Technical Field

[001] The technology relates to prebiotic honey. In particular the technology relates to prebiotic compositions comprising specific concentrations of certain oligosaccharides which can increase Lactobacilli and/or Bifidobacteria levels in the intestine of a mammal, which can lead to a subsequent suppression of Clostridia levels.

Related Application

[002] This application claims priority to US provisional patent application No 62/337,980 filed 18 May 2016 which is herein incorporated by reference in its entirety.

Background

[003] Worldwide, there has been a growing awareness of the relationship between diet and health, which has led to an increasing demand for food products that support health beyond simply providing basic nutrition. It is well recognised that the intestine harbours a diverse microbial population that impacts on the health of the host in many ways and is not limited to digestive tract health. Evidence is also accumulating that links the intestinal microbes with brain responses and hence neurological conditions including stress, anxiety and autism.

[004] Since diet influences the composition and function of the intestinal microbes, constituents of the diet can play a beneficial role in health by improving these microbes. The term "prebiotic" has been coined to refer to dietary ingredients that induce the growth or activity of some intestinal microbes and thereby induce specific changes in the composition and/or activity of these microbes, thus conferring benefits upon host health.

[005] The concentration of bacterial populations increases towards the distal regions of the intestine. Lactobacilli are the dominant bacterial species in the ileum, or distal small intestine. In addition to Lactobacilli, the genera Bifidobacterium and Bacteroides are also found, in lower numbers, in the jejunum and ileum. The dominant colonic microflora includes strict anaerobes (10 ° to 10 ¹¹ per gram) such as Bacteroides, Eubacterium, Bifidobacterium and Peptostreptococcus. Facultative anaerobes are found in lower numbers in the colon (1000-fold lower) than the strict anaerobes and include Enterobacteriaceae, Lactobacilli, Enterococci and Streptococci. Generally, Lactobacilli are present in the colon at a level of 10 ⁴ -10 ⁹ per g wet weight of the contents while Bifidobacterium constitute approximately 5-10% of the total bacterial flora in the large intestine. [006] The indigenous microbes contribute to the health of the host by aiding digestion, synthesis of vitamins, protection of the host from invasion of ingested pathogens and also stimulate the immune system.

[007] Dietary components which can impact on the composition or function of the intestinal microbiota can play a vital role in maintaining, establishing or restoring a healthy profile of microbes and hence contribute to health and well-being.

[008] The present inventors have found that compositions of selected component honeys provide health and wellness benefits that are correlated with particular profiles of certain oligosaccharides.

Summary

[009] In a first aspect there is provided a prebiotic composition for increasing Lactobacilli and/or Bifidobacteria levels in the intestine of an animal, the composition comprising: a) at least 0.5 mg/g of nigerose; and

b) a total concentration of about 4.0 mg/g to at least 250 mg/g of any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose.

[010] In an embodiment the prebiotic composition has a concentration of nigerose from about 0.5 mg/g to about 20 mg/g.

[01 1] The concentration of nigerose may be 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1 .4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 1 1.0 mg/g, 1 1.2 mg/g, 1 1.4 mg/g, 1 1.6 mg/g, 1 1.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, or 20 mg/g.

[012] In some embodiments the concentration of any one of trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose may be 0.1 mg/g, 0.5 mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, .2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 1 1.0 mg/g, 11.2 mg/g, 11.4 mg/g, 11.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, 20 mg/g, 21 mg/g, 22. mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, 35 mg/g, 36 mg/g, 37 mg/g, 38 mg/g, 39 mg/g, 40 mg/g, 41 mg/g, 42 mg/g, 43 mg/g, 44 mg/g, 45 mg/g, 468 mg/g, 47 mg/g, 48 mg/g, 49 mg/g, or at least about 50 mg/g.

[013] In an embodiment the prebiotic composition has a concentration of trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose independently from about 0.1 mg/g to about 50 mg/g.

[014] In some embodiments the total concentration of any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose may be about 4 mg/g, 6 mg/g, 8 mg/g, 10 mg/g, 12 mg/g, 14 mg/g, 16 mg/g, 18 mg/g, 20 mg/g, 22 mg/g, 24 mg/g, 26 mg/g, 28 mg/g, 30 mg/g, 32 mg/g, 34 mg/g, 36 mg/g, 38 mg/g, 40 mg/g, 42 mg/g, 44 mg/g, 46 mg/g, 48 mg/g, 50 mg/g, 52 mg/g, 54 mg/g, 56 mg/g, 58 mg/g, 60 mg/g, 62 mg/g, 64 mg/g, 66 mg/g, 68 mg/g, 70 mg/g, 72 mg/g, 74 mg/g, 76 mg/g, 78 mg/g, 80 mg/g, 82 mg/g, 84 mg/g, 86 mg/g, 88 mg/g, 90 mg/g, 92 mg/g, 94 mg/g, 96 mg/g, 98 mg/g, 100 mg/g, 102 mg/g, 104 mg/g, 106 mg/g, 108 mg/g, 110 mg/g, 112 mg/g, 114 mg/g, 116 mg/g, 1 18 mg/g, 120 mg/g, 122 mg/g, 124 mg/g, 126 mg/g, 128 mg/g, 130 mg/g, 132 mg/g, 134 mg/g, 136 mg/g, 138 mg/g, 140 mg/g, 142 mg/g, 144 mg/g, 146 mg/g, 148 mg/g, 150 mg/g, 152 mg/g, 154 mg/g, 156 mg/g, 158 mg/g, 160 mg/g, 162 mg/g, 164 mg/g, 166 mg/g, 168 mg/g, 170 mg/g, 172 mg/g, 174 mg/g, 176 mg/g, 178 mg/g, 180 mg/g, 182 mg/g, 184 mg/g, 186 mg/g, 190 mg/g, 192 mg/g, 194 mg/g, 196 mg/g, 198 mg/g, 200 mg/g, 202 mg/g, 204 mg/g, 206 mg/g, 208 mg/g, 210 mg/g, 212 mg/g, 214 mg/g, 216 mg/g, 218 mg/g, 220 mg/g, 222 mg/g, 224 mg/g, 226 mg/g, 228 mg/g, 230 mg/g, 232 mg/g, 234 mg/g, 236 mg/g, 238 mg/g, 240 mg/g, 242 mg/g, 244 mg/g, 246 mg/g, 248 mg/g or at least about 250 mg/g.

[015] In some embodiments the total concentration of any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose may be about 85 mg/g, 90 mg/g, 95 mg/g, 100 mg/g, 105 mg/g, 110 mg, 115 mg/g, 120 mg/g, or 125 mg/g. [016] In an embodiment the prebiotic composition comprises a mixture of honeys to obtain the concentrations of nigerose, trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose in the composition.

[017] In an embodiment the composition is a single source honey or blend of honeys. In some embodiments the composition comprises a single source honey or blend of honeys blend of honeys and at least one oligosaccharide selected from the group consisting of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose from an exogenous source. For example the oligosaccharide may be purified or isolated from a naturally occurring source such as honey or may be synthesised.

[018] The animal may be any domestic or wild animal. For example that animal may be a mammal or an insect. The mammal may be a porcine, bovine, ovine, avian, rodent, lagomorph or human. In some embodiments the insect may be a bee such as a honey bee (Apis mellifera).

[019] In some embodiments the prebiotic composition may additionally reduce the levels of Clostridia.

[020] In an embodiment the prebiotic composition comprises at least 1 mg/g of nigerose and reduces the level of Clostridia in a mammalian intestine.

[021] In an embodiment the prebiotic composition has a Prebiotic Activity of at least about 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 1 1 .0, at least 1 1.5, at least 12.0, at least 12.5, at least 13.0, at least 13.5, at least 14.0, at least 14.5, at least 15, at least 15.5, at least 16.0, at least 16.5, at least 17.0, at least 17.5, at least 18.0, at least 18.5, at least 19.0, at least 19.5, or at least 20.

[022] In a second aspect, there is provided a nutritional composition comprising prebiotic composition of the first aspect.

[023] The nutritional composition may be selected from a vitamin supplement, mineral supplement, herbal supplement, meal supplement, sports nutrition product or natural food supplement.

[024] In a third aspect there is provided use of the prebiotic composition of the first aspect or the nutritional composition of the second aspect to increase the level of Lactobacilli and/or Bifidobacteria in the intestine of a mammal in need thereof.

[025] The use may additionally reduce the levels of Clostridia.

[026] The level of Lactobacilli may be increased by 5%, 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least 1000% of the level before use of the composition. [027] Alternatively or additionally, the level of Bifidobacteria may be increased by 5%, 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least 1000% of the level before use of the composition.

[028] Alternatively or additionally the level of Clostridia may be decreased 5%, 10%, 50%, 100%, 200%, 300%, 400%, or at least 500% of the level before use of the composition.

[029] In a fourth aspect, there is provided a food product supplemented with prebiotic composition of the first aspect or a nutritional composition of the second aspect.

[030] The food product may be an infant formula, baby food, baked good (for example a bread, cake, biscuit or cookie, beverage (e.g. soft drink, flavored milk), breakfast food (e.g. cereal), muesli bar, tinned food, snack food (e.g. chips, crisps, corn snack, nuts, seeds), confection, condiment, marinade, dairy product, dip, spread or soup.

[031] The food product may contain less than about 1 % (w/w) of the prebiotic honey or about 1 % (w/w), or about 2% (w/w), or about 3% (w/w), or about 4% (w/w), or about 5% (w/w), or about 6% (w/w), or about 7% (w/w), or about 10% (w/w), or about 1 1 % (w/w), or about 12% (w/w), or about 13% (w/w), or about 14% (w/w), or about 15% (w/w) of the prebiotic composition.

[032] In a fifth aspect there is provided a pharmaceutical composition, complementary medicine or medical substance comprising the comprising prebiotic composition of the first aspect.

[033] In a sixth aspect there is provided a method of preparing the prebiotic composition of the first aspect from a plurality of component honeys, the method comprising:

assaying at least one component honey for a desired amount of the oligosaccharide nigerose;

assaying at least one component honey for at least one oligosaccharide selected from the group consisting of trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose;

selecting a number of component honeys on the basis of the oligosaccharide levels determined by the assays; and

mixing the component honeys to form the prebiotic composition.

[034] In a seventh aspect there is provided a method of preparing the prebiotic composition of the first aspect from a plurality of component honeys, the method comprising: assaying at least one component honey using an intestinal microcosm for a desired effect on Lactobacilli, Bifidobacteria and/or Clostridia levels;

selecting a number of component honeys on the basis of their effects on Lactobacilli, Bifidobacteria and/or Clostridia levels; and

mixing the component honeys to form the prebiotic composition.

[035] In an eighth aspect there is provided a method of determining the Prebiotic Activity of substance relative to a negative control, the method comprises:

establishing an intestinal microcosm;

adding a sample of the substance to the microcosm;

incubating the sample and the microcosm for a period of time;

measuring the levels of Lactobacilli, Bifidobacteria and Clostridia;

measuring the levels of Lactobacilli, Bifidobacteria and Clostridia in an intestinal microcosm to which a sample of the substance has not been added; and

calculating the Prebiotic Activity using the equation

(Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm), wherein Lac, Bif and Clo are the log counts of Lactobacilli, Bifidobacteria and Clostridia, respectively.

[036] In some embodiments the microcosm may be incubated for about 48 hours. In other embodiments the microcosm may be incubated for about 8 hours, about 16 hours, about 24 hours, about 32 hours, about 40 hours or about 48 hours. In other embodiments the microcosms may be incubated for more than 48 hours.

[037] The substance may be an oligosaccharide, mixture of oligosaccharides, honey or mixture of honeys.

[038] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[039] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

[040] As used herein the term "in need thereof" is a statement of purpose for which the method or use must be performed. For example, the term is to be interpreted to mean that the prebiotic honey is to be used to increase the level of Lactobacilli and Bifidobacteria and reduce the levels of Clostridia in the intestine of a mammal with a recognized need for modulation of the levels of these bacteria.

[041] Embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

Figure 1 shows the impact of honey on the lactobacilli, bifidobacteria and Clostridia levels after 48 hours incubation in a microcosm (in vitro) containing a final concentration of 1 % (w/v) honey. The honeys used were H1 , H2, H3 and H4 (the Four Honeys). Results expressed as the difference between the log cfu test honey microcosm and the negative control microcosm at 48 hours.

Figure 2 shows the relative counts of Lactobacillus (cfu/g) via microcosm assay (in vitro) containing a final concentration of 1 % (w/v) honey at 3 separate intervals (0, 15 and 33 days).

Figure 3 shows the relative counts of Bifidobacteria (cfu/g) via Microcosm assay (in vitro) containing a final concentration of 1 % (w/v) honey at 3 separate time intervals (0, 15 and 33 days).

Figure 4 shows the linear relationship between Lactobacillus and Bifidobacteria and total anaerobes following honey digestion (in vitro).

Figure 5. Bacterial counts (cfu/g) of Lactobacillus and Bifidobacteria in microcosms established with 24 different raw Australian honeys relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control.

Figure 6. Bacterial counts (cfu/g) of Clostridia in microcosms established with 24 different raw Australian honeys relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control.

Figure 7. Prebiotic activity for 24 raw Australian honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control. Results expressed as bacterial counts (cfu/g) according to the equation Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm).

Figure 8. Bacterial counts (cfu/g) of Lactobacillus and Bifidobacteria in microcosms established with the Four Honeys relative to the negative control (microcosm with no added honey).

Figure 9. Bacterial counts (cfu/g) of Clostridia in microcosms established with the Four Honeys relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control.

Figure 10. Prebiotic activity for the Four Honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey) and inulin as the positive control. Results expressed as bacterial counts (cfu/g) according to the equation Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm).

Figure 11. Bacterial counts (cfu/g) of Lactobacillus and Bifidobacteria in microcosms established with 20 different blended honeys relative to the negative control (microcosm with no added honey). Controls include H1 and H4, artificial honey (AH) with added inulin or FOS and inulin. Inulin concentrations used: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1.5 % total oligosaccharides in honey.

Figure 12. Bacterial counts (cfu/g) of Clostridia in microcosms established with 20 different blended honeys relative to the negative control (microcosm with no added honey). Controls include H1 and H4, artificial honey (AH) with added inulin or FOS and inulin. Inulin concentrations used: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1.5 % total oligosaccharides in honey.

Figure 13. Prebiotic activity of 20 different blended honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control. Results expressed as actual bacterial counts (cfu) according to the equation Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm).

Figure 14. Bacterial counts (cfu/g) of Lactobacillus and Bifidobacteria in microcosms established with 21 different imported honeys relative to the negative control (microcosm with no added honey). Controls include H1 and H4, artificial honey (AH) with added inulin or FOS and inulin. Inulin concentrations used: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1.5 % total oligosaccharides in honey. Figure 15. Bacterial counts (cfu/g) of Clostridia in microcosms established with 21 different imported honeys relative to the negative control (microcosm with no added honey). Controls include H1 and H4, artificial honey (AH) with added inulin or FOS and inulin. Inulin concentrations used: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1.5 % total oligosaccharides in honey.

Figure 16. Prebiotic activity for 21 different imported honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey) and 1 % inulin as the positive control. Results expressed as bacterial counts (cfu/g) according to the equation Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm).

Figure 17. Specific di- and tri-saccharides detected in 24 honeys. Results expressed as mg per g honey.

Figure 18. Average content of oligosaccharides in 24 different raw Australian honeys alongside the standard deviation. Results expressed as mg per g of honey.

Figure 19. Average content of oligosaccharides in 20 different Capilano processed and blended honeys alongside the standard deviation. Results expressed as mg per g of honey.

Figure 20. Oligosaccharides consumed by Lactobacillus fermentum PC 1 and

Bifidobacterium breve when grown in Basal Medium supplemented with 1 % of honeys H1 - H4. Honeys tested separately and bacteria tested separately and results combined and expressed as a percent (%) of the total amount of each saccharide.

Figure 21. Heat map of the 24 different raw Australian honeys showing the 10 saccharides which varied most across all honeys. Results presented as mg per g of honey. The branching groups similar amounts of saccharides.

Figure 22. Prebiotic activity for Capilano (CZ) honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey). Controls include H1 and H2, artificial honey (AH) with added inulin or FOS and inulin. Results expressed as mean ± SD from duplicate trials.

Figure 23. Bacterial counts (log cfu/ml) of bifidobacteria, lactobacilli and Clostridia in microcosms.

Figure 24. Prebiotic activity for different imported honeys (non-Australian honeys) enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey). Controls include artificial honey (AH) with added inulin or FOS and AH alone. Results expressed as mean + SD from duplicate trials. Figure 25. Bacterial counts (log cfu/ml) of bifidobacteria, lactobacilli and Clostridia in microcosms of imported honey (non-Australian honeys).

Figure 26. Prebiotic activity for raw Capilano honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey). Controls include H1 and H2, artificial honey (AH) with added inulin or FOS and inulin. Results expressed as mean ± SD from duplicate trials. Inulin concentrations used: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1 .5 % total oligosaccharides in honey.

Figure 27. Repeated analysis of the Prebiotic Activity of 5 different floral honeys (blue (bars 1 -5), yellow (bars 6-10), red (bars 1 1-15), green (bars 16-20), and orange (bars 21 and 22)) enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey). The honeys were tested at 1 % final concentration in microcosms established with adult human microbiota. Positive control is 1 % inulin (4 times recommended daily dose). Results expressed as mean values ± SD from four separate assays, all run in triplicate.

Figure 28. The average summed content of K8 individual oligosaccharides that correlate to an increasing Prebiotic Activity for the Four Honeys. The rising content of K8

oligosacharides is correlated with increasing prebiotic activity.

Figure 29. The average content (mg/g) of individual oligosaccharides (trehalulose, nigerose, maltulose, isomaltose, panose, turanose, koibiose, palatinose) that correlate to an increasing Prebiotic Activity of the Four Honeys. There is a linear (r ² =0.94) and exponential (r^O.88) relationship illustrated.

Figure 30. The combined average content (mg/g) of individual oligosaccharides

(trehalulose, nigerose, maltulose, isomaltose, panose, turanose, koibiose, palatinose) that correlate to an increasing Prebiotic Activity of the Four Honeys with the addition of four raw honeys with a Prebiotic Activity above 6.5. There is a linear (r ² =0.72) and exponential (r ² =0.81 ) relationship.

Figure 31. Prebiotic activity of the Four Honeys and Capilano raw honeys (9, 10, 7 and 13) enumerated using microcosms established with whole honey (1 % final concentration), relative to the negative control (microcosm with no added honey). Results expressed as mean ± SD from experiments performed in triplicate. Positive control (inulin) tested in three concentrations: 0.15, 2.5, and 5.0 mg/ml equivalent to 1 % of 1.5% total oligosaccharides found in honey, one serving of inulin (5g), and two servings of inulin (10g), respectively. Figure 32. The relationship between the concentration of maltulose, nigerose, turanose and kojibiose as detected in a range of different honeys honeys. Results expressed in mg per g of honey.

Figure 33. The relationship between maltulose and a combination of turanose and kojibiose as detected in a range of different honeys. Results expressed in mg per g of honey.

Figure 34. The relationship (r ² =0.97) between the concentration of maltulose and the summed concentration of K8 oligosaccharides (trehalulose, nigerose, maltulose, isomaltose, panose, turanose, koibiose, palatinose) detected in a range of different honeys. Results expressed in mg per g of honey.

Figure 35. The relationship (r ² =0.76) between the concentration of Maltulose and the concentration of total disaccharides detected in a range of different honeys. Results expressed in mg per g of honey.

Figure 36. The relationship between the concentration of nigerose and the summed concentration of K8 oligosaccharides (trehalulose, nigerose, maltulose, isomaltose, panose, turanose, koibiose, palatinose) detected in a range of different honeys. Results expressed in mg per g of honey.

Figure 37. The relationship between the concentration of nigerose (mg/g) and the total concentration of disaccharides detected in a range of different honeys. Results expressed in mg per g of honey.

Figure 38. Bacterial counts (log cfu/ml) of bifidobacteria, lactobacilli and Clostridia in microcosms.

Figure 39. Heat map of the numbers of the various bacterial groups in microcosms supplemented with raw honeys 1-24. Results presented as log cfu per ml of microcosm .

Figure 40. Flow chart of clinical study design to test 4 different honeys ( H1 -4). The study took 4 months to complete with two separate groups (Group 1 and 2) each of 20 subjects. Group 1 tested honeys 1 and 3. Group 2 tested honeys 2 and 4. There were four separate phases each one month long. Honey was excluded from the diet during phases 1 and 3. Stool samples (S1 , 2, 3 and 4) were collected at time points 0, 1 , 2, 3, 4, and 5 months.

Figure 41. The abundance of the major bacterial groups in samples from all subjects over the course of the study. Time point 1 to 2 = wash out before honey; point 2 to 3 = consumption of first honey; points 3 to 4 = wash out before second honey; point 4 to 5 = consumption of second honey. Results presented for all subjects separately as the log colony forming units (CFU) per gram of sample (wet weight). All subjects grouped for the first honey and also for the second honey even though two different honeys were tested.

Figure 42. The effect of honey consumption on the abundance of each bacterial group. Abundance is expressed as the log cfu per g in samples taken before and after 4 weeks honey consumption. Grey lines represent the slope of the line during the period of honey consumption for individual subjects and the black line is the mean of all subjects. P values were determined by ANOVA analysis of the slope.

Figure 43. The impact of the four different honeys (1 -4) on the abundance of (A) (B) lactobacilli, and (C) bifidobacteria and Clostridia. Results for two time points, namely before and after honey consumption are expressed as the mean with confidence intervals of all subjects (log cfu per gram wet weight). The means at each time point are joined by a dark line for clarity. Grey lines represent individual subjects. The figures are generated using the estimates from linear mixed effects model with subjects as a random factor. For each of the honeys, the abundance of lactobacilli and bifidobacteria were significantly higher after honey consumption (P<0.0001 ) and the abundance of Clostridia were significantly lower after honey consumption (P=0.001 ). There were no significant differences between the different honeys for each of the bacterial groups after honey consumption.

Figure 44. The impact of honey on the lactobacilli , bifidobacteria and Clostridia either (A) after 4 weeks of subjects eating 20 g of honey daily (in vivo study) or (B) after 48 hours incubation in an intestinal microcosm (in vitro) containing a final concentration of 1 % (w/v) whole honey (This is the same data as Figure 1 ). Results are expressed as the difference between the log cfu before and after the 4 weeks (in vivo clinical study) or the difference between the log cfu for the honey microcosms and the negative control microcosm.

Figure 45. The effect of various honeys on the bacterial populations in intestinal microcosms (in vitro testing). The honeys included those tested in the clinical study and four Beeotic® Capilano honeys. The microcosms were established with stool samples and whole honey (1 % final concentration) as well as increasing concentrations of inulin as a positive control or buffer alone (negative control). Counts of lactobacilli, bifidobacteria and Clostridia in the microcosms at time of establishment (initial) and after 48 hours incubation are expressed as log cfu/ml (mean of triplicate assays presented).

Figure 46. The effect of various honeys on the bacterial populations in intestinal microcosms (in vitro testing). The honeys included those tested in the clinical study and ten Beeotic® Capilano honeys. The microcosms were established with stool samples and whole honey (1 % final concentration) as well as increasing concentrations of inulin as a positive control or buffer alone (negative control). Counts of lactobacilli, bifidobacteria and Clostridia in the microcosms at time of establishment (initial) and after 48 hours incubation are expressed as log cfu/ml (mean of triplicate assays presented).

Figure 47. Box plots of the concentrations of sucrose, maltulose, nigerose, turanose, kojibiose, trehalulose, palatinose and isomaltose in each of the four honeys used in the clinical study (H1 -4). Honeys were analysed by gas chromatography. Results expressed as mg of saccharide per/g of honey (mean of duplicate analyses).

Figure 48. Box plots of the concentrations of tetra- and penta- saccharides (analysed by HPLC) and total saccharides (analysed by GC) for each of the honey used in the clinical study (H1 -4). Results expressed as mg per g of honey (mean of duplicate analyses).

Figure 49. Total disaccharide and trisaccharide content of the duplicate assays of clinical study honeys (samples H1 - H4), various Capilano Beeotic® honey blends (samples 121— 138) and raw unprocessed honeys (samples 1 -24). Results presented as the mean of duplicate analyses (mg/g).

Figure 50. The relationship between the increasing prebiotic activity of the honeys used in the clinical study and the summed concentration (expressed as mg/g honey) of the identified eight key saccharides (maltulose, nigerose, turanose, isomaltose, kojibiose, trehalulose, palatinose and panose). The analysis was performed at 0 months (A) and at 12 months (B) for each of the four honeys. The Prebiotic Activity of each honey sample was assessed by in vitro microcosm and calculated using the counts of the Lactobaccillus and Bifidobacterium and Clostridium relative to the negative control.

Figure 51. The relationship between maltulose concentration (mg/g) in the 42 honeys analysed and the sum of the identified eight key oligosaccharides (maltulose, nigerose, turanose, isomaltose, kojibiose, trehalulose, palatinose and panose) and total disaccharides in each honey.

Figure 52. Average content (mg/g) of saccharides and the standard deviations for (A) 32 raw honeys and (B) 10 Capilano Beeotic® processed honey blends. Results expressed as the mean of duplicate assays using gas chromatography.

Figure 53. Growth of (A) lactobacilli and (B) bifidobacteria in in vitro intestinal microcosms after 48 hours incubation with inulin added either alone (dark red or blue) or together with a honey (H7). Negative controls had buffer or or honey, but no added inulin. Results presented as the cfu per ml (mean of triplicate analyses). Description of Embodiments

[042] The prebiotic compositions described herein are useful for increasing the levels of Lactobacilli and/or Bifidobacteria in an animal intestine. In some embodiments the compositions are useful for reducing the levels of Clostridia in an animal intestine. The compositions have been developed based on the inventor's demonstration that there is a correlation between prebiotic activity and content of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose in the compositions. In particular, the previously unknown prebiotic activity of nigerose is demonstrated herein.

[043] In general the composition has a total composition of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose of between about 4.0 mg/g to at least 250 mg/g.

[044] The prebiotic composition typically consists of a plurality of component honeys which are sourced from a number of locations and combined into a bulk lot to form the prebiotic composition. The component honeys are selected on the basis of particular oligosaccharide levels such that when combined the prebiotic composition is formed.

Individual component honeys do not contain the combination of oligosaccharides of the prebiotic composition.

[045] In some embodiments the composition consists of a single source honey or blend of honeys. The composition can additionally comprise an oligosaccharide selected from the group consisting of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose. The oligosaccharide may be from an exogenous (i.e. non-honey source) or may be derived from a honey that is not a component honey of the composition. For example the oligosaccharide may be purified from a naturally occurring source such as honey or may be synthesised.

[046] The compositions are typically used to alter the levels of Lactobacilli, Bifidobacteria and/or Clostridia in an animal intestine. The animal may be any wild, domestic or commercially important mammal. For example that animal may be a mammal or an insect.

[047] The mammal may be a porcine, bovine, ovine, avian, rodent, lagomorph or human. The human may be an infant, child adult or elderly human.

[048] In one embodiment the avian may be a chicken, for example a chicken raised for egg or meat production.

[049] In some embodiments the insect may be a bee, for example a wild bee or a bee used for honey production such as Apis mellifera.

[050] The prebiotic composition may comprise any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose. Each of these oligosaccharides has been demonstrated herein to correlate with the prebiotic activity of the compositions.

[051] In one embodiment the prebiotic composition comprises nigerose. In this embodiment the prebiotic composition is useful for reducing the levels of Clostridia in a mammalian intestine.

[052] Other oligosaccharides that are typically present in the prebiotic composition are trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose, each of which has been demonstrated to correlate with the prebiotic activity of the composition.

[053] The composition has a minimum concentration of each oligosaccharide of at least about 0.1 mg/g.

[054] The concentration of nigerose may be about 0.5mg/g, 1.0 mg/g, 1 .2 mg/g, 1.4 mg/g, 1 .6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 1 1.0 mg/g, 1 1.2 mg/g, 1 1.4 mg/g, 1 1.6 mg/g, 1 1.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, or at least about 20 mg/g.

[055] In various embodiments the amount of each oligosaccharide (trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose) is about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 1 1.0 mg/g, 1 1.2 mg/g, 1 1 .4 mg/g, 1 1.6 mg/g, 1 1.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, 20 mg/g, 21 mg/g, 22. mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, 35 mg/g, 36 mg/g, 37 mg/g, 38 mg/g, 39 mg/g, 40 mg/g, 41 mg/g, 42 mg/g, 43 mg/g, 44 mg/g, 45 mg/g, 468 mg/g, 47 mg/g, 48 mg/g, 49 mg/g, or at least about 50 mg/g.

[056] In some embodiments the concentration of maltulose may be about 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 11.2 mg/g, 1 1.4 mg/g, 11.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, 20 mg/g, 21 mg/g, 22. mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, 35 mg/g, 36 mg/g, 37 mg/g, 38 mg/g, 39 mg/g, 40 mg/g, 41 mg/g, 42 mg/g, 43 mg/g, 44 mg/g, 45 mg/g, 468 mg/g, 47 mg/g, 48 mg/g, 49 mg/g, or 50 mg/g.

[057] In some embodiments the concentration of turanose may be about may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 11.2 mg/g, 11.4 mg/g, 1 1.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, about 20 mg/g, 21 mg/g, 22 mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, or at least about 35 mg/g. [058] In some embodiments the concentration of kojibiose may be about may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 11.2 mg/g, 11.4 mg/g, 1 1.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, about 20 mg/g, 21 mg/g, 22 mg/g, 23 mg/g, 24 mg/g, or at least about 25 mg/g.

[059] In some embodiments the concentration of trehalulose may be about may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 1 1.2 mg/g, 11.4 mg/g, 11.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, about 20 mg/g, 21 mg/g, 22 mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, 35mg/g, 36 mg/g, 37mg/g, 38 mg/g, 39 mg/g or at least about 40 mg/g.

[060] In some embodiments the concentration of palatinose may be about may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 1 1.2 mg/g, 11.4 mg/g, 11.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, or at least about 15.0 mg/g.

[061] In some embodiments the concentration of isomaltose may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 1 1.0 mg/g, 1 1.2 mg/g, 1 1.4 mg/g, 11.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, 15.0 mg/g, 15.2 mg/g, 15.4 mg/g, 15.6 mg/g, 15.8 mg/g, 16.0 mg/g, 16.2 mg/g, 16.4 mg/g, 16.4 mg/g, 16.8 mg/g, 17.0 mg/g, 17.2 mg/g, 17.4 mg/g, 17.6 mg/g, 17.8 mg/g, 18.0 mg/g, 18.2 mg/g, 18.4 mg/g, 18.6 mg/g, 18.8 mg/g, 19.0 mg/g, 19.2 mg/g, 19.4 mg/g, 19.6 mg/g, 19.8 mg/g, 20 mg/g, 21 mg/g, 22. mg/g, 23 mg/g, 24 mg/g, 25 mg/g, 26 mg/g, 27 mg/g, 28 mg/g, 29 mg/g, 30 mg/g, 31 mg/g, 32 mg/g, 33 mg/g, 34 mg/g, 35 mg/g, 36 mg/g, 37 mg/g, 38 mg/g, 39 mg/g, 40 mg/g, 41 mg/g, 42 mg/g, 43 mg/g, 44 mg/g, 45 mg/g, 468 mg/g, 47 mg/g, 48 mg/g, 49 mg/g, or at least about 50 mg/g.

[062] In some embodiments the concentration of panose may be about may be about 0.1 mg/g, 0.5mg/g, 1.0 mg/g, 1.2 mg/g, 1.4 mg/g, 1.6 mg/g, 1.8 mg/g, 2.0 mg/g, 2.2 mg/g, 2.4 mg/g, 2.6 mg/g, 2.8 mg/g, 3.0 mg/g, 3.2 mg/g, 3.4 mg/g, 3.6 mg/g, 3.8 mg/g, 4.0 mg/g, 4.2 mg/g, 4.4 mg/g, 4.6 mg/g, 4.8 mg/g, 5.0 mg/g, 5.2 mg/g, 5.4 mg/g, 5.6 mg/g, 5.8 mg/g, 6.0 mg/g, 6.2 mg/g, 6.4 mg/g, 6.6 mg/g, 6.8 mg/g, 7.0 mg/g, 7.2 mg/g, 7.4 mg/g, 7.6 mg/g, 7. 8 mg/g, 8.0 mg/g, 8.2 mg/g, 8.4 mg/g, 8.6 mg/g, 8.8 mg/g, 9.0 mg/g, 9.2 mg/g, 9.4 mg/g, 9.6 mg/g, 9.8 mg/g, 10.0 mg/g, 10.2 mg/g, 10.4 mg/g, 10.6 mg/g, 10.8 mg/g, 11.0 mg/g, 11.2 mg/g, 11.4 mg/g, 1 1.6 mg/g, 11.8 mg/g, 12.0 mg/g, 12.2 mg/g, 12.4 mg/g, 12.6 mg/g, 12.8 mg/g, 13.0 mg/g, 13.2 mg/g, 13.4 mg/g, 13.6 mg/g, 13.8 mg/g, 14.0 mg/g, 14.2 mg/g, 14.4 mg/g, 14.6 mg/g, 14.8 mg/g, or at least about 15.0 mg/g.

[063] In some embodiments the total concentration of any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose may be about 4 mg/g, 6 mg/g, 8 mg/g, 10 mg/g, 12 mg/g, 14 mg/g, 16 mg/g, 18 mg/g, 20 mg/g, 22 mg/g, 24 mg/g, 26 mg/g, 28 mg/g, 30 mg/g, 32 mg/g, 34 mg/g, 36 mg/g, 38 mg/g, 40 mg/g, 42 mg/g, 44 mg/g, 46 mg/g, 48 mg/g, 50 mg/g, 52 mg/g, 54 mg/g, 56 mg/g, 58 mg/g, 60 mg/g, 62 mg/g, 64 mg/g, 66 mg/g, 68 mg/g, 70 mg/g, 72 mg/g, 74 mg/g, 76 mg/g, 78 mg/g, 80 mg/g, 82 mg/g, 84 mg/g, 86 mg/g, 88 mg/g, 90 mg/g, 92 mg/g, 94 mg/g, 96 mg/g, 98 mg/g, 100 mg/g, 102 mg/g, 104 mg/g, 106 mg/g, 108 mg/g, 1 10 mg/g, 1 12 mg/g, 1 14 mg/g, 1 16 mg/g, 1 18 mg/g, 120 mg/g, 122 mg/g, 124 mg/g, 126 mg/g, 128 mg/g, 130 mg/g, 132 mg/g, 134 mg/g, 136 mg/g, 138 mg/g, 140 mg/g, 142 mg/g, 144 mg/g, 146 mg/g, 148 mg/g, 150 mg/g, 152 mg/g, 154 mg/g, 156 mg/g, 158 mg/g, 160 mg/g, 162 mg/g, 164 mg/g, 166 mg/g, 168 mg/g, 170 mg/g, 172 mg/g, 174 mg/g, 176 mg/g, 178 mg/g, 180 mg/g, 182 mg/g, 184 mg/g, 186 mg/g, 190 mg/g, 192 mg/g, 194 mg/g, 196 mg/g, 198 mg/g, 200 mg/g, 202 mg/g, 206 mg/g, 208 mg/g, 210 mg/g, 212 mg/g, 214 mg/g, 216 mg/g, 218 mg/g, 220 mg/g, 222 mg/g, 224 mg/g, 226 mg/g, 228 mg/g, 230 mg/g, 232 mg/g, 234 mg/g, 236 mg/g, 238 mg/g, 240 mg/g, 242 mg/g, 244 mg/g, 246 mg/g, 248 mg/g or at least about 250 mg/g.

[064] In preferred embodiments the total concentration of any combination of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose is between about 7.5 mg/g to about 185 mg/g. In some embodiments the minimum total concentration of the oligosaccharides is 17.0 mg/g.

[065] The Prebiotic Activity may be calculated relative to a negative control (no composition), using the equation:

Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm)

In this equation Lac, Bif and Clo are the log counts of Lactobacilli, Bifidobacteria and Clostridia, respectively.

[066] The prebiotic activity is typically calculated using a microcosm study such as that described in the examples.

[067] In general the higher the concentration of oligosaccharide the greater the Prebiotic Activity. Typically the prebiotic composition has a Prebiotic Activity of at least 4.0.

[068] In some embodiments the Prebiotic Activity of the composition is at least about 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 1 1.0, at least 1 1.5, at least 12.0, at least 12.5, at least 13.0, at least 13.5, at least 14.0, at least 14.5, at least 15, at least 15.5, at least 16.0, at least 16.5, at least 17.0, at least 17.5, at least 18.0, at least 18.5, at least 19.0, at least 19.5 or at least 20.

[069] The pH of the composition may be about 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8 or about 7.0. [070] The prebiotic compositions are useful to increase the level of Lactobacilli and/or Bifidobacteria in the intestine of an animal in need thereof. Increased levels of Lactobacilli and/or Bifidobacteria are associated with increased wellness.

[071] Upon ingestion the oligosaccharides are fermented in the colon and favour the increase of Lactobacilli and/or Bifidobacteria and in some cases the decrease of Clostridia thereby resulting in changes in gut microflora. The colon is a favorable environment for bacterial growth due to its slow transit time, readily available nutrients, and favorable pH. Generally, bacteria having an almost exclusive saccharolytic metabolism (i.e., no proteolytic activity) can be considered potentially beneficial. Such a metabolic profile is typical for Lactobacilli and Bifidobacteria. Together with the gut immune system, colon

microorganisms contribute significantly to a barrier that prevents pathogenic bacteria from establishing in the gastrointestinal (Gl) tract.

[072] The intestinal flora salvages energy through fermentation of prebiotics such as those in the presently described prebiotic compositions that are not digested in the upper gut. This fermentation can result in the production of butyrate which is preferentially used by colonic epithelial cells, even when competing substrates such as glucose are available. Butyrate is considered a key nutrient determining the metabolic activity and growth of colonocytes and may function as a primary protective factor against colonic disorders. Similarly, the fermentation of prebiotics can produce acetate and propionate. The brain, muscles, and tissues metabolize acetate systemically whereas propionate is cleared by the liver and may lower the hepatic production of cholesterol by interfering with its synthesis. Accordingly, an increase in Lactobacilli and/or Bifidobacteria in the colon is associated with increased wellness.

[073] Similarly, high levels of Clostridia have been associated with a number of conditions from allergies and atopic diseases to autism and therefore decreases in the level of Clostridia are also associated with increased wellness. In this regard the finding that the prebiotic composition described herein, particularly those containing at least 1 mg/g nigerose, can reduce the levels of Clostridia provides basis for the use of the prebiotic compositions to reduce the levels of Clostridia. Consequently wellness may be improved.

[074] The level of Lactobacilli may be increased by 5%, 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least 1000% of the level before use of the composition.

[075] Alternatively or additionally, the level of Bifidobacteria may be increased by 5%, 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least 1000% of the level before use of the composition. [076] Alternatively or additionally the level of Clostridia may be decreased 5%, 10%, 50%, 100%, 200%, 300%, 400% or at least 500% of the level before use of the composition.

[077] Typically the prebiotic compositions are consumed orally. A single dose is at least 1 g but may be up to about 50 g. For example a single dose may be 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g, 40 g, 41 g, 42 g, 43 g, 44 g, 45 g, 46 g, 47 g, 48 g, 49 g, or at least about 50 g.

[078] A subject may consume one or multiple doses per day. For example a subject may takel , 2, 3, 4 or 5 doses per day. Daily dosing is not necessarily required to alter the levels of Lactobacilli, Bifidobacteria and/or Clostridia. In some embodiments the dosing interval is selected from the group consisting of once per week dosing, twice per week dosing, three times per week dosing, four times per week dosing, five times per week dosing, six times per week dosing, weekly dosing, and twice-monthly dosing.

[079] Food products or animal feeds can be supplemented with the prebiotic composition.

[080] The food product may be an infant formula, baby food, baked good (for example a bread, cake, biscuit or cookie, beverage (e.g. soft drink or flavored milk), breakfast food (e.g. cereal), muesli bar, tinned food, snack food (e.g. chips, crisps, corn snacks, nuts, seeds), confection, condiment, marinade, dairy product, dips, spreads or soups.

[081] The animal feed may be a premix or a compound feed in the form of a meal, pellet or crumble.

[082] The food products may contain less than about 1 % (w/w) of the prebiotic composition or about 1 % (w/w), or about 2% (w/w), or about 3% (w/w), or about 4% (w/w), or about 5% (w/w), or about 6% (w/w), or about 7% (w/w), or about 10% (w/w), or about 1 1 % (w/w), or about 12% (w/w), or about 13% (w/w), or about 14% (w/w), or about 15% (w/w) of the prebiotic honey.

[083] In other embodiments there is provided a pharmaceutical composition,

complementary medicine or medical device or substance comprising or consisting of the prebiotic composition.

[084] For example the prebiotic composition can be used as a pharmaceutical composition or complementary medicine. For example the prebiotic composition may be applied directly to the skin of a patient or directly onto a wound. Alternatively the composition may be formulated into a wound gel, ointment or barrier cream. [085] The prebiotic composition may be added to a medical device, for example it can be incorporated into a wound dressing such as a bandage, hydrogel dressing, hydrocolloid dressing, alginate dressing, collagen dressing, or a tulle dressing.

[086] The prebiotic composition can be used as a component of a nutritional supplement. For example the nutritional supplement may be a vitamin supplement, mineral supplement, herbal supplement, meal supplement, sports nutrition product, natural food supplement, and other related products used to boost the nutritional content of the diet.

[087] Typically the prebiotic compositions are prepared from a plurality of component honeys by assaying a number of component honeys. Typically the method comprises: assaying at least one component honey for a desired amount of the oligosaccharide nigerose;

assaying at least one component honey for at least one oligosaccharide selected from the group consisting of trehalose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose,

selecting a number of component honeys on the basis of the oligosaccharide levels determined by the assays; and

mixing the component honeys to form the prebiotic composition.

[088] One or more of the oligosaccharides (trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose.) may be detected and their concentration measured using any method known in the art. For example they may be detected and their concentrations measured using gas chromatography and mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), high-performance liquid chromatography with charged aerosol detection (HPLC-CAD).

[089] In typical commercial production component honeys are sourced from a number of geographically diverse producers and the oligosaccharide content of at least some of these honeys are measured to determine which honeys can be combined to produce the prebiotic compositions described herein.

[090] The prebiotic composition can also be prepared from a plurality of component honeys on the basis of microcosm assays. In this embodiment the method comprises assaying at least one component honey using an intestinal microcosm for a desired effect on Lactobacilli, Bifidobacteria and/or Clostridia levels;

selecting a number of component honeys on the basis of their effects on Lactobacilli, Bifidobacteria and/or Clostridia levels; and mixing the component honeys to form the prebiotic composition.

[091] The microcosm assays utilize the microbial populations in faecal material from healthy human volunteers to allow examination of the effect of honeys on the entire intestinal microbial population in particular the levels of Lactobacilli, Bifidobacteria and/or Clostridia in an in vitro setting. Typically the microcosm assays are performed as set out in P.L. Conway, R Stern, L. Tran; (2010); The Value-adding Potential of Prebiotic Components of Australian Honey; Rural Industries Research and Development Corporation 09/179, Australian Government. The assays involve setting up the microcosms, measuring the levels of Lactobacilli, Bifidobacteria and/or Clostridia then applying samples of at least one of the honeys to the microcosms, incubating the microcosms for a predetermined period of time before again measuring the levels of Lactobacilli, Bifidobacteria and/or Clostridia in the microcosm to determine the effect of the component honey(s) on the levels of those groups of bacteria.

[092] Component honeys can then be mixed to form the prebiotic composition that will have the desired effect on Lactobacilli, Bifidobacteria and/or Clostridia levels. For example if one component honey results in a 15% increase in Lactobacilli levels and another results in a 10% increase these honeys can be mixed in equal portions to provide a prebiotic composition that will result in a 12.5% increase in Lactobacilli levels.

[093] In some embodiments mixing individual honeys results in a synergistic effect. For example if one component honey results in a 15% increase in Lactobacilli levels and another results in a 10% increase, these honeys can be mixed in equal portions to provide a prebiotic composition that will result in an increase in Lactobacilli levels that is greater than 12.5%.

[094] Similarly, in some embodiments some oligosaccharides have a synergistic effect on microbial levels when used together. For example if one oligosaccharide results in a 5% increase in Lactobacilli levels and another results in a 10% increase it would be expected that when used together these oligosaccharides would produce a 15% increase in

Lactobacilli levels. However, in this embodiment the use of multiple oligosaccharides will produce an effect that is greater than the additive effect of each in isolation.

[095] In some embodiments the pH and/or the temperature may be manipulated during production to optimize the level of at least one oligosaccharide selected from the group consisting of trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose.

[096] Without being bound by any particular mechanism of action it is believed that certain enzymes such as sucrase and lactase present in, or added to, the composition are able to utilize saccharides that may for example, be present in component honey to produce trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and/or palatinose. In one embodiment the pH or the prebiotic composition may be modulated to provide favorable conditions for the enzymes to produce one or more of the oligosaccharides. Typically, honey may have a pH as low as 4 which may prevent effective enzymatic activity from occurring. By raising the pH either temporarily or permanently, enzymatic activity that produces the oligosaccharides can be facilitated. For example the pH may be raised to about 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8 or about 7.0.

[097] The pH may be raised permanently or temporarily. For example the pH is typically raised for a period of time sufficient to facilitate a suitable level of enzyme activity. This may be from a few minutes up to several hours or a day.

[098] In some embodiments the pH is raised or lowered to a pH that is near to the optimum pH for the enzymes.

[099] The pH can be reduced by using an acid that is a GRAS (Generally Recognized As Safe) substance. For example, these include acetic acid, aconitic acid, adipic acid, ascorbic acid (vitamin C), benzoic acid, caprylic acid, cholic acid, citric acid, desoxycholic acid, erythorbic acid (D-isoascorbic acid);glutamic acid; glutamic acid hydrochloride; glycocholic acid; Hydrochloric acid; lactic acid; linoleic acid; malic acid; niacin; pectin; phosphoric acid; propionic acid; sorbic acid; stearic acid; succinic acid; tannic acid; tartaric acid; taurocholic acid or thiodipropionic acid.

[0100] The pH can be lowered by using a substance that is a GRAS (Generally Recognized As Safe) substance. For example, these include ammonium hydroxide, sodium carbonate; ammonium phosphate; magnesium phosphate or potassium phosphate.

[0101] Similarly the temperature of the composition can be modulated to facilitate enzymatic activity to produce trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and/or palatinose. The temperature may be raised from room temperature to 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C or higher. The temperature is typically raised for a period of time sufficient to facilitate a suitable level of enzyme activity. For example the temperature can be raised for a few minutes up to several hours or a day. In some embodiments the temperature is raised for up to a day.

[0102] In some embodiments the temperature is raised to a temperature that is near to the optimum temperature of the enzymes.

[0103] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0104] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

[0105] As used herein the term 'in need thereof is a statement of purpose for which the method or use must be performed. For example, the term is to be interpreted to mean that the prebiotic honey is to be used to increase the level of Lactobacilli and Bifidobacteria and reduce the levels of Clostridia in the intestine of a mammal with a recognized need for modulation of the levels of these bacteria.

[0106] In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

[0107] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

Example 1 : Prebiotic testing using in vitro microcosms

[0108] Intestinal microcosms were used to explore the prebiotic properties of Australian sourced honeys. The prebiotic effects of honey samples were compared to those of the same total concentrations of inulin and sucrose using intestinal microcosms containing the full complex microbial community of the human intestine. Microcosms were set up using faecal material from healthy human volunteers to allow examination of the effect of ingested honeys on the entire intestinal microbial population as previously described (P.L. Conway, R Stern, L. Tran; (2010); The Value-adding Potential of Prebiotic Components of Australian Honey; Rural Industries Research and Development Corporation 09/179, Australian Government.).

[0109] By calculating the difference between the test honey microcosm and the negative control microcosm it is possible to determine the prebiotic activity (see Example 9 below) of the test honey in the laboratory (in vitro). It is to be noted that the digestion of the honey to simulate the passage of the whole honey through the small intestine produced a comparable result to that for whole honey when tested in the in vitro microcosm by examining the lactobacilli, bifidobacteria and Clostridia response.

[01 10] The levels of lactobacilli, bifidobacteria and Clostridia levels after 48 hours incubation in vivo and in a microcosm (in vitro) containing a final concentration of 1 % (w/v) honey were examined to determine whether in vitro assays utilising human faecal material provided a useful and comparative in vivo model. Four honeys were used (H1 , H2, H3 and H4 - collectively referred to as the Four Honeys).

[01 11] As illustrated in Figure 1 , it was found that in vitro microcosm methodology may be effective in predicting human in vivo results from whole honey. Thus, enzymatic pre- digestion of oligosaccharides metabolised by humans was not necessary to obtain an indication of in vivo effects.

[01 12] The values obtained within a given test provide a useful comparison of results obtained with the same microcosm. However, as illustrated in Figure 2 (Lactobacillus), Figure 3 (Bifidobacteria) and Table 1 (Clostridia), reliable comparisons cannot be made between different microcosms due to the differences in donor profiles intrinsic to the assay. The difference in negative control value reported for bacterial counts (cfu) in Table 1 illustrates the variation observed between microcosms.

Table 1 - Relative counts (cfu) of Clostridia via Microcosm assay run on days 0, 15 and 33

SAMPLE Day 0 Day 15 Day 33

Initial 14 97 21

CZ0121 100 49

CZ0122 109

CZ0123 26

CZ0124 1 13 48

CZ0125 61 20

CZ0126 20 42

CZ0127 32

CZ0128 23

CZ0129 15

CZ0130 40

CZ0131 18 26 SAMPLE Day 0 Day 15 Day 33

CZ0132 27 29

CZ0133 44 38

CZ0134 24

CZ0135 15 18

CZ0136 133

CZ0137 33 26

CZ0138 20 35

CZ0139 55

CZ0140 40

CZ0141 / H3 23 22

CZ0142 / H2 105 58

CZ0143 / H4 87 105 29

CZ0144 / H1 23 76 29

Negative 2276 1353 2039

AH + inu 162

AH + fos 94

Inulin 0.25 % 72

inulin 0.15mg/ml 58 26

inulin 2.5mg/ml 43

inulin 5.0mg/ml 40

[01 13] As illustrated in Figure 4, the relationship between various sugars and bacterial species was also investigated and correlations were found between populations of various species and total anaerobes.

Example 2: Assessment of prebiotic activity in raw honeys using in vitro microcosms

[01 14] Prebiotic properties of 24 Capilano Australian raw honeys were studied using in vitro microcosms at 1 % honey. Microcosms with no added substrate (i.e. 1 % inoculum in medium only) were used as the negative controls and with 1 % inulin (commercial prebiotic), sucrose or glucose as the positive controls. Samples were taken after 48 hours incubation for enumeration of the major bacterial groups using anaerobic culture techniques and also for SCFA analysis and DNA molecular profiling using 16 s pyrosequencing using lllumina.

[01 15] Figure 5 illustrates the Lactobacillus and Bifidobacteria bacterial counts (cfu/g), and Figure 6 shows the Clostridia bacterial counts, in microcosms supplemented with the 24 raw Australian honeys.

[01 16] Figure 7 illustrates the Prebiotic Activity of the 24 raw honeys. The Prebiotic Activity of the raw honey varied, with honeys 9 and 10 most effective. These honeys increased the lactobacillus by at least log 2.4 and up till log 4.0 while an increase of only log 1.5 was noted for the positive control, inulin. In addition, the Clostridia were reduced with the honeys 9 and 10. These honeys are identified as 'Super Honey'.

[01 17] The prebiotic properties of the Four Honey were also tested using the same method (Figures 8-10).

Example 3: Assessment of prebiotic activity in blended and imported honeys using in vitro microcosms

[01 18] Prebiotic properties of a variety of other blended and imported honeys were also assessed using in vitro microcosms at 1 % honey.

Blended honeys

[01 19] Figure 11 illustrates the Lactobacillus and Bifidobacteria bacterial counts (cfu/g) in 20 different blended honeys, H1 (Jarrah honey) and H4 (Australian Yellow Box honey) respectively. Of the Four Honeys, H1 was the weakest performing promotor of Lactobacillus and Bifidobacteria growth, while H4 was the strongest performer.

[0120] The blended honeys were typically comparable with or outperformed H1 at promoting the growth of Lactobacillus bacteria, while 4 of the blended honeys showed decreased growth of Lactobacillus compared to H1.

[0121] For Bifidobacteria, the majority of blended honeys performed comparably with the raw Australian honeys, with only 2 of the blended honeys showing decreased levels of Bifidobaceria compared to H1 .

[0122] Figure 12 shows the Clostridia bacterial count in the same 20 blended honeys, H1 and H4. H1 appeared to suppress growth of Clostridia while H4 appeared to modestly promote growth. Only 4 blended honeys promoted the growth of Clostridia beyond the levels observed for H4, while 16 of the 20 blended honeys performed at least as well as H4 in suppressing Clostridia growth. Imported honeys

[0123] The in vitro performance of 21 imported honeys was also assessed using the microcosm assay. Figure 14 illustrates the Lactobacillus and Bifidobacteria bacterial counts (cfu/g) and Figure 15 shows the Clostridia bacterial counts for the imported honeys. Figure 16 shows the Prebiotic Activity of the 21 honeys tested.

[0124] None of the imported honeys outperformed the positive controls, as is typically the case for Australian honeys. One of the honeys (No. CZ106) was found to be approximately 10 times less effective at inhibiting Clostridia growth than the positive controls, but was 5 times more effective than the negative control (no honey added).

Comparative performance of honey groups

[0125] Comparison of the performance of the raw Australian, blended and imported honeys shows that the floral sources does not appear to determine or influence prebiotic activity, nor does the colour or source (single vs mixed) of the honey (Table 2).

[0126] In general, blended Australian honeys appear to have greater prebiotic activity than raw Australian honeys, which in turn have greater prebiotic activity than raw imported honeys. The blended honeys appear to provide greater uniformity across the sample set, which appears to correlate with the normalised oligosaccharide profiles discussed in Example 5.

Table 2 - Summary of qualitative comparative in vitro performance of various groups of honey

Honey group Compared with Compared with Compared with Comments negative control Australian honey positive control

All performed better All performed better All performed better Best overall

Australian

performance blends

All performed better (Not tested) Did not perform as Low counts

Imported

well as positive overall control

All performed better (Not tested) 50% of samples Wide variability in

Australian raw

performed better results

All performed better 50% performed Wide variability in

Four Honeys

better than H1 and results

H4 Example 4: Analysis of oligosaccharide content in raw Australian honeys

The oligosaccharide content of the 24 Capilano raw Australian honeys used in Example 3 were analysed using GC-MS for the di- and tri-saccharides and HPLC for the tetra- and penta-saccharides. Seventeen di-saccharides and eight tri-saccharides as well as several unidentified di- and tri-saccharide peaks were quantified. Noticeable differences among the samples were observed in the composition of individual carbohydrate oligosaccharides (Figure 17).

[0127] The oligosaccharide content of the Four Honeys was also tested by the same method.

[0128] It was found (see below) that in the Four Honeys, the oligosaccharides maltulose, nigerose, turanose, kojibiose, trehalulose, palatinose and isomaltose at between 17.0 mg/g and 183.2 mg/g correlated with an elevation of lactobacilli and that of these the nigerose, palatinose and isomaltose were most extensively utilised by the lactobacilli and

bifidobacteria. The concentration of these was a maximum of 14, 7 and 34 mg per g of honey, respectively.

[0129] Dominant sugars in the 24 Capilano raw honeys were consistent with those detected in the Four Honeys.

Example 5: Analysis of oligosaccharide content in other honeys

[0130] Various other blended and raw honeys were also assessed for their oligosaccharide content using GC-MS for the di- and tri-saccharides and HPLC for the tetra- and pentasaccharides.

[0131] No correlation was observed between the relative concentrations of

oligosaccharides between raw honeys (Figure 18) and blended honeys (Figure 19). Single floral source, raw honey and blended honeys all presented different oligosaccharide profiles. The only constant value identified was the complete absence of leucrose.

[0132] Blending of honey appeared to result in profound changes in the oligosaccharide profile. For example, the relative concentrations of various oligosaccharides between the honeys is more normalised, the concentration of sucrose is decreased and for a number of oligosaccharides (α,β-threhalose, maltulose, nigerose, kojibiose) the average concentration appears to increase, and standard deviation decreases, indicating that for this data set these constituents appear to increase and standardise.

The present inventors have postulated that blending of honeys results in an increased number of enzyme and substrate interactions occurring, resulting in progressing towards a new equilibrium between these natural constituents. In this proposed model, unblended honeys have likely reached an exhaustion of the necessary substrates for particular enzymes, but when blending occurs the substrates of each honey are utilised by the enzymes remaining in the other. Physical mixing and heating may also accelerate these reactions.

Example 6: Utilisation of saccharides by beneficial bacteria

[0133] To determine which oligosaccharides are utilised by lactobacilli and bifidobacteria the strains L. fermentum PC1 and B. breve M16V were grown in a Basal Medium (BM) supplemented with the Four Honeys and the resultant spent culture honey was analysed for saccharide content.

[0134] While some of the oligosaccharides were partially or completely utilised, there were increases in some indicating that larger oligosaccharides were being broken down and the end products not utilised. It was found that isomaltose and nigerose, which are in amounts greater than 0.5 mg per gram of honey, are also extensively utilised. Compilation of the data for the Four Honeys and both bacteria is presented in Figure 20.

[0135] Most of the oligosaccharides identified had about 40% utilisation, namely maltulose, turanose, kolibiose, trehalulose, while the remainder had higher amounts utilised with 55, 75, 85 and 95% of the palatinose, nigerose, panose and isomaltose, respectively. This shows that these four oligosaccharides play an active role in the prebiotic activity of the honeys.

Example 7: Quantification of Oligosaccharides in Capilano Raw Honeys

[0136] Analysis of the data showed the overall abundance of each detected

oligosaccharide across all 24 honeys (Figure 17). Ten oligosaccharides were detected at larger concentrations and these were erlose, nigerose, trehalulose, maltulose, kojibiose, sucrose, turanose and maltose. Most of the variation in honeys occurs in about 10 oligosaccharides and Figure 21 presents the heat map of these oligosaccharides for the 24 Capilano raw honeys. The 10 oligosaccharides showing most variation across all honeys are erlose, nigerose, trehalulose, maltulose, kojibiose, sucrose, turanose, maltose α,β- trehalose and isomaltose.

[0137] The amounts of the tetra-saccharides (DP 4) were low for all honeys with 0 - 2 mg/g of honey, except for honey 4 which had 8 mg/g. Only three honeys had detectable pentasaccharides, namely 2, 14 and 15 with honey 2 having considerably more pentasaccharides. [0138] For the Four Honeys it was found (see below) that the oligosaccharides maltulose, nigerose, turanose, kojibiose, trehalulose, palatinose and isomaltose correlated with an elevation of lactobacilli and that of these the nigerose, palatinose and isomaltose were most extensively utilised by the lactobacilli and bifidobacteria.

Example 8: Identification of Oligosaccharides of Interest for Prebiotic Activity

[0139] The di- and tri-saccharides in the Four Honeys and in the 24 Capilano raw

Australian honeys were quantified. These were sourced as pure analytical grade saccharides and tested for prebiotic activity in the in vitro microcosms by measuring the amounts of lactobacilli, bifidobacteria and Clostridia. In addition, they were evaluated in defined laboratory medium using pure cultures of these bacteria.

[0140] Results showed that most pure saccharides promoted the levels of lactobacilli and bifidobacteria and reduced numbers of Clostridia (Table 3). The effect was reduced when the pure saccharide was included with honey compared to the addition of the pure saccharide alone to the microcosm.

Table 3 - A summary of the impact of the various pure oligosaccharides on the bacterial populations in the intestinal microcosms supplemented with the saccharide as the sole added carbohydrate

Saccharide Cone used Cone in honey Lacto Bifi Clos Comments

(mg/100ml) (mg/g honey)

nigerose 4 4 U u D small benefit but good

overall

nigerose 7 4 U u D* small benefit but good palatinose 1 1 u u D good

palatinose 7 1 u u D good

isomaltose 2 2 u u D good

isomaltose 7.5 2 u u D good

maltose 7.5 14 u u D good

maltose 14 14 u u D ^* good

erlose 7.5 4 u u D# small benefit but good

overall

kojibiose 7.5 6.5 u u N small benefit, except for

Clostridia

α,β-trehalose 7.5 2 u u D# small benefit, better with honey poor for Lb & Bif; Saccharide Cone used Cone in honey Lacto Bifi Clos Comments

(mg/100ml) (mg/g honey)

good for clos

panose 7.5 1 N N# D#

1-kestose 7.5 1 U N D ^* small benefit for Lb and

Clostridia

raffinose 7.5 0.2 U ^* N D ^* marginal

meliobiose 7.5 0.05 N U ^* D marginal

trehalulose 7.5 4 U* U* D* good

maltulose 7.5 6 N U* N Only good for bifido turanose 7.5 16 N IP D ^* Bifido up and Clostridia down turanose 15 16 N U* D -

U = increase; D = decrease; N = no effect; * less when in honey; # better with honey

[0141] Pure cultures were used to illustrate that most of the tested saccharides could be utilised by lactobacilli. Most notably in pure culture, the combination of the pure oligosaccharides yielded more growth at the two time points studied than the pure saccharides alone. This would simulate the effect of the honey and highlights that the combination of oligosaccharides could be conferring biologically significant results on bacterial growth.

Example 9: Microcosm of Capilano Finished Good Products for Prebiotic Activity

[0142] Samples of twenty processed honeys from Capilano retention samples were tested in the microcosm to investigate the prebiotic activity of blended commercial Australian honeys produced exclusively by Capilano.

[0143] The positive control was inulin at a concentration of: 0.25 % (2.5 mg/ml) equivalent to One serving' of inulin, and 0.15 mg/ml equivalent to 1 % of 1.5 % total oligosaccharides in honey.

[0144] The prebiotic activity of the honeys was calculated relative to the negative control (no honey added), using the equation:

Prebiotic Activity = (Lac + Bif - Clo in honey microcosms) - (Lac + Bif - Clo in negative control microcosm)

In this equation Lac, Bif and Clo are the log counts of Lactobacilli, Bifidobacteria and Clostridia, respectively. [0145] Figure 22 details the Prebiotic Activity of the processed honeys tested using the microcosm assay, it includes H1 and H2. The Prebiotic Activity microcosm test demonstrated H1 to be superior than H2. Figure 24 shows the growth profile of the target bacterium. Table 2 (below) shows the bacterial counts (log cfu/ml) of bifidobacteria, lactobacilli and Clostridia that are represented graphically in Figure 26.

Table 4 Bacterial Counts (log cfu/ml) of bifidobacteria, lactobacilli and Clostridia for processed honeys

Bifidobacteria Lactobacilli Clostridia

Log SD Log SD Log CFU/ml SD

CFU/ml CFU/ml

Initial 5.55 0.17 5.31 0.06 2.61 0.12

CZ0121 7.32 0.14 5.43 0.04 4.61 0.34

CZ0122 7.71 0.06 7.21 0.00 4.69 0.32

CZ0123 7.60 0.05 6.45 0.04 3.25 0.78

CZ0124 7.45 0.11 5.88 0.08 4.73 0.13

CZ0125 7.42 0.08 5.81 0.08 4.11 0.83

CZ0126 7.79 0.08 7.33 0.14 3.00 0.67

CZ0127 7.50 0.03 6.84 0.20 3.47 0.49

CZ0128 7.56 0.06 5.71 0.02 3.13 0.61

CZ0129 7.37 0.00 5.27 0.06 2.70 0.00

CZO130 7.66 0.09 5.43 0.19 3.70 0.14

CZ0131 7.02 0.03 4.82 0.09 2.91 0.30

CZ0132 7.62 0.14 6.49 0.17 3.31 0.69

CZ0133 7.69 0.12 5.37 0.21 3.78 0.12

CZ0134 7.74 0.09 5.97 0.08 3.19 0.95

CZ0135 7.75 0.04 7.44 0.07 2.70 0.00

CZ0136 7.74 0.02 6.78 0.15 4.89 0.41

CZ0137 7.76 0.05 7.17 0.02 3.49 1.12

CZ0138 7.71 0.21 7.42 0.09 3.00 0.67

CZ0139 7.89 0.04 6.52 0.21 4.01 0.06

CZO140 7.98 0.06 6.20 0.08 3.70 0.00 Bifidobacteria Lactobacilli Clostridia

H1 7.78 0.00 6.97 0.02 3.13 0.61

H2 7.45 0.04 5.92 0.10 4.47 0.28

AH + inu 8.29 0.58 5.62 0.02 5.09 0.00

AH + fos 7.82 0.12 5.22 0.00 4.54 0.00

Inulin 8.09 0.07 5.95 0.21 4.27 0.01 0.25%

Inulin 7.76 0.12 5.11 0.06 4.06 0.09

0.15

mg/ml

Negative 5.99 0.02 4.05 0.07 7.73 0.19 control

[0146] The presence of honey and positive control prebiotics in the microcosm environment delivered increases in bifidobacteria, lactobacilli and supressed Clostridia. In comparison to the 'single serve' quantity of inulin that included as a commercial 'benchmark' it was found that:

• 13 (65%) of 20 commercial honeys received a Prebiotic Activity score greater than the Inulin positive control;

• 4 (20%) of 20 commercial honeys received a Prebiotic Activity score 20% better than the Inulin positive control.

Example 10: Microcosm of International Honeys for Prebiotic Activity

[0147] Table 5 and Figures 24 and 26 show the microcosm results for non-Australian honeys. With the exception of a single Chinese honey (No. CZ01 11 ) all imported honeys had a Prebiotic Activity below that of the inulin 'single serve' positive control (Prebiotic Activity 7.5). Table 5 - Prebiotic activity for different imported honeys enumerated using microcosms established with whole honey relative to the negative control (microcosm with no added honey). Controls include artificial honey (AH) with added inulin or FOS and AH alone.

Description of Origin Lab No. CZZ No. Prebiotic SD

activity

Brazilian Orange Blossom CZ0088 205034 4.4 0.1

Arg Eucalypt - B:825 CZ0101 205098 4.9 0.1 PO:32727

Arg Eucalypt - B:826 CZ0102 205099 6.8 0.4 PO:32728

Arg Eucalypt - B:830 CZ0103 205100 4.4 0.1 PO:32732

Arg Eucalypt - B:843 CZ0104 205101 5.9 0.2 PO:32736

Arg Eucalypt - B:846 CZ0105 205102 7.0 0.2 PO:32739

Arg Eucalypt - B:847 CZ0106 205103 5.4 0.0 PO:32740

Chinese Acacia - #15051 1 - CZ0107 205104 7.3 0.1 2

Hungarian Acacia - HA - 20 CZ0108 205105 6.5 0.1

Hungarian Acacia - HA - 27 CZ0109 205106 6.2 0.2

Chinese # 30 Feb 15 CZ01 10 205109 6.3 0.1

Chinese # 93 June 15 CZ01 1 1 2051 10 7.9 0.2

Chinese * 152 Oct 15 CZ01 12 2051 1 1 5.0 0.7

Argentine # 4 Feb 15 CZ01 13 2051 12 6.1 0.2

Argentine # 18 June 15 CZ01 14 2051 13 4.8 0.3

Argentine # 44 Oct 15 CZ01 15 2051 14 6.7 0.5

Mexican # 1 May 15 CZ01 16 2051 15 6.5 0.1

Mexican # 10 July 15 CZ01 17 2051 16 7.3 0.1

Mexican # 20 Sept 15 CZ01 18 2051 17 4.9 0.2 Description of Origin Lab No. CZZ No. Prebiotic SD

activity

Brazilian Org # 1 Feb 15 CZ01 19 2051 18 5.6 0.1

Brazilian Org # 14 June 15 CZ0120 2051 19 6.6 0.0

Positive Control 1 AH + inu AH + inu 6.6 0.1

Positive Control 2 AH + fos AH + fos 7.1 0.1

Positive Control 3 AH AH 7.0 0.3

Example 11 : Prebiotic Activity Data of Raw Capilano honeys and the Four Honeys tested for their Saccharide Content

[0148] Twenty raw honeys were tested in microcosm assays to determine bacterial growth responses and prebiotic activity (Figures 23 and 26). In addition, the Four Honeys were tested in the microcosm to investigate their prebiotic activity (Figure 27, Table 6).

Table 6 - Prebiotic activity (PA) of the Four Honeys.

Example 12: Correlations of Honey Oligosaccharide Content with Prebiotic Activity

[0149] Many of the oligosaccharides quantified had no correlation with Prebiotic Activity or the growth of Bifidobacteria and Lactobacilli, as set out in Table 7.

Table 7: Oligosaccharide Content (mg/g) of the Four Honeys and 24 raw Australian honeys

[0150] Figure 28 and Table 8 detail the average sum of those oligosaccharides in honeys whose detected content correlated to an increasing Prebiotic Activity.

Table 8 - Summary of the key oligosaccharide content (mg/g) of the Four Honeys and their percentage composition. The Linear R ² is included to illustrate the degree of correlation of the key oligosaccharides with the honeys Prebiotic Activity. An R ² = 1 indicates that the regression line perfectly fits the data, while an R ² of 0 indicates that the line does not fit the data at all.

[0151 ] The oligosaccharides detailed in Table 8 are responsible for the increasing Prebiotic Activity. Figure 29 shows the linear and exponential relationship of these summed identified oligosaccharides and the resulting increase in Prebiotic Activity. The relationship is stronger as a linear model.

[0152] Figure 30 shows the linear and exponential relationship of the summed

oligosaccharides found in the Four Honeys with an additional four raw Capilano honeys and the resulting increase in prebiotic activity. The exponential model relationship was a better correlation. Raw Capilano honeys tested with a Prebiotic Activity of less than 6.5 were excluded as there was a concern that the organic matter in raw honey had reduced the efficacy of oligosaccharide purification and quantification using analytical techniques (GS- MS). Example 13: Correlation of the Four Honeys with 8 oligosaccharides

[0153] The oligosaccharide profiles of the Four Honeys were extensively analysed and combined with metabolic characteristic of the various Lactobacillus, Bifidobacteria and Clostridia species to determine which oligosaccharides could be potentially therapeutic and/or characterising of prebiotic effect.

[0154] Examination of these profiles indicated that the relationship between 8 key oligosaccharides is highly conserved regardless of the concentration of the total oligosaccharides or the concentration of these combined oligosaccharides (Table 9).

[0155] These 8 oligosaccharides were identified as key to prebiotic activity and maintaining this relationship is key to optimising prebiotic effect. The 8 oligosaccharides (collectively referred to as K8) are maltulose, nigerose, turanose, kojibiose, trehalulose, palatinose, isomaltose and panose (Table 10). Each of these K8 oligosaccharides contains reducing sugars (Table 11 ).

Table 9 - Concentration of oligosaccharides in mg/g from the Four Honeys and their percentage composition.

SAMPLE H1 H2 H3 H4

Sucrose 0.2 0.1 31.8 29.1 0.2 0.1 0.6 0.2 α,α-Trehalose 0.1 0.3 0.1 0.1 0.1 0.2 0.1 0.3 α,β-Trehalose 3.3 5.1 0.8 4.1 3.2 6.2 3.0 6.0

Inulobiose 0.9 0.5 2.9 0.8 1.8 0.7 1.3 0.6

Cellobiose 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Laminaribiose 0.0 0.0 2.1 1.1 0.7 0.5 0.7 0.4

Maltulose 43.6 44.0 2.5 2.5 16.9 17.9 16.0 20.1

Nigerose 14.4 10.9 4.0 2.4 9.5 9.5 10.0 8.6

Turanose 28.1 24.7 5.5 10.0 24.0 29.7 26.6 28.7

Maltose 11.4 17.7 14.1 10.7 7.1 5.9 10.7 12.9

Kojibiose 19.0 14.6 2.6 2.3 16.2 15.3 13.8 15.6

Trehalulose 36.3 35.6 1.7 2.1 8.8 7.1 11.5 15.2

Palatinose 7.5 8.2 0.2 0.1 1.1 1.3 2.4 3.0

Gentiobiose 0.2 0.5 0.4 0.5 0.1 0.3 0.1 0.2

Isomaltose 34.4 37.0 0.5 0.3 7.2 5.8 7.8 10.0

Melibiose 0.2 0.0 0.0 0.0 0.1 0.0 0.1 0.0

Unknown DS 2.1 2.2 6.1 5.0 2.7 3.3 2.4 10.1

Raff i nose 0.2 0.2 0.1 0.1 0.0 0.0 0.0 0.1

1-Kestose 0.6 0.7 0.7 0.5 0.3 0.3 1.2 1.3 SAMPLE H1 H2 H3 H4

Erlose 0.2 0.1 0.9 0.7 0.5 0.5 3.4 2.8

Melezitose 0.2 0.2 0.1 0.1 0.2 0.1 0.4 0.3

Theanderose 0.2 0.1 0.0 0.0 0.1 0.1 0.2 0.2

Maltotriose 0.3 0.3 0.1 0.1 0.2 0.2 0.5 0.3

Panose 1.0 1.3 0.0 0.0 0.2 0.3 0.4 0.6

Unknown TS 2.8 3.7 0.2 0.0 0.5 0.5 0.8 0.9

TOTAL 207.2 208.1 77.5 72.6 101.8 105.9 114.0 138.4

TOTAL DS 201.8 201.5 75.3 71.2 99.8 103.9 107.2 131.9

TOTAL TS 5.4 6.6 2.2 1.4 2.0 2.0 6.8 6.5

Total less human 193.2 192.9 36.1 37.4 95.3 99.7 106.3 123.3 digestible

T8 184.3 176.3 17.1 19.7 84.0 86.9 88.5 101.8

Table 10 - Concentration of the 8 key oligosaccharides (K8) for which the relative ratio is highly conserved in prebiotic honey

Sample % Total % Total % Total % Total % Total % total % Total % total Maltulose Nigerose Turanose Kojibiose Trehalulose Palatinose Isomaltose Panose

CT1 23.68 7.81 15.25 10.28 19.71 4.07 18.64 0.57

CZ141 24.98 6.15 14.02 8.25 20.20 4.68 20.96 0.75

CT2 14.81 23.30 32.08 15.22 10.19 1.17 2.93 0.29

CZ142 12.84 11.98 50.89 11.68 10.84 0.51 1.27 0.00

CT3 20.16 11.29 28.62 19.28 10.53 1.25 8.63 0.25

CZ143 20.60 10.91 34.17 17.55 8.15 1.50 6.73 0.39

CT4 18.07 11.28 30.09 15.58 13.04 2.65 8.86 0.42

CZ144 19.77 8.46 28.19 15.27 14.91 2.95 9.87 0.59

Average 19.4 11.4 29.2 14.1 13.4 2.3 9.7 0.4

Std Dev 4.1 5.2 11.5 3.7 4.5 1.5 6.9 0.2

CV 0.2 0.5 0.4 0.3 0.3 0.6 0.7 0.6 Table 11 - Reducing sugars present in K8 oligosaccharides

[0156] Each of the Four Honeys has different effects on the in vivo promotion of

Lactobacillus, promoting Bifidobacteria and suppressing Clostridia (Figure 1 ).

[0157] The ability of humans, Lactobacillus, Bifidobacteria and Clostridia to digest each of the oligosaccharides indentified in the Four Honeys was investigated. Oligosaccharides capable of being digested by humans (referred to as 'human digestible' oligosaccharides), including sucrose, α,α-trehalose, cellobiose, maltose, palatinose (aka isomaltulose) and maltotriose were not considered in the investigation of stimulation or suppression of various gut biota. Oligosaccharides identified via literature review as having prebiotic effect included isomaltose, kojibiose and erlose.

[0158] Lactobacillus appears to have enzymes that offer it exclusive access to inulobiose, 1 -Kestose and raffinose, and produces 17 enzymes that can utilise 13 of the

oligosaccharides (leucrose, trehalulose & maltulose are not digested).

[0159] Bifidobacteria appears to have enzymes the offer exclusive access to maltulose and trehalulose, and can utilise of 1 1 oligosaccharides; using just 6 enzymes (α,β-trehalose, laminaribose, maltulose, nigerose, turanose, trehalulose, gentiobiose, melibiose, raffinose, isomaltriose and panose).

[0160] Clostridia appears to be able to utilise 6 oligosaccharides; with 4 enzymes, 1 of which is inhibited by nigerose. Two of the enzymes are shared by Lactobacillus; thus in an environment when Lactobacillus and Bifidobacteria predominate Clostridia will have preferential access only to leucrose (which is not digested by either Lactobacillus or Bifidobacteria). Example 14: K8 Oligosaccharide ratio is highly conserved in prebiotic honey

[0161] The ratio of K8 oligosaccharides is highly conserved between all honeys. The Four Honeys appear to have the greatest variation in the relative ratio of K8 oligosaccharides (Table 12). Blended honeys have the highest degree of consistency in the ratios (CZ121 to CZ138, Table 13). The Four Honeys were not blended or commercially processed (they were however heated and filtered at bench top scale).

Table 12 - Relative ratio of the K8 oligosaccharides in the Four Honeys.

Table 13 - Relative ratio of the K8 oligosaccharides in blended honeys

Sample % Total % Total % Total % Total % Total % total % Total % total Maltulo Nigerose Turanose Kojibiose Trehalulose Palatinose Isomaltose Panose se

CZ121 17.83 12.54 35.39 17.81 8.03 1.57 6.16 0.67

CZ124 19.00 10.98 28.99 19.55 9.41 1.63 9.80 0.64

CZ125 18.81 9.23 35.54 15.30 12.67 2.11 5.80 0.54

CZ126 16.31 12.76 40.59 14.79 8.40 1.58 4.88 0.68

CZ131 16.98 15.73 31.81 20.27 6.75 1.43 6.32 0.70

CZ132 17.80 15.16 37.83 16.35 7.55 1.66 4.21 0.49

CZ133 18.70 14.41 34.26 17.93 7.20 1.43 5.60 0.47

CZ135 16.61 8.16 35.51 20.54 9.41 1.53 7.55 0.68

CZ137 16.60 14.49 30.87 19.93 8.75 1.55 6.96 0.85

CZ138 16.99 11.36 39.65 15.89 9.65 1.94 4.12 0.41

Averag 17.56 12.48 35.05 17.84 8.78 1.64 6.14 0.61 e

Std 1.00 2.54 3.73 2.17 1.68 0.22 1.70 0.13 Dev

CV 0.06 0.20 0.11 0.12 0.19 0.13 0.28 0.22

[0162] While turanose is typically the most abundant of the K8 oligosaccharides, its relative concentration varies considerably with each honey (Figure 32). The concentration of turanose does however appear to be proportional to the combined concentrations of nigerose and kojibiose (Figure 33).

[0163] The concentration of maltulose is indicative of the overall concentration of the K8 oligosaccharides (R ² = 0.97108) (Figures 34 and 35). While not the most abundant, Maltulose is the most highly conserved of the 8 key oligosaccharides, and can be used to predict the concentration of the remaining seven key oligosaccharides (R ² = 0.97108) and the total concentration of disaccharides (R ² = 0.76339). The combined concentration of turnarose and kojibiose closely mimics the concentration of maltulose.

[0164] Nigerose is the second most highly conserved K8 oligosaccharide (after maltulose). nigerose concentration can be used to predict the K8 (Figure 36) and total disaccharide (Figure 37) concentration in honey. There also appears to be a strong relationship between the concentration of nigerose in mg/g and the magnitude of combined Lactobacillus and Bifidobacteria growth in vitro . Thus, nigerose may be predictive of the magnitude of combined response of Lactobacillus and Bifidobacteria in in vitro assays. In addition to supporting growth of Lactobacillus and Bifidobacteria, maltulose, turnarose, panose and kojibiose appear to be linked with the suppression of Clostridia. High concentrations of turnarose are also associated with declines in Clostridia populations.

[0165] While it has previously been theorised that nigerose can inhibit enzymatic processes in Clostridia, the inhibition of Clostridia growth by erlose, melibiose, α,β-trehalose and maltulose has not previously been published. The oligosaccharide profile of these various honeys indicates that the relationship between K8 oligosaccharides is highly conserved regardless of the actual concentration of the total oligosaccharides (or the concentration of these combined oligosaccharides), and that maintaining this relationship is key to optimising prebiotic effect. The optimal ratio is found most frequently in blended honey. When the optimal relative ratio of the 8 key oligosaccharides is maintained, prebiotic effects are observed in vivo, regardless of overall concentration. Table 14 - Relative ratio of K8 oligosaccharides for achieving optimal prebiotic effect

Example 16: Comparing commercial doses of inulin to Honey using microcosms

[0166] The Prebiotic Activity testing of the commercial prebiotic inulin against raw honeys and the Four Honeys by microcosm demonstrated that some honeys (H1 , raw honey CZZ 9, raw honey CZZ 10) out-performed the high microcosm inoculation of inulin, which was based on a daily human dose regime (Figure 31 ).

Example 17: Clinical Study

[0167] Following the In vitro assessment of the Four Honeys (H1-H4) each honey was tested in a clinical study for their prebiotic potential by quantifying the major bacterial groups and the short chain fatty acids (SCFAs) in stool samples after daily consumption of 20 g honey for four weeks. The major bacterial groups were evaluated in the microcosms (above) and the results compared to those from the clinical study.

[0168] The oligosaccharides (di-, tri-, tetra- and penta-saccharides) in the tested honeys were determined and correlated with the changes in the bacterial populations in the clinical study samples and in the microcosms. To further assist in the identification of

oligosaccharides that were utilised by the lactobacilli and bifidobacteria, a lactobacillus strain and a bifidobacterium strain, both of intestinal origin, were grown in honey supplemented growth medium and the saccharide content determined before and after growth. Several di- and tri-saccharides that correlated with creating a desirable bacterial profile in the microcosms were examined in more detail. The individual saccharides were added to in vitro microcosms with and without added honey to ascertain the impact of the individual oligosaccharides on the bacterial populations. In addition, the growth of various intestinal lactobacilli, bifidobacteria and Clostridia pure cultures was studied in a basal medium supplemented with either the individual saccharides alone or with added honey.

[0169] The study investigated the prebiotic potential of H1 -H4 by profiling the intestinal microbes in volunteers consuming one of the honeys daily for 4 weeks. The rational for honey selection was based on the assessment of the Prebiotic Index and butyrate production in vitro.

[0170] H1 1 was sampled from the bulk containers in which they were delivered by beekeepers, and were not further processed before testingH2-H4 were warmed below 45°C for eight to ten hours and then filtered through a 100 micron filter. Honeys were aliquoted (20g) into single serve containers so that each subject was to consume the contents of one serve per day.

[0171] The study was conducted according to the guidelines laid down in the Declaration of Helsinki and approval was obtained from the University of New South Wales (UNSW) Human Research Ethics Committee. A double blind cross-over design with washout was used. Subjects that were included in the study were aged 20-50 years of age and free of chronic diseases of the digestive tract or the cardiovascular system, and were not diabetic, obese, pregnant or allergic to honey. The recruitment officer recorded details about the volunteer, namely, age, sex, dietary preferences, and honey and probiotic consumption prior to commencement. Subjects were randomised into groups.

[0172] The study design is included in the flow chart in Figure 40. In order to reduce subject variability, subjects (n=40) were divided into 2 groups (groups 1 and 2) and each group of 20 subjects tested 2 different honeys. The study took 4 months to complete. Group 1 tested honeys 1 and 3. Group 2 tested honeys 2 and 4. There were four separate phases each one month long. Honey was excluded from the diet during phases 1 and 3. Stool samples (S1 , 2, 3 and 4) were collected at time points 0, 1 , 2, 3, 4, and 5 months. Of the 40 subjects, 16 were male, 4 vegetarian, and 20 ate honey regularly and these were evenly distributed across the two groups of 20. The recruiting officer assigned subject numbers to all volunteers and all other study documentation only referred to the subjects by their assigned number. The study was conducted as a double blind study with neither the subjects nor those analysing the samples and results having knowledge of which honey was being consumed. A wash out control one month period was used rather than a placebo control utilising a glucose + fructose mixture in order to see the effect of whole honey rather than the oligosaccharides of honey, as would be the case if one used the placebo control, since the primary outcome was to determine if honey ingestion altered the microbial profile. [0173] A total sample size of twenty subjects per two honeys in a cross-over design enabled effect sizes greater than 0.7 to be detected as statistically significant (two-tailed a 1/4 0-05) with 80% power.

[0174] The study was divided into four phases each of which was four weeks in duration:

Phase 1. Honey excluded from the diet

Phase 2: Each day consumption of 20g of honey (either honey 2 or 3) Phase 3: Honey excluded from the diet

Phase 4: Each day consumption of 20g of honey (either honey 1 or 4).

[0175] The phases 1 and 3 served as a wash out period to remove the effects of previously ingested honey. Participants were asked to try to maintain a stable dietary regime during the study and should major deviations occur, to record the event. Antibiotic usage was to be avoided, but if necessary was to be recorded. Compliance was monitored at the end of each phase and major deviations from protocol resulted in subjects being discontinued. Participants were their own controls and group allocations ensured uniform distribution of age, male/females and diet.

[0176] Freshly void faecal samples were collected at the beginning of phase 1 and at the end of each phase (5 in total) and delivered to a 5°C cold room prior to being transferred to -80°C for storage prior to analysis. While it is recognised that the freezing could have yielded slightly lower numbers of the more sensitive bacteria such as bifidobacteria, results were calculated as the change from before to after honey consumption rather than absolute numbers, to ensure possible losses due to the freezing did not impact on the conclusions draw. The faecal samples were analysed for microbial diversity by quantifying the colony forming units (cfu) of the major bacterial groups, namely the total anaerobes (Wilkin Chalgren anaerobic agar), bacteroides (Kanamycin-Vancomycin agar), lactobacilli (Rogosa agar), bifidobacteria (Aniline blue dicloxacilin agar), enterics (MacConkey No 3 agar), enterococci (M17 agar at pH 9.6) and Clostridia (Columbia horse blood agar plus heat treatment of faecal suspension) using the respective selecting growth media as previously described (Conway et al. 2010). Results were expressed as the mean cfu per gram wet weight +SD for triplicate analyses. In addition, the diversity was examined by molecular profiling using Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis for samples from two of the honeys tested. Briefly, DNA was extracted from faecal suspensions using the Isolate Fecal DNA Kit from Bioline. The extracted DNA was labelled using standard PCR conditions. The PCR product was purified using DNA Clean and

Concentrator kits (Zymo Research). Purified PCR products were digested with two restriction enzymes, Mspl and/or Rsal (New England Biolabs) as per manufacturer's instructions and the resultant DNA was purified using Zymo DNA Clean and

ConcentratorTM -5 kits. Sample analysis was conducted by the Ramaciotti Centre for Gene Function Analysis (University of New South Wales).

[0177] SCFA levels in the faecal suspensions were determined by gas chromatography using an HP-FFAP column (J&W 50m, 0.2 mm, 0.332 pm; Agilent Technologies). Analyses were performed on triplicate samples with each analysed in triplicate.

[0178] The effects of the honeys were calculated as the change from the beginning to the end of the 4 week honey consumption (change between phases 2 and 3 as well as the change between phases 4 and 5). The slope of the change from before and after honey consumption was plotted for each subject and the mean slope calculated using the linear mixed effects model with subjects as the random factor. This model takes into account that the same subject was used before and after honey consumption. An ANOVA analysis of the slope data was used to show the statistical difference between the before and after honey consumption results.

[0179] The major bacterial groups in the stool samples were quantified by culturing on selective media and results expressed as the mean + standard deviation of triplicate samples, each analysed in triplicate and presented as colony forming units per gram (wet weight).

[0180] The abundance of the major bacterial groups was plotted against time for each subject and this illustrates the change in the various bacterial groups thorough-out the experiment. Results were shown to change over the 5 sampling times of the study and these are presented in Figure 41. During both wash out periods, namely between time point 1 and 2 as well as between 3 and 4, subjects were not to consume probiotic or prebiotic products or any honey. It is interesting to note that during this time there is a decrease in lactobacilli and bifidobacteria and an increase in Clostridia.

[0181] During time points 2 and 3, subjects consumed honey 2 or 3. By combining all subjects for honey A means that subjects consuming floral honey 2 were combined with those consuming floral honey 3. Similarly between time point 4 and 5, subjects consuming floral honey 1 were combined with those consuming floral honey 4. There is a clear trend that during these time spans, the lactobacilli and bifidobacteria increased and the Clostridia decreased. These changes corresponded with honey consumption.

[0182] The consumption of 20g of honey per day:

• increased lactobacilli from 2.2 x 104 cfu/g to 5.75 x 105 cfu/g;

• increased bifidobacteria from 5.37 x 106 cfu/g to 5.25 x 107 cfu/g;

• decreased Clostridia from 2.88 x 106 cfu/g to 9.12 x 105 cfu/g. [0183] The data for all subjects and honey types were combined and means of replicates converted to log 10 for averaging of all subjects. All statistical analyses were carried out using log data and above real numbers were generated by conversion from log values.

[0184] The change in abundance of all bacterial groups during honey consumption was examined by determining, for each subject, the slope of the line during the period of honey consumption and then taking the mean of all subjects. Initially all honeys were grouped (Figure 42) and in the figure the grey lines represent each subject while the heavy black line is the mean of the slope for all subjects. An ANOVA analysis of this slope confirmed that the increases in the lactobacilli and bifidobacteria were significant with P< 0.0001 . The decrease in Clostridia was also significant (P =0.0012). An increase (P=0.003) was noted for the total anaerobes which would reflect the increase in the lactobacilli and bifidobacteria. No significant changes in the bacteroides, enterics or enterococci were noted. The statistical analysis used the linear mixed effects model to take into consideration that the same subject was used for the data at the before and after time point.

[0185] The data for individual honeys were plotted to present the abundance of the lactobacilli, bifidobacteria and Clostridia before and after honey consumption and the means and confidence intervals are presented in Figure 43. In the Figure, the means are connected with a line for clarity (black line) with the data for individual subjects included as grey lines. An ANOVA analysis of deviance (Type II tests) for each major bacterial group, namely lactobacilli, bifidobacteria and Clostridia also revealed a significant difference between the before and after honey consumption time points for each of the four honeys, but no significant difference between H1-H4. For each honey, significant increases in the lactobacilli and bifidobacteria, (P<0.0001 ) were noted and a significant decrease in the Clostridia (P=0.001 ) was noted. There were no significant differences between the different honeys for each of the bacterial groups.

[0186] The TRFLP analyses were carried out for samples from subjects before and after consumption of honeys H2 (10 subjects) and H3 (8 subjects). There was a natural variability in the abundance associated with each subject in the before honey samples (phase 2 and 4) and hence also in the changes in specific genera and species after honey consumption. For H2, an increase in lactobacilli and bifidobacteria was observed across all subjects which is consistent with the culture studies, and a decrease in Clostridia was noted for most subjects. For H3, the increase in lactobacilli and bifidobacteria was again demonstrable while Clostridia were decreased in two thirds of the subjects. As expected, there were changes in bacteria which could not be classified. For the lactobacilli, L casei, L fermentum and L acidophilus were seen to increase with consumption of honey while for the bifidobacteria, bands consistent with B breve were noted after honey consumption. The results support the findings of the culture studies and future work on the samples will reveal further information about the particular genera which were not cultured in this study.

[0187] The levels of acetic, propionic and butyric acid in the stool samples were quantified by gas chromatography and no pattern of elevation or decrease across the various subjects during consumption of the individual honeys was noted for any of these acids (data not included). While generally there were slight increases in the levels of butyrate after ingestion of the various honeys, there was some variability between subjects. For example, in one subject (S30) a four-fold decrease in butyrate was noted during consumption of H4 while in two others (S25 and 46), a five and an eight fold increase was noted for S25 and 46, respectively, for the same honey. For most subjects and all honeys, there was no significant increase in butyrate concentrations with honey ingestion. However, it is important to flag that in two subjects (S23 and 24) a large increase in butyrate was noted after ingestion of H1 , namely 20 and 100 fold increase, respectively.

Example 18: Comparison of in vivo and in vitro assessment of clinical study honeys

[0188] In order to investigate the mechanisms associated with the enhancement of the lactobacillus and bifidobacterium populations in the clinical study, a single stage in vitro microcosm assay utilising human faecal inoculum was utilised. Initially, the four honeys tested in the clinical study were tested in the in vitro microcosm, and the results of both digested and whole honey tested in the in vitro microcosm were compared to the results obtained from the clinical study. As can be seen in Figure 44 (which includes Figure 1), a consistent profile for the three bacterial groups relative to each other was obtained for the four honeys when tested in vivo and in vitro for both digested and whole honeys.

Interestingly, the digested honey enriched microcosms had slightly lower levels of lactobacilli relative to the bifidobacteria than in those supplemented with whole honey.

Example 19: In vitro assessment of various honeys

[0189] Figure 45 illustrates the conformity of bacterial growth profiles between the four clinical study honeys and four Capilano Beeotic® proprietary processed honeys. In all cases, the growth of Lactobacillus and Bifidobacterium was higher in the honey inoculated microcosms than the negative control. Concurrently, the growth of Clostridium was notably higher in the negative control when compared to the honeys. The honeys delivered comparable outcomes to that of the commercial prebiotic inulin, which was added at a concentration of up to 5mg/ml.

[0190] In the replication of this experiment, whereby more Capilano Beeotic® honeys were included, the outcomes with regard to the growth profiles of Lactobacillus, Bifidobacterium and Clostridium were replicated. The results presented in Figure 46 illustrate some variability in the prebiotic properties of different honeys, but the effect appears to be relatively conserved. In many instances, the honeys performed better than inulin positive controls at 0.15mg/ml to 10mg/ml.

Example 20: Oligosaccharide profile analysis

[0191] The dominant di- and tri-saccharides detected in the clinical study honeys are presented in Figure 47. The tetra- and penta-saccharides and total saccharides are presented in Figure 48. It can be seen that H1 was the honey with the highest amount of total carbohydrates, followed by H4. Noticeable differences among the samples were observed for the composition of individual carbohydrates. The highest content of glucosyl- fructose disaccharides (such as maltulose and trehalulose) and isomaltose was detected in H1. Sucrose was found to be extremely high in H2 whereas H4 was characterized by the highest content of erlose. No penta-saccharides were detected in H2 and H3. Tetra- saccharides were highest in H1 and H4.

[0192] Despite the clinical efficacy of all of the honeys from the clinical study, considerable variability in the saccharide content of these honeys was noted.

[0193] The oligosaccharide content for the four clinical study honeys were analysed over a 12 month period, with the results shown in Table 15. Table 16 has the oligosaccharide content (mg/g) in various Capilano Beeotic® processed proprietary honey blends and Table 7 (above) contains the oligosaccharide content (mg/g) of raw unprocessed Australian honeys.

Table 15. Saccharide content (mg/g) for honeys used in the clinical study. The analysis was performed at t=0 and again 12 months later (t=1212).

Time

0 12 0 12 0 12 0 12 (months)

Honey

SAMPLE Honey 1 Honey 1 Honey 2 Honey 2 Honey 3 Honey 3 Honey 4

4

Sucrose 0.20 0.08 31.75 29.05 0.20 0.10 0.60 0.20 α,α-

0.10 0.35 0.10 0.15 0.10 0.18 0.09 0.29 Trehalose

α,β- 3.25 5.09 0.75 4.11 3.20 6.25 2.95 5.99 Trehalose

Inulobiose 0.95 0.50 2.89 0.78 1.75 0.74 1.31 0.58

Cellobiose 0.15 0.12 0.12 0.08 0.12 0.09 0.10 0.06

Laminaribios 0.00 0.00 2.06 1.12 0.69 0.55 0.70 0.38

Maltulose 43.64 44.05 2.53 2.53 16.94 17.90 16.00 20.13

Nigerose 14.39 10.85 3.98 2.36 9.49 9.48 9.99 8.62

Turanose 28.10 24.72 5.48 10.03 24.05 29.69 26.65 28.70

Maltose 11.43 17.69 14.05 10.70 7.11 5.86 10.67 12.94

Kojibiose 18.95 14.55 2.60 2.30 16.20 15.25 13.80 15.55 Trehalulose 36.33 35.62 1.74 2.14 8.85 7.08 11.55 15.18

Palatinose 7.50 8.25 0.20 0.10 1.05 1.30 2.35 3.00

Gentiobiose 0.15 0.50 0.40 0.50 0.07 0.30 0.10 0.20

Isomaltose 34.35 36.95 0.50 0.25 7.25 5.85 7.85 10.05

Melibiose 0.20 0.00 0.00 0.00 0.10 0.00 0.10 0.00

Unknown DS 2.10 2.17 6.13 5.00 2.69 3.29 2.41 10.07

Raffinose 0.15 0.19 0.08 0.12 0.03 0.04 0.03 0.05

1-Kestose 0.58 0.68 0.71 0.45 0.27 0.30 1.15 1.31

Erlose 0.19 0.13 0.92 0.69 0.51 0.49 3.45 2.76

Melezitose 0.20 0.18 0.10 0.07 0.15 0.14 0.36 0.33

Theanderose 0.16 0.12 0.04 0.02 0.08 0.06 0.19 0.17

Maltotriose 0.35 0.31 0.15 0.07 0.21 0.16 0.48 0.35

Panose 1.05 1.33 0.05 0.00 0.21 0.34 0.37 0.60

Unknown TS 2.76 3.71 0.17 0.03 0.49 0.48 0.81 0.89

TOTAL 207.18 208.11 77.46 72.63 101.78 105.92 114.01 138.37

TOTAL DS 201.75 201.47 75.26 71.18 99.83 103.90 107.18 131.92

TOTAL TS 5.43 6.64 2.20 1.45 1.95 2.02 6.83 6.46

Table 16. Saccharide content (mg/g) in various Capilano Beeotic® processed honey blends.

BLEND CZ1

CZ121 CZ124 CZ125 CZ126 CZ131 CZ132 CZ133 CZ135 CZ137 SAMPLE 38

14.6

Sucrose 6.00 0.31 1.65 5.10 8.00 5.50 1.90 0.50 0.23

0 a,a-Trehalose 0.20 0.23 0.19 0.19 0.15 0.12 0.13 0.12 0.15 0 13 a^-Trehalose 4.01 6.48 5.43 5.28 4.69 3.92 3.82 4.54 5.57 3 83

Inulobiose 0.70 0.95 0.80 0.90 0.90 0.77 0.76 0.75 0.73 0 93

Cellobiose 0.05 0.08 0.07 0.06 0.07 0.05 0.06 0.09 0.06 0 06

Laminaribiose 0.06 0.00 0.43 0.36 0.25 0.11 0.00 0.00 0.21 0 18

Maltulose 7.96 16.86 10.70 10.87 7.12 9.09 9.17 9.79 12.29 7 43

Nigerose 5.60 9.75 5.25 8.50 6.60 7.74 7.07 4.81 10.73 4 97

1 7.3

Turanose 15.80 25.74 20.22 27.04 13.34 19.32 16.81 20.93 22.85

5

24.6

Maltose 20.43 20.47 12.97 22.08 26.01 25.57 23.29 18.56 14.02

6

Kojibiose 7.95 17.35 8.70 9.85 8.50 8.35 8.80 12.10 14.75 6 95

Trehalulose 3.59 8.36 7.21 5.60 2.83 3.86 3.54 5.55 6.48 4 22

Palatinose 0.70 1.45 1.20 1.05 0.60 0.85 0.70 0.90 1.15 0 85

Gentiobiose 0.20 0.25 0.35 0.20 0.25 0.10 0.15 0.20 0.10 0 10

Isomaltose 2.75 8.70 3.30 3.25 2.65 2.15 2.75 4.45 5.15 1 80

Melibiose 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.00 0 00

Unknown DS 2.33 3.45 5.02 4.12 3.44 2.12 1.89 1.33 2.36 1 99

Raffinose 0.10 0.03 0.04 0.05 0.05 0.05 0.04 0.59 0.02 0 06

1 -Kestose 0.94 1.09 0.46 0.98 0.96 1.13 0.85 2.45 1.10 0 90

Erlose 3.13 1.17 1.39 4.72 4.86 3.02 3.07 8.18 1.00 3 08 Melezitose 0.56 0.13 0.31 0.30 0.22 0.20 0.12 0.22 0.13 0.55

Theanderose 0.07 0.07 0.05 0.08 0.09 0.05 0.07 0.10 0.07 0.07

Maltotriose 0.27 0.22 0.23 0.38 0.29 0.26 0.24 0.31 0.26 0.21

Panose 0.30 0.57 0.31 0.45 0.29 0.25 0.23 0.40 0.63 0.18

Unknown TS 0.36 0.66 0.32 0.47 0.30 0.29 0.29 0.32 0.66 0.23

95.3

TOTAL 84.03 124.33 86.58 111.85 92.44 94.86 85.74 97.36 100.70

0

90.0

TOTAL DS 78.31 120.41 83.47 104.43 85.38 89.61 80.83 84.80 96.82

3

TOTAL TS 5.72 3.93 3.11 7.43 7.06 5.25 4.91 12.56 3.88 5.27

[0194] There was no clear correlation between the relative concentrations of

oligosaccharides across the various honeys, whether they are from a single floral source, raw honeys or blended processed honeys, apart from the complete absence of leucrose.

[0195] For the raw honeys ten saccharides were detected at larger concentrations and these were eriose, nigerose, trehalulose, maltulose, kojibiose, sucrose, turanose and maltose. Most of the variation in honeys seemed to be occurring in about 10 saccharides, with those showing the most variation across all honeys being eriose, nigerose, trehalulose, maltulose, kojibiose, sucrose, turanose, maltose, α,β-trehalose and isomaltose. The amounts of the tetra-saccharides (DP 4) were low for all honeys with 0 - 2 mg/g of honey, except for honey 4 which had 8 mg/g. Only three honeys had detectable pentasaccharides, namely 2, 14 and 15 with honey 2 having considerably more pentasaccharides.

[0196] The disaccharide and trisaccharide content of all of the honeys that were analysed are illustrated in Figure 49. H2 had the lowest concentration of total saccharides at 77.46 and 72.63 mg/g for duplicate analyses. All of the Capilano Beeotic® processed proprietary honey blends had total saccharide contents above this. Interestingly, these processed blends were more uniform when compared to the variation in clinical study honeys and the 24 raw unprocessed honeys (Figure 49).

[0197] The saccharides identified in Australian honeys were examined to determine the susceptibility of each saccharide to digestion by the host digestive enzymes and by the bacteria of the genera Lactobacillus, Bifidobacterium and Clostridium. The data are presented in Table 17 Table 17. Susceptibility of saccharides in honey to enzymatic digestion by humans, Clostridia, lactobacilli and bifidobacteria and the likely prebiotic potential based on that susceptibility.

[0198] The individual saccharide concentrations (mg/g) are tabulated together with the calculated Prebiotic Activity for each of the four honeys tested in the clinical study (Table 18). The Prebiotic Activity was calculated from data generated using the in vitro by microcosm as the sum of the numbers of Lactobacillus and Bifidobacterium and Clostridium in the test microcosm relative to the negative control. In addition, the table contains the R ² value or coefficient of determination which is a statistical measure of how close the data is to the fitted linear regression line. This analysis was used to assess the influence of each saccharide on the calculated prebiotic activity. An R ² of 1 indicates that the regression line perfectly fits the data. The R ² values were then used together with data presented in Table 4 to identify the honey saccharides most likely to be contributing to the prebiotic activity of the honey (Table 19).

Table 18. Concentration of saccharides (mg/g) for the clinical study honeys in relation to the Prebiotic Activity of the honey. The Prebiotic Activity of each honey was assessed using data from the in vitro microcosm (see above). Key Eight - refers to the sum of maltulose, nigerose, turanose, kojibiose, trehalulose, palatinose, isomaltose, panose.

ANALYSIS

R ² TIME 0 12 0 12 0 12 0 12

Value (MTHS)

Honey Honey Honey Honey Honey Honey Honey Honey

SAMPLE

1 1 2 2 3 3 4 4

Prebiotic

9.20 9.20 7.50 6.40 8.10 8.10 8.60 7.10 1.00 Activity

Sucrose 0.20 0.08 31.75 29.05 0.20 0.10 0.60 0.20 0.43 α,α-

0.10 0.35 0.10 0.15 0.10 0.18 0.09 0.29 0.00 Trehalose

α,β-

3.25 5.09 0.75 4.11 3.20 6.25 2.95 5.99 0.00 Trehalose

Inulobiose 0.95 0.50 2.89 0.78 1.75 0.74 1.31 0.58 0.02 Cellobiose 0.15 0.12 0.12 0.08 0.12 0.09 0.10 0.06 0.53

Laminaribios 0.00 0.00 2.06 1.12 0.69 0.55 0.70 0.38 0.35

Maltulose 43.64 44.05 2.53 2.53 16.94 17.90 16.00 20.13 0.68

Nigerose 14.39 10.85 3.98 2.36 9.49 9.48 9.99 8.62 0.75

Turanose 28. 0 24.72 5.48 10.03 24.05 29.69 26.65 28.70 0.31

Maltose 11.43 17.69 14.05 10.70 7.1 1 5.86 10.67 12.94 0.03

Kojibiose 18.95 14.55 2.60 2.30 16.20 15.25 13.80 15.55 0.47

Trehalulose 36.33 35.62 1.74 2.14 8.85 7.08 11.55 15.18 0.60

Palatinose 7.50 8.25 0.20 0.10 1.05 1.30 2.35 3.00 0.59

Gentiobiose 0.15 0.50 0.40 0.50 0.07 0.30 0.10 0.20 0.09

Isomaltose 34.35 36.95 0.50 0.25 7.25 5.85 7.85 10.05 0.62

Melibiose 0.20 0.00 0.00 0.00 0.10 0.00 0.10 0.00 0.33

Unknown DS 2.10 2.17 6.13 5.00 2.69 3.29 2.41 10.07 0.52

Raffinose 0.15 0.19 0.08 0.12 0.03 0.04 0.03 0.05 0.11

1-Kestose 0.58 0.68 0.71 0.45 0.27 0.30 1.15 1.31 0.00

Erlose 0.19 0.13 0.92 0.69 0.51 0.49 3.45 2.76 0.04

Melezitose 0.20 0.18 0.10 0.07 0.15 0.14 0.36 0.33 0.08

Theanderose 0.16 0.12 0.04 0.02 0.08 0.06 0.19 0.17 0.27

Maltotriose 0.35 0.31 0.15 0.07 0.21 0.16 0.48 0.35 0.36

Panose 1.05 1.33 0.05 0.00 0.21 0.34 0.37 0.60 0.56

Unknown TS 2.76 3.71 0.17 0.03 0.49 0.48 0.81 0.89 0.60

TOTAL 207.18 208.11 77.46 72.63 101.78 105.92 114.01 138.37 0.59

TOTAL DS 201.75 201.47 75.26 71.18 99.83 103.90 107.18 131.92 0.59

TOTAL TS 5.43 6.64 2.20 1.45 1.95 2.02 6.83 6.46 0.28

KEY EIGHT ¹

OLIGOSAC 184.28 176.30 17.06 19.70 84.02 86.89 88.54 101.81 0.68 CHARIDES

Table 19 - Data from tables 17 and 18 to identify honey saccharides likely to contribute to prebiotic activity.

[0199] From the collective data in Tables 17,18 and 19 the following eight saccharides were identified as the key contributors to the prebiotic activity in honey: maltulose, nigerose, turanose, isomaltose, kojibiose, trehalulose, palatinose and panose.

[0200] Turanose was the most abundant of the eight key saccharides in all honeys with one exception, and that one honey had more maltulose than turanose. The concentration of turanose was proportional to the other saccharides, such as nigerose, maltulose and kojibiose.

[0201] Using the duplicate chemical analyses of the honeys (at 0 months and at 12 months) used in the clinical study, and the values for the corresponding duplicate prebiotic activity assessment from the in vitro microcosms, the relationship between the increasing prebiotic activity of the honey and the summed concentration of the identified eight key saccharides was plotted (Figure 50). The summed concentration of the eight selected saccharide content and the prebiotic activity, expressed as the R ² value, was relatively consistent across the two samples with R ² =0.92 and R ² =0.82 for the 0 month and 12 month analyses, respectively.

Example 21 : Correlation between the eight key saccharides and overall saccharide content in the honeys

[0202] Eight key saccharides selected because of the observed correlation between concentration and the prebiotic activity of the clinical study honeys (Figure 51 ), were further examined in the Capilano Beeotic® processed as well as raw unprocessed honeys ( total honeys in analysed=42). The coefficient of determination as a statistical measure of how close the data fitted the linear regression, or R ² value, was calculated for the sum of the amounts of the eight key saccharides (maltulose, nigerose, turanose, isomaltose, kojibiose, trehalulose, palatinose and panose) and di- and tri-saccharides in the 42 tested honeys. Results are presented in Table 20.

Table 20. The coefficient of determination (R ² value) of the individual saccharide contents of the 42 honeys analysed from this study with the sum of the identified eight key oligosaccharides (maltulose, nigerose, turanose, isomaltose, kojibiose, trehalulose, palatinose and panose) and the sum of total disaccharides (DS) and trisaccharides (TS).

Saccharide OLIGOSACCHARID TOTAL DS TOTAL TS Total DS & KEY Type E TS EIGHT

R ² value R ² value

R ² value R ² value

Summed TOTAL KEY EIGHT 1 .000

Summed TOTAL DS 1.000 0.804

Summed TOTAL TS 0.068 1.000 0.014

Summed TOTAL DS & TS 0.991 0.123 1.000 0.777

Disaccharide Sucrose 0.019 0.075 0.026 0.077

Disaccharide α,α-Trehalose 0.31 1 0.003 0.287 0.344

Disaccharide α,β-Trehalose 0.270 0.000 0.253 0.309

Disaccharide Inulobiose 0.024 0.108 0.033 0.027

Disaccharide Cellobiose 0.159 0.075 0.171 0.074

Disaccharide Laminaribiose 0.034 0.154 0.047 0.048

Disaccharide Maltulose 0.763 0.005 0.731 0.971

Disaccharide Nigerose 0.528 0.000 0.496 0.751

Disaccharide Turanose 0.552 0.100 0.565 0.520

Disaccharide Maltose 0.094 0.134 0.1 1 1 0.001

Disaccharide Kojibiose 0.475 0.001 0.451 0.726

Disaccharide Trehalulose 0.733 0.008 0.704 0.902

Disaccharide Palatinose 0.702 0.01 1 0.678 0.867

Disaccharide Gentiobiose 0.117 0.016 0.102 0.097

Disaccharide Isomaltose 0.646 0.002 0.616 0.859

Disaccharide Melibiose 0.025 0.1 12 0.035 0.105

Disaccharide Unknown DS 0.081 0.027 0.068 0.066

Trisaccharide Raffinose 0.000 0.355 0.006 0.007

Trisaccharide 1-Kestose 0.019 0.443 0.039 0.005

Trisaccharide Erlose 0.000 0.881 0.008 0.034

Trisaccharide Melezitose 0.142 0.268 0.173 0.050

Trisaccharide Theanderose 0.418 0.470 0.481 0.337

Trisaccharide Maltotriose 0.370 0.200 0.401 0.379

Trisaccharide Panose 0.686 0.030 0.673 0.867

Trisaccharide Unknown TS 0.607 0.009 0.586 0.805 [0203] The notable correlations illustrated by Table 20 include:

• total disaccharides that represent the largest composition of the saccharides

detected in honey correlated with total disaccharides and trisaccharides detected in the honey (R ² =0.991 );

• the sum of the key eight disaccharides represent a major component of the

disaccharides detected, and hence their sum also correlated with total disaccharides detected (R ² =0.777);

• maltulose, which constituted a relatively large component of the detected

saccharides in the honeys (Table 1 ,2 & 3), correlated with the sum of the key eight disaccharides (R ² =0.971 ), total disaccharides (R ² =0.763) and total disaccharides and trisaccharides (R ² =0.731 );

• despite the fact that palatinose concentrations in honey are low (Table 1 ,2 & 3), its concentration correlated with the sum of the key eight disaccharides (R ² =0.867) and total disaccharides (R ² =0.702);

• panose, the only trisaccharide included as one of the key eight saccharides,

correlated with the key eight oligosaccharides (R ² -0.867) and total disaccharides (R2=0.686), even though it was detected in relative low concentrations in the honeys.

• erlose was the most abundant trisaccharide detected and was the best indicator of total trisaccharides, as can be expected (R ² =0.881 ).

[0204] Across the 42 honeys chemically analysed (Table 20), the summed concentration of the key eight saccharides correlated with total disaccharides (R ² =0.804) and maltulose was the single best saccharide indicator of total trisaccharides (R ² =0.731 ), total disaccharides (R ² =0.763) and for the sum of the key eight oligosaccharides (R ² =0.971 ), as shown in Figure 51.

Example 22: Saccharide components of the raw honeys and Capilano Beeotic® processed honey blends

[0205] The saccharide profiles of the Capilano Beeotic® processed proprietary honey blends and the unprocessed raw honeys as presented in Tables 15 and 16, respectively, were plotted to investigate the individual key saccharides (Figure 52). Some interesting characteristics in the processed honey blends were identified when compared to the raw honeys:

• the relative concentrations of the various saccharides were more normalised;

• the concentration of sucrose was lower, as was the standard deviation; • the concentration of a, -trehalose was notably higher;

• the standard deviation of the average concentration of maltulose, a notable

contributor to total disaccharides, was markedly lower;

• nigerose concentration was -50% greater, while the standard deviation was similar;

• the concentration of turanose was greater in the processed and blended honeys;

• the concentration of maltose appears to nearly double, while the standard deviation was similar;

• the concentration of kojibiose was -25% greater while the standard deviation was slightly less;

• the standard deviations of trehalulose, palatinose, isomaltose and erlose were lower for the processed honeys, indicating a standardisation of these constituents;

• for a number of saccharides (α,α-trehalose, α,β-trehalose, maltulose, nigerose, kojibiose) the average concentration was greater, and the standard deviation lower, indicating these constituents appear to be greater and become more uniform in concentration in Capilano Beeotic® processed proprietary honey blends.

Example 23: Utilisation of specific saccharides from clinical study honeys by beneficial bacteria

[0206] In order to further identify which of the saccharides in the honeys could be utilised by the beneficial bacteria, pure cultures of lactobacilli and bifidobacteria were inoculated into honey enriched growth media based on the four honeys tested in the clinical study. Each of the four clinical study honeys were individually tested in the honey enriched growth media with an intestinal strain of Lactobacillus fermentum (PB/C2) and Bifidobacterium breve (PB/C 12). After growth the saccharide contents of the honey enriched growth media were analysed and the change in the dominant saccharides quantified. Values of 100% utilisation were noted for several saccharides, but many were present in very low concentrations in honey, such as melibiose, raffinose, theanderose and inulobiose.

Interestingly, isomaltose and nigerose that are in amounts greater than 0.5 mg/g of honey were also extensively utilised. Most of the saccharides tested had about 40% utilisation, namely maltulose, turanose, kolibiose, trehalulose, while greater utilisation was noted for the palatinose, nigerose and isomaltose, with 55, 75 and 95%, respectively.

[0207] In order to study the utilisation of the saccharides in the individual honeys by the lactobacillus and bifidobacteria separately, a statistical heat map was used. The consumption patterns of the major saccharides appeared quite similar for the lactobacillus and bifidobacterium strains tested. While most saccharides were utilised to some extent, the concentrations of maltotriose and maltose increased in the honey enriched broths after growth of these beneficial bacteria. This increase can be attributed to the soluble starch which was added to the Basal Medium for these studies to ensure initial good growth of the lactobacilli and bifidobacteria. The amylose in the soluble starch would have been broken down by the bacterial amylases to the maltose and maltotriose, consequently the increase in these saccharides would not have been associated with a conversion from honey saccharides.

Example 24: Impact of pure saccharides of interest on in vitro bacterial growth both in the microcosm and in pure culture

[0208] The individual saccharides identified in the honeys were sourced as pure chemicals and each tested separately for their capacity to support the growth of lactobacilli, bifidobacteria and Clostridia, both in the mixed culture of the in vitro microcosms and in pure culture using laboratory defined media.

[0209] The microcosms were established using stool samples and the saccharides were added at the concentrations found in honey both alone or together with honey. Inulin was used as the positive control and tested at a range of concentrations corresponding to the levels of the saccharides in honey and also the level corresponding to the use of inulin as a prebiotic, namely 100mg/ml. Negative controls contained buffer or honey alone. The inulin was less effective at promoting the lactobacilli with less than a log increase in lactobacilli and almost a 3 log increase in bifidobacteria at the concentrations consistent with those in honey (ie. 2 -14 mg per g honey)(Figure 53). In addition, the inulin was also less effective at promoting the lactobacilli than the honey alone as seen in the honey negative control.

[0210] The effects of the eight key saccharides described above on the bacterial populations in the microcosms were tested at two concentrations, one which corresponds to the average concentration of that particular sugar found in honey and 7.5mg as that was the average concentration of all the saccharides in the honeys. The mg added corresponded to, for example, 7.5 mg per g of honey and the honey was used at a final concentration of 1 % in the microcosm. The data for panose, maltulose, trehalulose and kojibiose are included in Figure 53 and these saccharides were only tested at 7.5 mg per g honey.

[0211] The addition of palatinose and isomaltose increased the lactobacilli in the microcosms when added alone, an effect that was enhanced when added with honey. The minor elevation of lactobacilli with added nigerose was improved when added with honey. The bifidobacterium levels were increased in the microcosms with added palatinose, isomaltose, nigerose, turanose, maltulose, trehalose, kojibiose, however, this effect was not further enhanced when combined with honey as noted for the lactobacilli. The levels of Clostridia in the microcosms decreased when isomaltose, nigerose, panose and trehalulose was added. Turanose also reduced Clostridia when used at the concentration found in honey, but not at the lower concentration tested. All eight key saccharides exerted desirable effects on the microbial populations in the microcosms, and interestingly the effects on the lactobacilli were enhanced when added with honey.

[0212] The addition of the pure saccharides to defined culture medium with no added carbohydrates was also tested for a number of pure cultures. It was shown that most of the tested saccharides could be utilised by the lactobacilli. All tested saccharides were utilised by Lactobacillus fermentum strain PB/C 2 with the best growth obtained for palatinose, nigerose and isomaltose, the three which also favoured the growth of lactobacilli in in vitro microcosm experiments.

Discussion

[0213] Like humans, bees have developed a symbiotic relationship with their gut biota that includes Bifidobacterium and Lactobacillus species. These bacteria play a key role in honey production and bee food storage preservation. Their presence and enzymatic action, in conjunction with enzymes added by the bee, is responsible for the introduction of prebiotic oligosaccharides to honey, which then selectively stimulate beneficial bacteria of the human colon following ingestion. The present inventors have found that the di- saccharide and tri-saccharide content of Australian honeys vary considerably.

[0214] The Four Honeys tested promoted the growth of the beneficial Lactobacillus and Bifidobacterium and suppressed the less desirable Clostridium (n= 20 per honey). It was concluded that each of the Four Honeys tested had demonstrable prebiotic activity as shown by the changes in the lactobacilli, bifidobacteria and Clostridia, which were significantly altered (P<0.05).

[0215] The Four Honeys and Capilano Beeotic® proprietary processed honey blends tested using the microcosm presented favourable growth profiles of lactobacilli, bifidobacterium and Clostridia. When inulin was inoculated into the microcosm there was some effect of dose at levels 0.15mg/ml to 10mg/ml, but these were not of the same magnitude as the concentration increases, suggesting that the prebiotic effect is not dose dependent.

[0216] The Prebiotic Activity of each honey was established using a calculation of bacterial growth against a negative control. This measure enabled the ranking and comparison of honeys. Raw honeys had lower average Prebiotic Activity results and lower average oligosaccharide concentrations, which can be attributed to one or more of the following factors:

I. the presence of organic matter impedes the purification and chemical analysis of the honey;

II. the presence of organic matter contains increased antimicrobials reducing

microcosm growth profiles; III. the heating, blending and filtering of different raw honeys into a processed blend increases the variability in the profile of beneficial oligosaccharides that improve the growth response of beneficial bacteria;

IV. heating of honey during processing increases the presence of beneficial

oligosaccharides from chemical reactions or increased enzymatic activity before any degradation or bacterial mortality;

V. bacterial and enzymatic action increases the presence of beneficial oligosaccharides with time with the blending of different honeys permitting further availability of precursor substrates for enzymes that assemble beneficial oligosaccharides;

VI. bacterial and enzymatic action was degraded and eliminated by honey processing's heat application resulting in a stable, improved compositional profile of

oligosaccharides within a bacteriostatic environment.

[0217] From the imported honeys, only 5 of the 21 (23.8%) tested had a Prebiotic Activity above or equal to the lowest positive control, and there was no correlation with country of origin (Mexico, China, Argentina, Brazil). This reinforces the theory with respect to the prebiotic activity originating from the bee and its gut biota, thus being somewhat irrelevant of the floral origin or country of origin unless the flora delivers a nutritional boost to increase bee gut bacterial activity.

[0218] 13 of 20 (65%) blended processed Capilano honeys delivered a Prebiotic Activity in microcosm greater than that of the inulin positive control, with 4 (20%) of the honeys delivering a 20% increase on the Prebiotic Activity of the positive control (inulin). It was shown that Australian processed honeys have a heightened incidence of Prebiotic Activity than those of imported honeys.

[0219] The Four Honeys were subject to heating and filtration as per normal industry standards, however their oligosaccharide contents were reviewed relative to the raw honeys analysed. A correlation between the increasing prebiotic activity and oligosaccharide content was identified between trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose (Table 4). When these identified beneficial

oligosaccharides were pooled for each of the Four Honeys a strong linear (r ² =0.94) and exponential (r^O.88) relationship with increasing content of oligosaccharides and increasing Prebiotic Activity was demonstrated.

[0220] The Capilano Beeotic® proprietary processed honey blends have total

oligosaccharide contents above the lowest efficacious clinical study honey, which contained total saccharides of over 72.6 mg/g.

[0221] The Capilano Beeotic® proprietary processed honey blends have oligosaccharide profiles that were more normalised with notably lower variability in standard deviations. Of the raw honeys that were tested, the average oligosaccharide content was 74.7 mg/g with a standard deviation of 29.2 mg/g (Table 3); this compares with the processed honey blends which had a higher average content of 97.3 mg/g saccharides and a lower standard deviation of 12.5 mg/g (Table 2). It is hypothesised that the blending and heating of honeys results in an increased number of enzyme and substrate interactions occurring; resulting in a progression towards a new maximised equilibrium between these natural compositional components. Unblended honeys may have reached an exhaustion of the necessary substrates for particular enzymes, but with the blending of honey from geographically diverse apiaries, the substrates of each honey are utilised by the enzymes remaining in the other. Physical mixing and heating of the honeys may also accelerate these reactions to deliver a renewed elevated content of saccharides.

[0222] Pure cultures showed the key 8 beneficial oligosaccharides identified were utilised in pure culture growth of the desirable bacteria, with nigerose, panose and isomaltose readily consumed. Assessment of the enzymatic mode of action ensured that the identified 8 oligosaccharides have appropriate prebiotic potential and would be available in the lower intestine without human host degradation. Nigerose was shown to be an effective prebiotic.

[0223] It has been shown that when intestinal microbes are supplemented with the complex oligosaccharides found in Australian honeys, beneficial species can be selectively enhanced. The microbial profiles are consistent with those associated with health benefits (increased Bifidobacterium and Lactobacillus and decreased Clostridium levels). While many publications have shown that beneficial microbes in pure culture can utilise natural honey, this work has been extended to demonstrate an effect on a complex microbial population, as found in the human large intestine and our in vitro microcosm. In addition, 8 specific oligosaccharides were identified that increase the Prebiotic Activity of honey as their individual and summed content in honey increases.

Conclusions

• A Prebiotic Activity microcosm assay and measurement scale was developed to assess a honey's prebiotic potential. This assessment assay can be effectively used to screen honeys to produce a beneficial commercial product rated against a negative control sample added to each assay. The assessment against a negative control per assay is included to mitigate the intrinsic microbial variability.

• Raw honeys have been shown to have lower average Prebiotic Activity than processed mixed honey blends, meaning that the process in preparing Capilano honeys influences the resulting prebiotic activity. • A correlation between the Prebiotic Activity of a given honey and the individual and summed oligosaccharide content was identified between trehalose, nigerose, maltulose, isomaltose, panose, turanose, kojibiose and palatinose.

• The honeys were effective if they contained a minimum of 17.06 mg/g of the identified 8 oligosaccharides.

• Nigerose has been discovered as a prebiotic oligosaccharide.

• Honey with above 1 mg/g of nigerose will assist in inhibiting Clostridium's digestive processes, thus reducing the growth of this undesirable bacterium.

• The intrinsic composition of honey delivers a variable combination of

oligosaccharides that enhances the prebiotic effect of the product as it facilitates a range of enzymatic functions that enhance the growth of both Bifidobacterium and Lactobacillus beneficial species.

• Honey containing key beneficial oligosaccharides selectively supports the growth of beneficial intestinal microbes such as bifidobacteria and lactobacilli, whilst suppressing Clostridia, which is a genus containing known pathogens. It has been shown that some mono- and disaccharides in honey are likely to be absorbed rapidly in the upper gastrointestinal tract and the non-digestible oligosaccharides reach the lower tract to selectively influence the colonic microbes.

• The clinical in vivo response and accompanying in vitro studies illustrate beneficial gut microbiota outcomes with honey attributable to the oligosaccharides present in the honey. These honey oligosaccharides triggered a proliferation of beneficial bacteria, and a suppression of undesirable bacteria. Consequently, certain honey blends selectively support the growth of bifidobacteria and lactobacilli in the gastrointestinal tract and inhibit growth of undesirable Clostridia.