciminis (optionally in combination with L. rhamnosus and/or one or more metabolites) and a prebiotic. The prebiotic may be administered simultaneously with (e.g. in admixture together with or delivered simultaneously by the same or different routes) or sequentially to (e.g. by the same or different routes) the pet food (or constituents thereof) according to the present invention. Other components of the combinations of the present invention include polydextrose, such as Litesse®, and/or a maltodextrin and/or lactitol. These other components may be optionally added to composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) to assist the drying process.

In some embodiments it may help the survival of viable cells comprised therein.

Further examples of other suitable components include one or more of: thickeners, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles, excipients, diluents, lubricating agents, flavouring agents, colouring matter, suspending agents, disintegrants, granulation binders etc. These other components may be natural. These other components may be prepared by use of chemical and/or enzymatic techniques.

In one embodiment the composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) may be encapsulated. In one embodiment the composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) as a dry powder or granule as described in WO2007/044968 (referred to as TPT granules) - reference incorporated herein by reference.

In one preferred embodiment composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) for use in the present invention may be used in combination with one or more lipids.

For example, the composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) for use in the present invention may be used in combination with one or more lipid micelles. The lipid micelle may be a simple lipid micelle or a complex lipid micelle.

The lipid micelle may be an aggregate of orientated molecules of amphipathic substances, such as a lipid and/or an oil.

As used herein the term "thickener or gelling agent" refers to a product that prevents separation by slowing or preventing the movement of particles, either droplets of immiscible liquids, air or insoluble solids. Thickening occurs when individual hydrated molecules cause an increase in viscosity, slowing the separation. Gelation occurs when the hydrated molecules link to form a three-dimensional network that traps the particles, thereby immobilising them.

The term "stabiliser" as used here is defined as an ingredient or combination of ingredients that keeps a product (e.g. a pet food product) from changing over time. The term "emulsifier" as used herein refers to an ingredient (e.g. a pet food ingredient) that prevents the separation of emulsions. Emulsions are two immiscible substances, one present in droplet form, contained within the other. Emulsions can consist of oil-in-water, where the droplet or dispersed phase is oil and the continuous phase is water; or water-in-oil, where the water becomes the dispersed phase and the continuous phase is oil. Foams, which are gas-in-liquid, and suspensions, which are solid-in-liquid, can also be stabilised through the use of emulsifiers.

As used herein the term "binder" refers to an ingredient (e.g. a pet food ingredient) that binds the product together through a physical or chemical reaction. During "gelation" for instance, water is absorbed, providing a binding effect. However, binders can absorb other liquids, such as oils, holding them within the product. In the context of the present invention binders would typically be used in solid or low-moisture products for instance baking products:

pastries, doughnuts, bread and others.

"Carriers" or "vehicles" mean materials suitable for administration of the composition (e.g. comprising at least L. farciminis (optionally in combination with L. rhamnosus)) and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

Examples of excipients include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of: polyvinylpyrrolidone,

hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesium stearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are in admixture together or even when they are delivered by different routes) or sequentially (e.g. they may be delivered by different routes). NUCLEOTIDE SEQUENCE

In some embodiments nucleotide sequences (e.g. primers) may be used, particularly in the "Intact Cell Assay" taught herein.

The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or

recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.

The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA.

Typically, the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).

PREPARATION OF THE NUCLEOTIDE SEQUENCE

The nucleotide sequence may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al., (1981 ) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801 -805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors. The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al., {Science (1988) 239, pp 487- 491 ). HYBRIDISATION

The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto. The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.

Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g. 50°C and 0.2xSSC {1 xSSC = 0.15 M NaCI, 0.015 M Na ₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e.g. 65°C and O.l xSSC {1xSSC = 0.15 M NaCI, 0.015 M Na ₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 50°C and 0.2xSSC).

In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g. 65°C and O.l xSSC). RECOMBINANT

In one aspect the sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1 -3, Cold Spring Harbor Laboratory Press. GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1 -3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.

Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

ADVANTAGES

Surprisingly it has been found that Lactobacillus farciminis (optionally in combination with L. rhamnosus) can be used in the manufacture of an extruded pet food and is able to still provide probiotic effects even after the extrusion manufacturing process is completed. It is surprising that a substantial proportion of the cells are able to remain intact.

Advantageously, Lactobacillus farciminis (optionally in combination with L. rhamnosus) is able to still provide a probiotic effect when administered in an effective amount to a pet following manufacturing processes comprising extrusion. This is highly surprising, especially as many extrusion processes require the use of high temperatures and/or pressure and thus negatively impact bacteria in the extruder.

It has surprisingly been found that the bacterial fermentates as disclosed herein when used before or during extrusion are able to retain their probiotic effectiveness post-extrusion. The bacteria and/or fermentates as disclosed herein post extrusion may comprise intact nonviable and/or intact non-cultivable cells which have surprisingly been found to be stable during extrusion and still have the ability to provide the probiotic effect to a pet administered with a pet food comprising the bacteria and/or fermentate.

Additionally, we have found that the use of L. farciminis (optionally in combination with L. rhamnosus) in a non-viable form provides the advantage of not needing manufacturing facilities suitable for handling live microbial cells. Still further we have found that the use of non-viable L. farciminis (optionally in combination with L. rhamnosus) provides the advantage of prolonging shelf life for the probiotic functionality in the pet food composition by not relying on living probiotic cells. Still further, we have found that the use of non-viable L. farciminis (optionally in combination with L. rhamnosus) provides the advantage that the probiotic functionality can be introduced into pet food at any step of the extrusion manufacturing process.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR

BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991 ) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.

The term "protein", as used herein, includes proteins, polypeptides, and peptides.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the lUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bacteria" or "a bacterium" includes a plurality of such candidate agents and reference to "the pet food ingredient" includes reference to one or more pet food ingredients and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The invention will now be described, by way of example only, with reference to the following Figures and Examples. EXAMPLES

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Example 1 : Extruded pet food comprising non-viable probiotic microorganism of Lactobacillus rhamnosus and Lactobacillus Farciminis

Extrusion of dog treats (model system) containing heat treated fermented milk product which is a co-culture of Lactobacillus rhamnosus CNCM I-3698 and Lactobacillus farciminis CNCM I-3699 (hereinafter named Sample A).

Objective Test of stability of intact non-viable probiotic microorganism of Lactobacillus rhamnosus and Lactobacillus farciminis during extrusion of a pet food treat composition.

Experimental set up

Extrusion was done on a Clextral BC 45 co-rotating twin-screw extruder with 5 barrel sections. Section no 4 was a vacuum section used for evaporation of steam during the process. This evaporation served to remove water from the extrusion dough and to lower temperature in the final section of the extruder.

Screw speed for the extrusion process was 150 rpm and the temperatures were as shown in the Table 1 :

The above recipe was mixed and fed into section 1 of the extruder by a volumetric feeder. Feed rate of dry mix was 36 kg/h. Water for the process was added into section 1 of the extruder by a piston pump at a rate of 6.5 kg/h.

The components tested in the model system were:

1 . 1 % Sample A + 9% cereal carrier 2. 10% Cereal carrier (reference)

In each case the powdery component was mixed 1 :1 with glycerol w/w, heated to 37°C and added by a mono-pump into the final part of section 4 of the extruder. Feed rate for this 1 :1 mixture was a total of 8.8 kg/h to achieve a concentration in final product of 10% of the specific component.

The product was extruded through a single die opening of 12 mm 0. Final product was left at room temperature for approximately 30 minutes to cool off before being cut into pieces of 1 cm. Water content at this stage was measured to around 15%. Various types of drying were tested to obtain a final water content of 7-10%.

The test sample and the reference sample were processed at the extrusion parameters listed above.

Process data and samples

500 g samples were collected for all three batches before and after extrusion

Test of samples

Water content

Cell count by the "Intact Cell Assay" taught herein

The variation of the "Intact Cell Assay" taught herein is + / - 0,5 Log.

Quantification of cells was performed using the "Intact Cell Assay" as taught herein.

Results

Water content after drying was 7-10%

Quantification of intact bacteria cells showed the results listed below in Table 3:

Example 2: Extruded pet food comprising probiotic microorganism of Lactobacillus rhamnosus and Lactobacillus farciminis

Extrusion of dog treats (model system) containing fermented milk product which is a co- culture of Lactobacillus rhamnosus CNCM I-3698 and Lactobacillus farciminis CNCM I-3699 (hereinafter named Sample B).

Objective Test of stability of probiotic microorganism of Lactobacillus rhamnosus and Lactobacillus farciminis during extrusion of a pet food composition containing same.

Experimental set up

Extrusion was done on a Clextral BC 45 co-rotating twin-screw extruder with 5 barrel sections. Section no 4 was a vacuum section used for evaporation of steam during the process. This evaporation served to remove water from the extrusion dough and to lower temperature in the final section of the extruder.

Screw speed for the extrusion process was 150 rpm and the temperatures were as shown Table 4 below:

The above recipe was mixed and fed into section 1 of the extruder by a volumetric feeder. Feed rate of dry mix was 36 kg/h. Water for the process was added into section 1 of the extruder by a piston pump at a rate of 6.5 kg/h.

The components tested in the model system were: 1 . 0.2% Sample B + 9.8% cereal carrier

2. 10% Cereal carrier (reference)

In each case the powdery component was mixed 1 :1 with glycerol w/w, heated to 37°C and added by a mono-pump into the final part of section 4 of the extruder. Feed rate for this 1 :1 mixture was a total of 8.8 kg/h to achieve a concentration in final product of 10% of the specific component.

The product was extruded through a single die opening of 12 mm 0. Final product was left at room temperature for approximately 30 minutes to cool off before being cut into pieces of 1 cm. Water content at this stage was measured to around 15%. Various types of drying were tested to obtain a final water content of 7-10%.

The test sample and the reference were processed at the extrusion parameters listed above Process data and samples

500 g samples were collected for all three batches before and after extrusion

Test of samples

Water content

Cell count by a the "Intact Cell Assay" taught herein

The variation of the "Intact Cell Assay" taught herein is + / - 0,5 Log.

Quantification of cells was performed using the "Intact Cell Assay" as taught herein.

Results

Water content after drying was 7-10%

Quantification of intact bacteria cells showed the results listed in Table 6 below:

viable: 50% intact non-viable cells, suitably at least 75% viable: 25% intact non-viable cells. * ^** These samples comprise at least intact non-viable cells.

Example 3: Quantification of L. farciminis and/or L. rhamnosus before and after extrusion at high temperatures

Extrusion dry pet food (model system) at high temperature and pressure containing fermented milk product which is a co-culture of Lactobacillus rhamnosus CNCM I-3698 and Lactobacillus farciminis CNCM I-3699 (hereinafter named Sample C). Objective

Test of stability of non-viable probiotic microorganism of Lactobacillus rhamnosus and Lactobacillus Farciminis during extrusion of a pet food composition.

Experimental set up

Extrusion was done on a Clextral BC 45 co-rotating twin-screw extruder with 5 barrel sections. Sample C was admixed with the dry pet food ingredients prior to extrusion. Screw speed for the extrusion process was 150 rpm and the temperatures were as shown below:

The above recipe was mixed and fed into section 1 of the extruder by a volumetric feeder. Feed rate of dry mix was 36 kg/h. Water for the process was added into section 1 of the extruder by a piston pump at a rate of 6.5 kg/h.

The components tested in the model system were: 1 . 0.91 % of Sample C + 8.19% cereal carrier

2. 9.1 % Cereal carrier (reference)

The product was extruded through a single die opening of 12 mm 0. Final product cut off by a knife mounted on at the extruder exit. The expanded kibbles was left at room temperature for approximately 30 minutes before water content was measured. Water content at this stage was measured to around 15%. Various types of drying were tested to obtain a final water content of 7-10%.

The test sample and the reference sample were processed using these extrusion parameters.

Process data and samples

500 g samples were collected for all three batches before and after extrusion

Test of samples

Water content

Cell count by the "Intact Cell Assay" taught herein.

The variation of the "Intact Cell Assay" taught herein is + / - 0,5 Log.

Quantification of cells was performed using the "Intact Cell Assay" as taught herein.

Quantification of total bacteria from the "Intact Cell Assay" taught herein

Quantification of the total number of intact non-viable cells before and after extrusion was calculated and the results are presented in the Table 7 below:

As can be seen from the results in Table 7, surprisingly extrusion even at high temperatures does not substantially reduce the number of intact non-viable L. rhamnosus cells. A similar analysis was performed for intact non-viable L. farciminis cells and the data are presented below in Table 8:

As can be seen from the results in Table 8, surprisingly extrusion even at high temperatures does not substantially reduce the number of intact non-viable L. farciminis cells.

Example 4: Probiotic effects of intact non-cultivatable cells and intact non-viable cells on attachment of pathogenic bacteria on intestinal mucus in vitro

The ability to inhibit adhesion of pathogenic bacteria on intestinal mucus would hinder the colonization, and thus reduce the risk of illness. The aim of this study was to start to elucidate the potential anti-adhesive properties of the products, as one mechanism of their health promoting effects in monogastric animals. The functionality of the test products was studied in vitro, applying an assay adapted from Conway P. L, Welin A., Cohen P. S. 1990.

Presence of K88-Specific Receptors in Porcine Ileal Mucus Is Age Dependent. Infection and Immunity 58(10) p. 3178-3182 which is incorporated herein by reference.

Test products

The test products originate from a fermentation containing a mixture of L. rhamnosus CNCM I-3698 and Lactobacillus farciminis CNCM I-3699. The whole fermentate in Sample D is a co- culture of L. farciminis and L. rhamnosus comprising both viable and intact non-viable cells, suitably at least 50% viable: 50% intact non-viable cells, suitably at least 75% viable: 25% intact non-viable cells. Out of the viable cells approximately 25-30% may be intact non- cultivatable cells. Sample E is a co-culture of L. farciminis and L. rhamnosus comprising only intact non-viable cells and does not comprise any viable cells.

Table 9. Name and identification data of the tested products.

Product name Product batch FU /g product

Sample D production date 1.00E+08 10.02.2010

Sample E production date 5.00E+08

09.02.2010

Due to the carrier matrix, the products could not be tested as total suspensions, and a pretreatment was needed. Thereby, the product was mixed 1 :5 (Sample D) or 1 :10 (Sample E) with the buffer (pH 7.4), extracted by shaking on a horizontal position for 10 minutes at 200 rpm rate, after which the larger matrix particles were removed by centrifugation at 200xg for 15 minutes. The supernatant (containing LAB and soluble components) was collected and diluted further for the tests. This soluble fraction was tested corresponding to the following product concentrations in reaction well.

• Sample D: 4%, 1 % and 0.1 % (0.04, 0.01 and 0.001 g product/ml buffer, respectively) · Sample E: 2%, 0.2% and 0.02% (0.02, 0.002 and 0.0002 g product/ml buffer,

respectively)

Difference in concentration (w/v) of the test products originates from the method to dose probiotic candidates based on product cell count. Thus, the sample dilutions were prepared according to that. However, the results may be more understandable and comparable with other type of products when presented as weight per volume concentrations. Link between the weight per volume dose and cell count (fluorescent units) for these products is shown in Table 10. For Sample D the 4.0E+06 FU/ml level was practically the highest that could be tested.

Table 10. Dose of the products in test: correlation between cell count and weight per volume (%).

Target level (cells/ml in reaction well)

1.00E+08 1.00E+07 4.00E+06 1.00E+06 1.00E+05

Product FU/g product Corresponding concentration (%; w/v) in reaction well

Sample D 1.00E+08 4 ϊ 5ΤΪ

Sample E 5.00E+08 2 0.2 0.02

Pathogenic bacteria in test

E. coli K88, C. perfringens ATCC 3626 (type B), C. perfri ngen s ATCC 13124 (type A) and S. typhimurium ATCC 23564

Adhesion surfaces

Solutions of mucus (0.1 mg protein/ml) isolated from 21 -day old broiler jejunum, ileum and caecum were immobilized on polyethylene terephthalate wells.

Assay (competition) To the appropriate wells, the test product and the [ ³ H]thymidine -labeled bacterial suspension of adjusted density (A600=0.25) were added. After 1 -hour incubation, unbound bacterial cells were washed away and the amount of adhered cells determined by scintillation counter. The effect of test product on adherence of pathogen bacteria on mucus was calculated as percentage of the maximum pathogen adherence (maximum 100% = non- inhibited; i.e. no test product added).

Each combination (mucus, sample dilution, pathogen) was tested as three replicate reaction wells per run, and total of three replicate independent runs per combination were performed.

Results and discussion

Sample D

The effect of Sample D on attachment of the tested pathogens on intestinal mucus in vitro is shown in Figure 1 as mean ± SD (N=3). The soluble fraction of Sample D (containing LAB and soluble components) at the highest concentration, corresponding to 4% (w/v) of Sample D in reaction, reduced significantly (Table 1 1 ) the adherence of all tested pathogens on all mucus preparations. When the soluble fraction of the product was diluted to 1 % (w/v) level, still less than 50% of the C. perfringens ATCC 3626 and S. typhimurium ATCC 23564 cells were able to adhere mucus in vitro.

In addition, inclusion of Sample D showed a clear dose response effect with Clostridia and salmonella. In contrast this was not seen with E. coli K88. Origin of the mucus had no significant effect on the inhibitory effect of Sample D.

For comparison, based on our earlier studies, for example with the best performing commercial feed yeast products, the presence of whole suspension at concentrations around 0.3-1 % in reaction, on average 20-40% of the E. coli K88 -bacteria can adhere on mucus in vitro.

Table 11. For each mucus type and pathogen, significance of difference (P-values; 95% conf. level) in comparisons between non-inhibited reaction (maximum adherence) and Sample D effect is shown. The tested soluble fraction of Sample D corresponded to product concentration of 0.1 %, 1 % or 4% (w/v) in reaction. jejunum

Sample E

The effect of Sample E on adherence of the tested pathogens on intestinal mucus in vitro is shown in Figure 2 as mean ± SD (N=3). The soluble components in Sample E decreased significantly attachment of C. perfringens ATCC 3626 at the highest product concentration, corresponding to 2% (w/v) of Sample E in reaction. Additionally the reducing effect on adherence of S. typhimurium on jejunum and ileum mucus was statistically significant. Table 12. For each mucus type and pathogen, significance of difference (P-values; 95% conf. level) in comparisons between non-inhibited reaction (maximum adherence) and

Sample E effect is shown. The tested soluble fraction of Sample E corresponded to product concentration of 0.02%, 0.2% or 2% (w/v) in reaction.

jejunum

Sample E E. coli K88 C. perf 3626 C. perf 13124 S. typh 23564

0.02 % 0.921 0.956 0.263 0.013

0.2 % 0.191 0.914 0.289 0.011

2 % 0.884 < 0.001 0.114 0.209

ileum

Sample E E. coli K88 C. perf 3626 C. perf 13124 S. typh 23564

0.02 % 0.039 0.684 0.548 0>011

0.2 % 0.074 0.912 0.032 0.023

2 % 0.649 < 0.001 0.206 0.367

caecum

Sample E E. coli K88 C. perf 3626 C. perf 13124 S. typh 23564

0.02 % 0.562 0.231 0.667 0.480

0.2 % 0.149 0.386 0.560 0.214

2 % 0.248 < 0.001 0.259 0.468 Conclusion

The study result indicates that Sample D product has a very strong potential to inhibit the mucosal adherence of enteric pathogens in the intestines. The outcome is supported by Sample E results, showing a capability to inhibit especially attachment of C. perfringens ATCC 3626 strain. In a way this also suggests that part of the effect originates from the fermented metabolites or the carrier matrix of the product. Without wishing to be bound by theory, it is believe that at the molecular level, the detected inhibitory effect may be based on aggregation with the pathogenic micro-organisms or competition on the mucosal binding sites.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

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