The present invention relates to a foodstuff which comprises the probiotic Lactobacillus acidophilus strain deposited under the accession number NCMB 41117, in combination with one or more prebiotic substrate of galactooligosaccharide, biotose or gentose.

Background of the Invention

The commensal gut microflora are a vital factor in gastrointestinal (GI) health, aiding the host in digestion, nutrient metabolism and vitamin production, and restricting colonisation by pathogenic bacteria. Microbial populations in the gut are however, susceptible to change and these variations may impact on the health of the host. The commensal microflora comprises both beneficial (lactobacilli and bifidobacteria) and potentially detrimental (Clostridia, enterococci, staphlococci) bacteria. Imbalances in the numbers of beneficial and detrimental bacterial species canbe induced by various factors including poor nutrition, stress, GI infection, antibiotic administration, pre¬ disposing illness and immunosuppression.

Over recent years methods of enhancing the GI microflora by increasing the proportion of beneficial bacterial species have been developed in the form of probiotics and prebiotics. Probiotics are defined as 'live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial ° balance. Although the mechanisms are as yet unclear, probiotic bacteria may also stimulate host immune function, thereby reducing the potential for gastrointestinal infection.

Prebiotics are short chain carbohydrates (<60 degrees of polymerisation) that are not digestible by the host and therefore pass into the small intestine and colon, where they are selectively metabolised (fermented) by beneficial bacterial species. Prebiotics therefore provide a source of nutrition specific for lactic acid bacteria and hence may substantially alter the GI microflora of the host. Synbiotic preparations include both aprobiotic bacterial species and aprebiotic carbohydrate. Therefore, they provide both the beneficial organisms for colonisation of the gastrointestinal tract and the nutrients required to support their growth and antipathogenic effects.

The development of effective synbiotic combinations therefore requires information on the nutrient preference of the probiotic species.

The probiotic strain deposited under the accession number NCIMB 4117 has proven efficacy in improving gastrointestinal and immune health in mammalian animals.

It would be of benefit to identify which prebiotic compounds would enhance the effectiveness of this probiotic and hence enhance the endowed health properties with a synbiotic foodstuff.

The Invention

The present invention provides as a first aspect a foodstuff comprising the probiotic Lactobacillus acidophilus strain deposited under the accession number NCIMB 41117, in combination with one or more prebiotic substrates of galactooligosaccharide (GOS), biotose or gentose. The foodstuff is a synbiotic.

The probiotic microorganism Lactobacillus acidophilus strain of the invention has been deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedure on 10 October 2001 under number NCIMB 41117 and a copy of the deposit receipt is part of this application.

The prebiotic substrate can be supplied in any format. It may be in a dried or liquid format, for example. Typical formats for GOS include a syrup form (75% minimum) or dry product (50% GOS, 50% whey protein concentrate). The GOS may be purified or part purified to reduce the monosacharide content. However, the GOS will generally be provided as unpurified GOS. The animal gut, in vivo generally "purifies" the GOS by removing/degrading small sugars which are present. The biotose and/or gentose may also be provided in any format.

The foodstuff may be any. In particular, it may be any foodstuff which pet animals consume as part of their diet. Thus, the invention covers standard food products, as well as food snacks (for example snack bars, biscuits and sweet products) and food supplements.

The foodstuff is preferably a cooked product. It may incorporate meat or animal derived material (such as beef, chicken, turkey, lamb, blood plasma, marrowbone etc, or two or more thereof). The foodstuff may alternatively be meat free (preferably including a meat substitute such as soya, maize gluten or a soya product) in order to provide a protein source. The product may contain additional protein sources such as soya protein concentrate, milk proteins, gluten etc. The product may also contain a starch source, such as one or more grains (e.g. wheat, corn, rice, oats, barley etc) or may be starch free. The foodstuff may be a dry, semi-dry , moist or liquid product. Moist products include food which is sold in tins or foil/plastic containers. Moist food usually has a moisture content of 70 to 90%. Dry products include food which have a similar composition, but with 5 to 15% moisture and presented as biscuit-like kibbles. The foodstuff is preferably packaged. A typical dry commercial dog or cat foodstuff contains about 30% crude protein, about 10-20% fat and the remainder being carbohydrate, including dietary fibre (4-60%) and ash. A typical wet or moist product contains (on a dry matter basis) about 40% fat, 50% protein and the remainder being fibre and ash. The present invention is particularly relevant for a foodstuff as herein described which is sold as a diet, foodstuff or supplement for a domestic dog or a domestic cat.

In the present text, the terms "domestic" dog and "domestic" cat mean dogs and cats, in particular Felis domesticus and Canis domesticus. The foodstuff may also be a human foodstuff, in particular snacks and treats, such as muesli bars, chocolate bars and candy, biscuits, cakes, yoghurts and desserts as well as all other foodstuffs. The human foodstuff of the invention may be a food supplement, such as a powder to be added to another food or drink.

Theprobiotic and the prebiotic may be provided as a combined powder. Such a combined powder may include other ingredients, such as a sugar or free flow agents. The powder may be fed directly to the animal, but it is preferred that it first be admixed or added to a separate food or liquid component.

Thus, the powder combination canbe added to water, milk, or other liquid or added to a dry, wet or semi-moist separate food before being fed to the animal.

The foodstuff of the invention may be a dry kibble with the probiotic present on the surface. The probiotic may, for all possible foodstuff inclusions, be stabilised, e.g. by encapsulation, freeze-dried, present in a fat or oil. The probiotic may be present in a liquid or fat or cream-like format, for example in the centre of a kibble or as a coating (e.g. in fat or oil). In such combinations, the prebiotic may be present as part of the main foodstuff to which the probiotic is applied or it may be present in combination with the probiotic for addition to the main foodstuff. One or more of the specified prebiotics may be used, optionally in combination with one or more other prebiotics. Further, one or more other fibres may also be present in the foodstuff.

The invention applies to human animals, non-human animals, pet animals, such as cats, dogs, birds, guinea pigs, rabbits, hamsters and the like.

In the foodstuff of the present invention, the probiotic may be present in any amount which contributes to the improved health of the GI tract. Typical levels include around 1 x 104 to 1 x 1012 cfu/kg foodstuff. More preferably, the levels are from 1 x 107 to 1 x 109 cfu/kg foodstuff for cats and from 1 x 108 to 1 x 1010 cfu/kg foodstuff for dogs. In general, dogs and cats are fed twice daily. Volumes vary, but as a guide, approximately 50-90 g dry food/day for cats (more if wet food) and 200-800 g dry food/day for dogs (more if wet food).

The prebiotic may be present in the food product at any suitable level, depending, to some extent, on the animal in question. Suitable ranges of prebiotics in the foodstuff are:

cat feed 1-100 g/kg foodstuff dog feed 0.1-150 g/kg foodstuff human 0.5-500 g/kg foodstuff others 0.0025-10 g/kg foodstuff supplement 1-999.9 g/kg foodstuff

The foodstuff of the first aspect is useful in maintaining or improving the gastrointestinal health and/or immune system of an animal. This is the second aspect of the invention. In particular, the probiotic of the synbiotic produces acidic metabolites. These acidic metabolites reduce the pH of the colon, favouring growth of other lactic acid bacteria, as well as inhibiting the growth of GI pathogens and putrefying bacteria by competitive exclusion. GI pathogens which are excluded are those such as pathogenic E. coli, Campylobacter spp and Salmonella enterica. In addition, many probiotic strains produce antimicrobial peptides that directly target bacterial pathogens such as E. coli and Salmonella spp. Although mechanisms are still unclear, probiotic bacteria may also stimulate host immune function, thereby reducing the potential for GI infection.

AU preferred features of the first aspect of the invention also apply to the second aspect. The present invention also provides as a third aspect a method for the manufacture of a foodstuff of the first and second aspects of the invention. The method comprises combining the ingredients, with optional heating/cooking. The combining of the ingredients may be in accordance with standard methods and practices as known in the art. The prebiotic may be admixed with the other food ingredients or supplied as a powdered supplement/liquid. The probiotic may also be admixed with the other food ingredients. However, if the food is to be heated/cooked, the probiotic is preferably added after any heating/cooking in order to maintain the viability of the probiotic. The probiotic may be sprayed onto the surface of a foodstuff. It may be spray dried onto the surface, so as to adhere to it. Alternatively, it may be sprinkled onto the food, just before eating. Thus, the foodstuff comprising the prebiotic may be supplied separately from the probiotic. The probiotic is then added to the pet food product at some point before ingestion. The pro and prebiotic may be in the form of a foodstuff which is sprinkled to other food products before being fed to the animal.

A fourth aspect of the invention provides the use of the probiotic Lactobacillus acidophilus strain deposited under the accession number NCIMB 41117, in combination with one or more prebiotic substrates of galactooligosaccharide, biotose or gentose, in the manufacture of a foodstuff for improving the gastrointestinal health and/or immune system of an animal;

All preferred features of the first to third aspects of the invention also apply to the fourth aspect.

A fifth aspect of the invention provides a method of improving the gastrointestinal health and/or immune system of an animal, the method comprising feeding to saidpet animal, a pet food product according to the first aspect of the invention.

AU preferred features of the first to fourth aspects of the invention also apply to the fifth aspect. Examples 1 and 2

Assessment of the growth (Example 1) and anti-pathogenic (Example 2)

characteristics of L. acidophilus deposited under the accession number NCIMB 41117

grown on various carbohydrate sources.

Materials and Methods

Utilisation of the prebiotic compound by L. acidophilus NCIMB 41117 strain to

support growth was assessed by standard growth curve, while inhibition of bacterial

pathogens was assessed by an assay described below. The prebiotic compounds

assessed are described in Table 1 and included a variety of commercially available

and inexpensive products. The prebiotics used display various degrees of

polymerisation and are contaminated by varying levels of monosaccharides and

disaccharides that would be absorbed by the gastrointestinal tract and therefore would

not be available for bacterial metabolism in the small intestine. Therefore, where

possible products contaminated with high levels of simple sugars were treated by

nanfiltration to remove these sugars.

Chemicals were supplied by BDH or Oxoid (media) unless otherwise stated.

Table 1. Prebiotic compounds assessed during the study (glucose control)

Prebiotic Chemical No.* 1 ' Structure and Composition Supplier (Tradename) '■ state 1 Raftiline HP 100% inulin, (long chain [>23] oligofructose) liquid Orafti 95% Oligofructose (oligomer of 2-7 repeating units), 2 Raftilose P-95 powder Orafti 5% Glucose, Fructose and Sucrose. 98% O-α-D-Galactopyranosyl-(1→6)-α-D- 3 Raffinose powder Meiji glucopyranosyl β-D-fructofuranoside 95% 4-O-β-D-GalactopyranosyI-D-fructofuranose 4 Lactulose powder Solvay 4-0-β-D-Galactopyranosyl-D-fructose Fnesland, Borculo 5 Vivinal GOS Galactooligosaccharide syrup liquid domo Ingredients 6 IMO P-900 Isomaltooligosaccharides Showa Sangyo Soy bean oligosaccharide syrup, contains the galactose Calpis Co. Ltd., 1 SOY-OS liquid tri- and terra- saccahrides raffinose and stachyose Tokyo, Japan Suntory , Osaka, 8 XOS Xylooligosaccharides Japan Melibiose 98% 6-O-α-D-Galactopyranosyl-D-glucose α-D- 9 powder Sigma Melibiose hydrate 98% β-D-Glc-(1→4)-D-GIc Cellobiose powder Sigma 4-O-β-D-Giucopyranosyl-D-glucose D-(+)-Maltose >97% Fisher Bioreagents D-(+)-Glucose >99.5% Sigma Nihon Shokuhm Biotose #50 Branched maltooligosaccharides syrup liquid Kako Co., Ltd. Nihon Shokuhin Nisshoku Panoiich High Panose syrup liquid Kako Co., Ltd. Nihon Shokuhin Gentose #45 Gentiooligosaccharides syrup liquid Kako Co., Ltd. Shintoa Lactosucrose LS-55L Lactosucrose Syrup, 40% minimum liquid Corporation Modified by Purified Vivinal GOS As 5 with reduced monosaccharide component liquid University of Reading Purified Panorich As 14 with reduced monosaccharide component liquid As above

Growth Studies - Example 1

Reduced carbohydrate MRS liquid media lacking the standard 2% glucose was

prepared (Appendix I) and dispensed in to 8.8 ml volumes and equilibrated to

anaerobic conditions (10% H2, 10% CO2, 80% N2). Sterile 10% (w/v or v/v)

solutions of the appropriate prebiotic were added to a final concentration of 1%.

Finally, a 200μl inoculum of L. acidophilus NCIMB 41117 strain (overnight culture

incubated in standard MRS broth) was added to the MRS broth containing prebiotics.

Broth cultures were incubated under anaerobic conditions at 37°C for 13h with optical

density measured hourly by spectrophotometry at 590nm wavelength. The effect of

each prebiotic on the growth of the probiotic strain was assessed in triplicate. Growth

media with no additional carbon source and glucose, which is readily metabolised by

many bacterial species and would be absorbed by the host, were used as experimental

controls.

Pathogen Inhibition - Example 2

L. acidophilus NCIMB 41117 was incubated under anaerobic conditions at 37°C in

standard MRS broth for 18h. MRS agar lacking glucose was prepared (Appendix I)

and dispensed into 28ml volumes. A 7ml volume of 10% prebiotic was added to give

a final prebiotic concentration of 2% prior to pouring into 90mm dishes (Bibby

Sterilin). The prepared agar was inoculated centrally with lμl 18h L. acidophilus NCMB 41117 culture and incubated at 37°C for 18h under anaerobic conditions. Meanwhile, 50ml volumes of nutrient broth were inoculated with Escherichia coli HE320 (canine EPEC isolate) or Salmonella enteήca serotype Typhimurium 7127 and incubated at 37°C for 18h under anaerobic conditions. The E. coli or S. enterica broth cultures were standardised to 0.4 ODsgonm and 300μl of the adjusted culture was added to 25ml molten nutrient agar cooled to 500C. The seeded agar was then poured gently over the L. acidophilus NCIMB 41117 inoculated MRS agar plate and allowed to set prior to incubation at 37°C for 18h under anaerobic conditions. The anti-pathogenic activity of the probiotic strain was assessed by measurement of the zone of clearing around the central L. acidophilus NCIMB 41117 inoculum. The assay was repeated in triplicate for each prebiotic compound being repeated for filter sterilised and autoclaved prebiotics.

Statistical Analysis

A one-way ANOVA (Student-Newman-Keuls method, Statgraphics Plus 4.1) was utilised for statistical analysis on the six most effective prebiotics for each assay. The group of top performers was selected to ensure validity of the analysis was not restricted by the large number of prebiotics tested compared to the replicates of each assay. The P value between the prebiotics analysed in each assay was <0.001.

Results Growth Studies - Example 1

The effect of the various probiotic compounds was assessed on both the maximum growth rate supported and the maximum optical density reached. Growth rate was found not to differ substantially as a result of growth on the various prebiotics (Figure 1). However, maximum optical density or maximum growth yield was affected by the prebiotic compound utilised (Figure 2). The best performing prebiotic was nanofiltrated galactooligosaccharides (GOS; prebiotic number [pn] 17) which was utilised byL. acidophilus NClMB 41117 to a growth density of ODsgonm 1-32, over three times the maximum optical density of the least efficient carbon source. Lactosucrose (pn 16), gentose (pn 15), soy-OS (pn 7) and biotose (pn 13) were the second best performing prebiotics (no significant difference between prebiotics in this group) with probiotic growth reaching 1.21, 1.20, 1.19 and 1.17 OD59onmrespectively, after 13h. GOS (impure form; pn 5) also supported a good growth yield of 1.08 OD59Qnm, although this was significantly less than the previously stated prebiotics. lhulin (pn 1) was a particularly poor performer and supported growth to a density of only 0.39 ODsgonm after 13 h. hi addition, the probiotic strain had entered the decline phase of the growth curve by 10 h.

In addition to maximum yield, the characteristic growth curve ofL. acidophilus NCMB 41117 strain varied depending on the prebiotic compound (Figure 3). Nanofiltrated GOS, lactosucrose, gentose, soybean oligosaccharide (soy-OS), biotose and GOS (impure form) all supported rapid logarithmic growth over a prolonged period, grown on these substrates the probiotic strain continued in the rapid growth phase up to 13 h. Other prebiotic compounds either produced lower growth rates of the probiotic or the growth phase terminated before 13h. Cellobiose, melibiose, Raftiline and xylo-oligosaccharides (XOS) performed particularly poorly with only slightly higher growth than the basal medium and declining cell numbers after 7-9 hours.

Pathogen Inhibition

The inhibition of an agar overlay seeded with E. coli HE320 or S. enterica serotype Typhimurium 7127 by a focal inoculum ofL. acidophilus NCIMB 41117 strain was quantified in mm. Growth ofS. enterica 7127 was inhibited most efficiently by L. acidophilus NCIMB 41117 grown on soy-OS (pn 7) with a mean zone of inhibition of 19.3 mm (Figure 4). GOS (pn 5), biotose (pn 13), gentose (pn 15), lactosucrose (pn 16), nanofiltrated GOS (pn 17) andpanorich (pn 14) were also able to support a good level ofS. enterica 7127 inhibition which ranged between 18.4 mm and 14.7 mm (mean values). Raftiline (inulin; pn 1), XOS (pn 8) and melibiose (pn 9) were not able to support any detectable anti-pathogen activity. Inhibition of the enteropathogenic E. coli strain HE320 also varied dependent on the prebiotic carbon source. Similarly to the S. enterica inhibition study raftiline (inulin; pn 1), XOS (pn 8) and melibiose (pn 9) did not support detectable inhibition ofE. coli HE320 by the probiotic strain. L. acidophilus NCMB 41117 strain was best able to inhibit growth ofE. coli HE320 when grown on GOS (non-filtrated; pn 5), biotose (pn 13), soy-OS (pn 7) and lactosucrose (pn 16) withno statistically significant difference between the performance of these prebiotics. Gentose (pn 15) was the next best able to support anti-pathogenic activity against E. coli with panorich (pn 14) and lactulose (pn 4) providing significantly less inhibition activity.

Discussion Growth Effects — Example 1

Growth is a vital factor in the colonisation of the gut by probiotics. Growth of lactic acid bacteria such as L. acidophilus NCIMB 41117 strain results in a lowered pH within the colon and hence enhancement of the growth of beneficial bacteria in the gut, thus aiding competitive inhibition of detrimental species. The growth rate of the probiotic strain was largely unaffected by the prebiotic compound present in the growth media. This is likely to be due to the specific growth rate of the bacterial species, which is determined by the maximum rate of DNA replication and protein production.

Ih contrast, maximum growth yield was effected by the prebiotic substances. Nanofiltrated GOS supported the highest maximum growth yield, with this compound supporting higher growth yields at 13 h than the glucose control. The basic GOS product was not able to support such a high growth yield suggesting that the relatively large proportion of contaminating mono- and di-saccharides found in GOS may suppress growth, or may provide less efficient carbon sources reducing growth compared to the pure product. Due to absorption in the gut, the pure form would be that encountered by lactobacilli (including the probiotic strain) in the colon, therefore the purified product best represents the prebiotic compound encountered in vivo. GOS, lactosucrose, gentose, soy-OS and biotose all supported prolonged growth, with the logarithmic (rapid growth) phase continuing until the termination of the experiment at 13 h. This suggests that on consumption and passage in to the gut these prebiotics may support growth of the probiotic strain over a longer period than the other prebiotics tested. Growth over this period may potentially support growth in the GI tract over a whole 24 h period were two evenly spaced feeds given in this time.

Of the other prebiotics tested, maltose, lactulose and raffinose also gave prolonged growth over the whole 13 h period, but showed slightly lower growth rates and hence reduced maximum yields compared to GOS, lactosucrose, gentose, soy-OS and biotose. The remaining prebiotics (panorich, cellobiose, mellibiose, XOS, isomalto- oligosaccharides, raftilose and raftiline) were not able to support good growth yields ofL. acidophilus NCIMB 41117 strain mainly due to growth terminating at between 5 and 9 h (stationary phase).

Anti-pathogen Activity - Example 2

The inhibition of bacterial pathogens such as E. coli and Salmonella spp. is important in the efficacy of probiotic strains in the prevention gastrointestinal infections. These bacterial pathogens are responsible for a considerable proportion of gastrointestinal infections in cats and dogs. The enteropathogenic E. coli strain was isolated from a clinical canine infection, while the S. enterica isolate represents a clinical isolate of a particularly virulent pathogen infecting humans and various animals including cats and dogs.

Soy-OS, GOS (non-filtrated) and biotose were the three prebiotics best able to support the anti-pathogenic activity of the probiotic strain against the pathogens tested. Lactosucrose was also able to support a relatively good level of inhibition of both the E. coli and Salmonella isolate. Nanofiltrated GOS did not provided such an efficient carbon source as the unfilitered GOS for the production of the inhibitory substance(s) suggesting that contaminating simple sugars that would be absorbed through the intestinal tract added to the inhibitory effect. The nanofiltrated GOS promoted a good level of inhibition ofS. enterica 7127, but only an intermediate level of inhibition ofE.coli HE320. L. acidophilus NCJMB 41117 strain grown onraftiline (inulin), XOS or melibiose did not inhibit growth of either pathogen, however it should be noted that the assay has a minimum detectable zone of inhibition of 4 mm and therefore some low level of inhibition may have occurred.

The extent of inhibition and the pattern of inhibition supported by the prebiotic compounds were largely similar between the pathogenic E. coli and Salmonella strains challenged, therefore although the mechanism is unknown it is likely that the mode of inhibition is common to both pathogens.

Summary — Examples 1 and 2

Since growth is a vital factor controlling colonisation of the gastrointestinal tract by a probiotic strain, enhancement of this parameter was defined as the most important feature of the prebiotic compounds. Rapid or prolonged growth resulting in high cell yields would be expected to lead to greater lactic acid production with a consequent reduction in colonic pH, increases in the growth of other lactic acid bacteria and decreased putrifying species. While anti-pathogenic activity is an important feature of probiotic strains, the desirable health effects occur mainly on ingestion of pathogenic bacteria or during periods of imbalance in the GI microflora. The anti-pathogen effect was therefore selected as the second most desirable effect.

Of the prebiotic compounds tested, nanofiltrated GOS was identified as significantly better than any other compound (including the glucose control) in supporting growth of the probiotic strain. This product was also able to support a good level of inhibition ofS. enterica 7127 and an intermediate level of inhibition in E. coli HE320. Unfiltered GOS gave reduced growth rate (and hence maximum yield) compared to the filtered product and increased anti-pathogenic activity. These effects therefore originate from contaminating simple sugars that would not reach the colon in vivo. Lactosucrose and soy-OS allowed the probiotic strain to reach high maximum growth yields and supported good levels of pathogen inhibition. However these products contain a large proportion of contaminating sugars, the effects observed in vitro are

therefore unlikely to represent the in vivo situation.

Appendix I Media Recipes

A) Reduced carbohydrate MRS liquid media (without glucose)

Peptone 5.0 'Lab-Lemco' Powder 4.0 Yeast Extract 2.0 Tween 80' 1.0 Di-Potassium Hydrogen Phosphate 2.0 Sodium Acetate Tri-Hydrate 5.0 Tri-Ammonium Citrate 2.0 Magnesium Sulphate Hepta- 0.2 Hydrate Manganese Sulphate Tetra-Hydrate 0.05 Resazurin 0.001 Cysteine Hydrochloride 0.5

> MRS Agar (without glucose) Cόhcentration {g/800ml) «

Peptone 10.0 'Lab-Lemco' Powder 8.0 Yeast Extract 4.0 Tween 80' 1.0 Di-Potassium Hydrogen Phosphate 2.0 Sodium Acetate Tri-Hydrate 5.0 Tri-Ammonium Citrate 2.0 Magnesium Sulphate Hepta- 0.2 Hydrate Manganese Sulphate Tetra-Hydrate 0.05 Reverse osmosis water to volume 800ml Agar 1.0% Adjust pH before autoclaving to 6.5