Compositions that reduce sugar bioavailability and/or have a

prebiotic effect

Field of the invention

The present invention relates to compositions that reduce sugar bioavailability, lower calorific effect and/or improve prebiotic function, processes for preparation of said compositions and methods of their use. For example, the present invention relates to methods of improving intestinal probiotics and/or reducing sugar bioavailability by consuming compositions of the invention.

Background of the invention

Eating a diet composed of energy dense and highly processed foods, as well as emulsifiers and artificial sweeteners, appears to compromise the barrier lining the gut. Artificial sweeteners may also change gut microflora and products formulated with these products may need to contain laxative warnings. There is rapidly expanding evidence that insufficient or poorly constituted gut microflora is a factor in some diseases. For example, diarrhoea (infectious, travellers’ or antibiotic-associated diarrhoea), dysbiosis and gut health associated conditions, osteoporosis, colon cancer, cardiovascular disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, Alzheimer’s disease and various mood disorders/mental illnesses have been linked to either damaged gastro-intestinal lining or poor quality gastro-intestinal microbiome. Studies are also increasingly linking gut health to mood and mental health.

Eating large quantities of sugar has a negative impact on the gastro-intestinal microbiome. Refined white sugar is almost completely sucrose. Following consumption sucrose passes through the stomach and into the small intestine where the enzyme sucrase hydrolyses the sucrose into glucose and fructose. The glucose and fructose then cross the lining of the small intestine. This process is relatively rapid such that the sugar has left the intestine near the beginning of the small intestine and does not travel through the small intestine and into the large intestine or the colon. This means that the sucrose in refined white sugar does not remain in the intestine long enough to provide a food source for the gastro-intestinal microbiome. It also means that essentially all of calories in white refined sugar are absorbed into the blood stream, posing a greater risk of diabetes. The gastro-intestinal microbiome is improved by the use of probiotic foods and supplements (ie that contain live beneficial bacteria). Increasing reports suggest that supplementing with Bifidobacterium and Lactobacillus bacterial strains for 1-2 months can improve anxiety, depression, autism, obsessive-compulsive disorder (OCD) and memory. The gastro-intestinal microbiome is also improved by consuming unrefined foods and purified sources of prebiotic fibres such as inulin, lactulose, fructo- oligosaccharide (FOS), b-ga!aclo-oligosaccharides (GOS) and Trans- galactooligosaccharides (TOS). When prebiotic fibres are consumed they pass along the small intestine undigested and are used by the beneficial bacteria in the gastro- intestinal microbiome as fuel when they reach the large intestine/colon. Unfortunately, the western diet often contains more refined foods and is deficient in prebiotic foods.

There is a need for alternative prebiotic foods and supplements. There is also a need for food and supplements that include sugars, wherein the sugars are less damaging to the microbiome. It would also be useful to develop versions of foods that traditionally have negative effects on the gastro-intestinal microbiome but instead benefit the gastro- intestinal microbiome.

There is also a need for reducing the amount of sugar absorbed/bioavailable and/or the speed of the absorption of sugar, which could minimise the risks of diabetes and obesity associated with high sugar diets and/or minimise the negative impact of such a diet on the gastro-intestinal microbiome.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention provides compositions that reduce the bioavailability of sugar.

The present invention also provides compositions that are prebiotic. Understanding of prebiotics remains incomplete. Food sources of prebiotics include chicory root,

Jerusalem artichoke, dandelion greens, garlic, leek, asparagus, onions, and bananas. Bagasse is used in industry and for animal feed. Bagasse has been identified as a new source of prebiotic food. While probiotics have been in use for some time, the

identification of prebiotic foods (that is foods that have a beneficial effect on the gastro- intestinal microbiome, in particular, the microbiome of the colon) is relatively recent. Relatively few prebiotics substances are known and the identification of a new prebiotic is significant.

In addition, the sugar compositions of the invention have been found to have reduced sugar bioavailability. This is of particular interest as the sugars are suitable for substitution for traditional white sugar, lowering the risk of diabetes upon consumption.

Further, when testing the bioavailability of the sugar compositions of the invention, the sugar compositions of the invention were found to have prebiotic effect. The negative impact of white sugar on the gastro-intestinal microbiome means it is very surprising that a sugar, which is still largely sucrose, can have a positive impact on the gastro- intestinal microbiome. This is of particular value where the sugar remains palatable and/or substitutable for traditional white sugar.

Prebiotic composition with two or more of hemicellulose, cellulose and lignin (eg bagasse)

In a first aspect, the present invention provides a method of increasing the quantity of probiotics in the colon of a mammal comprising consumption by the mammal of at least 3 g of a composition comprising two or more of hemicellulose, cellulose and lignin (such as sugar cane bagasse). Optionally, the probiotic bacteria increased is one or more of lactic acid bacteria, Bifidobacteria, Bacteroidetes, Baciullus, Streptococcus, Escherichia, Enterococcus, Anaeropstipes, Bacillus, Odoribacter, Victivallis (phylum Lentisphaerae) and unclassified Lentisphaerae, unclassified Marinilabiaceae and Anaerophaga (family Marinilabiaceae), Anaerophaga, Cerasicoccus, Roseburia, and/or Shigella species.

In an alternative first aspect, the present invention provides a method of increasing the quantity of probiotics in the colon of a mammal comprising consumption by the mammal of at least 3 g of a bagasse. Sugar cane, sugar beet and sorghum stalk bagasse is suitable. In preferred embodiments the bagasse is sugar cane bagasse.

Ammonia is a metabolite produced by the microbial fermentation of nitrogen containing molecules (such as protein, peptides, peptone and urea). Optionally, the increase in probiotic bacteria is measured by increase in ammonia. Optionally, the increase in ammonia in the colon following consumption of the composition is significant when compared to the ammonia following consumption of cellulose at 32, 56 or 80 hours. It is difficult to test the increase in bacteria and/or ammonia in the colon in vivo. Instead, testing is conducted using in vitro models such as the model used in Example 8. Optionally, the composition further includes ash.

In some embodiments of the first and alternate first aspect of the invention, the composition or bagasse is about 20-25% w/w hemicellulose, about 45-55% w/w cellulose and about 18-24% lignin. Optionally, the composition or bagasse is also about 1 -4% ash.

Optionally, at least 3.5 g or at least 7 g of the composition or bagasse is consumed. Optionally, the Rosebuira was increased over 3-fold (eg about 3.5-fold) 80 hours following consumption of 3.5 g of the composition. Optonally, the Roseburia was increased over 7-fold (eg about 7.5-fold) 80 hours following consumption of 7 g of the composition.

In some embodiments, the composition of the invention is sugar cane fibre or sugar beet fibre.

Optionally, the composition further comprises probiotics.

In some embodiments, the method is for the treatment of dysbiosis or diarrhoea

(infectious, travellers’ or antibiotic-associated diarrhoea). Alternatively, the method is for prevention of colon cancer.

In another alternative first aspect, the present invention provides a method comprising:

- selecting a mammal in need of treatment for dysbiosis or diarrhoea (infectious, travellers’ or antibiotic-associated diarrhoea), or weight management; and

- administering at least 3 g of bagasse or composition comprising two or more of hemicellulose, cellulose and lignin to that mammal;

wherein the method increases the quantity of probiotic bacteria in the colon of the patient.

Optionally, 7 g bagasse or composition is administered. Optionally, administration occurs at least once a day for 7 to 30 days. The bagasse or composition may be in the form of a capsule (eg similar to probiotic capsules) or in the form of a food.

In a second aspect, the present invention provides a method of weight management for a mammal comprising consuming at least 3 g per day of a composition comprising two or more of hemicellulose, cellulose and lignin (such as sugar cane bagasse). The composition used in the second aspect of the invention is optionally as described in the first aspect of the invention. Sugar compositions of the invention

Reduced compositions

In a third aspect, the present invention provides a sugar composition comprising sucrose and at least about 20 mg CE polyphenol / 100g carbohydrates (ie about 16 mg GAE polyphenol / 100 g carbohydrate) and about 0 to 0.3% w/w ash, wherein 90% or less of the sucrose is bioavailable to a mammal following consumption of the sugar. Optionally, 85% or less, or 80% or less, of the sugar is bioavailable. Optionally, the sugar contains about 0.05 to 0.3% w/w ash. In some embodiments, the sugar

composition has about 20 to 45 mg CE polyphenols / 100 g carbohydrates. In alternate embodiments, the sugar composition has about 46 to 100 mg CE polyphenols / 100 g carbohydrates. The sugar is optionally also low glycaemic or very low glycaemic. The sugar is optionally low to very low glycaemic.

Sucrose sugars with 20 to 45 mg CE polyphenols / 100 g carbohydrates (16 to 36 mg GAE polyphenol / 100 g carbohydrate) and 0 to 0.5 g/100 g reducing sugars are low glycaemic (see international patent application no. PCT/AU2017/050782). Sucrose sugars with 46 to 100 mg CE polyphenols / 100 g carbohydrates (37 to 80 mg GAE polyphenol / 100 g carbohydrate) and 0 to 1.5% w/w reducing sugars (with not more than 0.5% w/w fructose and 1 % w/w glucose) are also low glycaemic (see Singaporean patent application nos. SG 10201807121 Q & PCT/SG2019/050416). However, it was thought that all the sugar from these compositions was bioavailable to the mammal consuming the composition, that is, it was thought that while the polyphenols slowed the absorption of the sugar resulting in a lower glycaemic index, all of the sucrose was absorbed and bioavailable to the mammal consuming the composition. Surprisingly, that is not the case. For sugars containing sucrose and 20 to 45 mg CE polyphenols / 100 g carbohydrates only about 80% of the sucrose is bioavailable to the mammal consuming the composition. The higher amount of polyphenols in sucrose sugars with 46 to 100 mg CE polyphenols / 100 g carbohydrates also results in reduced bioavailability of the sucrose. A similar result is also expected for amorphous sucrose sugars such as those described in Singaporean patent application no SG 10201800837 U, which also contain polyphenols and are low glycaemic.

In some embodiments, the sugar composition of the third embodiment of the invention is also prebiotic. Prebiotic sugar compositions

The low glycaemic sugars referred to in the reduced bioavailability sugar compositions section above, are also prebiotic. It was thought that, while the polyphenols slowed the absorption of the sugar resulting in a lower glycaemic index, all of the sucrose and polyphenols were absorbed from the intestines of the mammal consuming the sugar (as occurs with other known sugars). Surprisingly, that is not the case and both sugar and polyphenols progress along the intestines and into the colon of the mammal.

In an alternate third aspect, the present invention provides a sugar composition comprising sucrose and at least 16 mg GAE polyphenol / 100 g carbohydrate and about 0 to 0.3% w/w ash, wherein the sugar is prebiotic. Optionally, there is about 16 mg GAE to about 36 mg GAE polyphenols / 100 g carbohydrates. Optionally, the sugar comprises about 0.05 to 0.3% w/w ash.

In an embodiment, the sugar composition is food grade and comprises sucrose crystals, reducing sugars and polyphenols, wherein the sugar particles comprise about 0 to 0.5g/100g reducing sugars and about 20mg CE/100g to about 45mg CE/100g

polyphenols and wherein a first proportion of the polyphenols are entrained within the sucrose crystals and a second proportion of the polyphenols is distributed on the surfaces of the sucrose crystals.

In a further alternate third aspect, the present invention provides a sugar composition comprising sucrose and about 37 to 80 mg GAE polyphenols/100 g carbohydrate, wherein the sugar is prebiotic. Optionally, the sugar has about 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose. This composition optionally also comprises 0 to 0.3% w/w ash.

In some embodiments of the third, alternate third and further alternate third aspects of the invention or their embodiments, the sugar composition of the invention has a first proportion of the polyphenols entrained within the sucrose crystals and a second proportion of the polyphenols is distributed on the surfaces of the sucrose crystals. The first portion of the polyphenols is endogenous to sugar cane and has been retained within the sucrose crystals during preparation of the sugar, for example, by incomplete washing of the massecuite. The second portion of polyphenols may be but is not required to be endogenous to sugar cane. Part or all of the second portion may be added to the sugar product by spraying polyphenols onto the sucrose crystals. The total amount of polyphenols is efficacious for achieving a low glycaemic sugar in the presence of low reducing sugar content, particularly low glucose content, as described elsewhere.

Referring to a first proportion and a second proportion of polyphenols does not imply that these proportions have a different source; in fact, in preferred embodiments the polyphenols in the first proportion and the second proportion are those originally in the massecuite (from which the sugar is prepared). The amount of polyphenols is efficacious for achieving a low Gl (as defined below) in a sugar particle with low reducing sugar content described elsewhere.

In some embodiments of the present invention of the third, alternate third and further alternate third aspects of the invention or their embodiments, the sugar has about 60% to 100% of the polyphenols outside sugar crystals and about 0% to 40% of the polyphenols within the sucrose crystals (ie up to 40% entrained). Alternatively, about 70% to 100% of the polyphenols are on the outside of the sugar particles and about 0% to 30% of the polyphenols are within the sucrose crystals, about 70% to 95% of the polyphenols are on the outside of the sugar particles and about 5% to 30% of the polyphenols are the sucrose crystals are within the sucrose crystals.

Preparation of these crystalline sugars can be conducted in accordance with the process described in international patent application no PCT/AU2017/050782 and Singaporean patent application nos SG 10201807121 Q & PCT/SG2019/050416.

However, for sugar compositions comprising ash, the ash level of the composition also needs to be tested.

Without being bound by theory, it is thought that the polyphenols slow the metabolism and/or absorption of the sucrose through the intestinal wall resulting in reduced bioavailability. In addition, this allows undigested sugar and polyphenols to pass through the stomach and along the small intestine and enter the colon to feed the gastro- intestinal microbiome. The development of a sugar that: remains largely sucrose, retains a sweet palatable taste with polyphenols, and has reduced sugar bioavailability is highly surprising. It is even more surprising that a sugar has been developed that, when consumed, benefits the gastro-intestinal microbiome (in particular, the colon microbiome). The development of a new version of traditional sugar that has a prebiotic effect and is low Gl is a significant advance likely to be of significant benefit to human and animal health. In a fourth aspect, the present invention provides a sugar composition comprising either:

- one or more sugars and bagasse; or

- one or more sugars, and two or more of hemicellulose, cellulose and lignin;

wherein the composition is prebiotic.

The one or more sugars is optionally a sugar composition of the third or alternate third aspects of the invention or their embodiments.

Optionally, the sugar composition of the fourth aspect of the invention further comprises at least 20 mg CE polyphenol / 100g carbohydrates and about 0 to 0.3% w/w ash and 90% or less of the sucrose is bioavailable to a mammal following consumption of the sugar. Optionally, 85% or less, or 80% or less, of the sugar is bioavailable. Optionally, the sugar contains about 0.05 to 0.3% w/w ash. In some embodiments, the sugar composition has about 20 to 45 mg CE polyphenols / 100 g carbohydrates. In alternate embodiments, the sugar composition has about 46 to 100 mg CE polyphenols / 100 g carbohydrates. The sugar is optionally also low glycaemic or very low glycaemic. The sugar is optionally low to very low glycaemic.

The one or more sugars are optionally one or more sugars as described in international patent application no. PCT/AU2017/050782, Singaporean patent application nos. SG 10201807121 Q & PCT/SG2019/050416 and Singaporean patent application no SG 10201800837 U.

The composition may further comprise ash and/or a source of prebiotic fibres such as hi-maize, fructo-oligosaccharide or inulin, digestive resistant dextrin derivatives or digestive resistant maltodextrin. One advantage of use of a digestive resistant starch is an improvement in anti-caking when using industrial quantities of the sugar.

In all aspects of the invention, the composition is suitable for consumption by a mammal. The mammal is optionally human. Alternatively, the mammal is livestock such as cattle, pigs or sheep. Alternatively, the mammal is a domestic pet such as a cat or dog. Embodiments of the third, alternate third, further alternate third and fourth aspects of the invention

The sugar in the third, alternate third, further alternate third and fourth aspects of the invention can be either amorphous or crystalline. The sugar optionally further comprises reducing sugars such as fructose and/or glucose. The sugar optionally has a maximum of 1 g polyphenols CE/100 g carbohydrate. The sugar optionally has about 0 to

0.5g/100g reducing sugars and a glucose based glycaemic index of less than 55.

The polyphenols in the sugars of the invention are optionally polyphenols that are found endogenously in sugar cane. The polyphenols in the sugars of the invention may be synthetic or isolated from a plant, for example, sugar cane. Preferably, the polyphenols are isolated from sugar cane or a sugar cane derived product, such as a sugar processing waste stream. The polyphenols preferably include flavonoids. Preferably, the polyphenols include one or more of tricin, luteolin and apigenin. Alternatively, the polyphenols include tricin. Optionally, the sugar composition of the third or fourth aspect of the invention comprises about 20 mg CE polyphenols / 100 g carbohydrate to about 1 g CE polyphenols / 100 g carbohydrate, about 20 mg CE polyphenols / 100 g

carbohydrate to about 800 mg CE polyphenols / 100 g carbohydrate, about 20 mg CE polyphenols / 100 g carbohydrate to about 500 mg CE polyphenols / 100 g

carbohydrate, about 30 mg CE polyphenols / 100 g carbohydrate to about 200 mg CE polyphenols / 100 g carbohydrate, or about 20 mg CE polyphenols / 100 g carbohydrate to about 100 mg CE polyphenols / 100 g carbohydrate.

There are multiple options for the measurement of polyphenol content. One option is to measure milligrams catechin equivalents (CE). An alternative is to measure gallic acid equivalents (GAE). Amounts in mg CE/100 g can be converted to mg GAE/100 g by multiplying by 0.81 ie 60 mg CE/100g is 49 mg GAE/100g.

The quantities of polyphenols can also be about 37 mg GAE/100 g to about 80 mg GAE/100 g, about 38 mg GAE/100 g to about 70 mg GAE/100 g, about 39 mg GAE/100 g to about 60 mg GAE/100 g, about 40 mg GAE/100 g to about 55 mg GAE/100 g or about 45 mg GAE/100 g to about 55 mg CE/100 g. In preferred embodiments of the invention, the polyphenol content is about 45 mg GAE /100 g to about 55 mg GAE /100 g. In preferred embodiments, the polyphenol content is about 50 mg GAE/100 g of the sugar. In these embodiments, the sugar composition is optionally very low glycaemic. Optionally, the reducing sugar content of the sugar composition of the third and fourth aspects of the invention is about 0.001 % to 1 .5%, 0.001 % to 1 .2%, 0.001 % to 1 %, 0 to 0.6%, 0.001 % to 0.5%, 0 to 0.3%, 0.001 % to 0.2%, 0 to 0.15%, 0.001 % to 0.15%, 0.01 to 0.1 % w/w of the sugar. Optionally, the reducing sugars are glucose and fructose. Optionally, the glucose to fructose ratio is 0.8 to 1 .2. Optionally, the reducing sugar is not more than 50% glucose. In preferred, embodiments the quantity of fructose is not more than 0.5% w/w or 0.3% w/w of the sugar.

Optionally, the sugar composition of the third, alternate third, further alternate third and fourth aspects of the invention comprises 0 to 1 % w/w reducing sugars of which 0 to 0.5% w/w is fructose and 0 to 0.5% w/w is glucose. Alternatively, the sugar composition comprises 0 to 1 .5% w/w reducing sugars of which 0 to 0.5% w/w is fructose and 0 to 1 % w/w is glucose. Alternatively, the sugar of the invention comprises 0 to 0.6% w/w reducing sugars of which 0 to 0.3% w/w is fructose and 0 to 0.3% w/w is glucose.

In some embodiments of the third, alternate third, further alternate third and fourth aspects of the invention, the sugar is 85% or more w/w sucrose, 90% or more w/w sucrose, 95% or more w/w sucrose. Alternatively, the sugar is 98% or more w/w sucrose.

Optionally, the sugar composition of the third, alternate third, further alternate third and fourth aspects of the invention has about 0 to 0.3% w/w moisture content. Alternatively, the sugar composition has about 0 to 10% w/w moisture content, about 0.1 to 8% w/w moisture content or about 0.1 to 5% w/w moisture content.

It is preferred that the sugar particles have moisture content of about 0.0.3% and a moisture content of about 0.02% to 1 %, about 0.02% to 0.8%, about 0.02% to 0.6%, about 0.1 % to 0.5%, about 0.1 % to 0.4% or about 0.2% to 0.3% w/w after 6 months storage at room temperature and 40% relative humidity in a low density plastic bag or, alternatively, after 12 months storage at room temperature and 40% relative humidity in a low density plastic bag. Alternatively, the increase in moisture content of the sugar particles is a maximum of 0.3% over the 2 year bagged shelf life for the sugar particles. The sugar particles of the invention retain the above low moisture content after storage because of their low hygroscopicity. Without being bound by theory, the lower hygroscopicity is thought to be a result of the low reducing sugar content (in particular the fructose content) of the sugar particles of the invention. Optionally, the sugar composition has low hygroscopicity eg 0 to 0.2% moisture at 50% relative humidity. Optionally, the sugar composition has high solubility eg >95% in water at 25 °C.

Low hygroscopicity is important because hygroscopicity makes the sugar difficult to use and store. This is particularly disadvantageous in an industrial setting because of the tendency for the sugar to clump and stick to equipment. Working with hygroscopic sugar in an industrial setting may require, for example, equipment operating under nitrogen to minimise the quantity of sugar that clumps or sticks to the equipment. Hygroscopic sugars can be sold in small retail products but they are not ideal for industrial use in the preparation of foods, such as, chocolate, beverages, cereals, confectionary, bakery goods and other retail foods containing sugar.

Optionally, the reducing sugars are 0% to 4% w/w, 0.1 % to 3.5% w/w, 0% to 3% w/w,

0% to 2.5% w/w, 0.1 % to 2% w/w of the sugar.

Optionally about 20% less sugar is metabolised from the amorphous sugar of the invention compared to metabolism of white refined sugar.

Optionally, the sugar composition further comprises probiotics.

The sugars of the invention are solids. However, syrup and liquid sugars are also contemplated. In liquid or syrup versions of the sugar of the invention the amount of sucrose is measured by solid weight and equivalent to the w/w% amounts for the solid sugars of the invention. Syrup and liquid versions of the sugars of the invention can be prepared by the addition of solvents such as water to the sugars of the invention. It is also possible to prepare a liquid or syrup sugar composition with the sucrose and polyphenol quantities described for the solid sugars of the invention and optionally the reducing sugar, glucose, fructose and pesticide/herbicide levels described for the solid sugars of the invention. These are liquid or syrup sugars of the invention.

In all aspects of the invention, the composition is suitable for consumption by a mammal. The mammal is optionally human. Alternatively, the mammal is livestock such as cattle, pigs or sheep. Alternatively, the mammal is a domestic pet such as a cat or dog.

Prebiotic compositions

In a further aspect, the present invention provides a prebiotic composition comprising a sugar composition of the invention and one or more further prebiotics. Probiotic compositions

In a further aspect, the present invention provides a probiotic composition comprising a sugar composition of the invention and one or more probiotics. Consumption of a composition comprising both probiotics and prebiotics is one way to ensure that beneficial probiotics are present to benefit from the prebiotics ingested.

Foods and beverages

In a further aspect, the present invention provides a food or beverage comprising one or more of the sugar compositions of the invention, the prebiotic composition of the invention or the probiotic composition of the invention. The food or beverage is optionally prebiotic and/or probiotic.

In a further aspect, the present invention provides a method of preparing a food or beverage comprising combining a sugar composition of the present invention, a prebiotic composition of the invention or a probiotic composition of the invention with one or more ingredients suitable for consumption.

Suitable foods include bread, cereal, chocolate and confectionary. Suitable beverages include fruit juices, tea-based drinks, milk-based drinks, soy milk-based drinks, nut juice-based drinks (eg almond milk) and soft drinks.

Methods of using the sugars of the invention

In a fifth aspect the present, invention provides a method of increasing the quantity of probiotic bacteria in the colon of a mammal comprising consumption by the mammal of at least 3 g of a composition according to the third or fourth aspects of the invention.

In an alternate fifth aspect, the present invention provides a method of increasing the quantity of probiotic bacteria in the colon of a mammal comprising consumption by the mammal of a prebiotic sugar composition, prebiotic composition and/or probiotic composition of the invention.

Optionally, the probiotic bacteria increased is one or more of lactic acid bacteria, Bifidobacteria, Bacteroidetes, Baciullus, Streptococcus, Escherichia, Enterococcus, Anaeropstipes, Bacillus, Odoribacter, Victivallis (phylum Lentisphaerae) and

unclassified Lentisphaerae, unclassified Marinilabiaceae and Anaerophaga (family Marinilabiaceae), Anaerophaga, Cerasicoccus, Roseburia, and/or Shigella species. Optionally, the increase in probiotic bacteria is measured by increase in ammonia.

Optionally, the increase in ammonia in the colon following consumption of the

composition is significant when compared to the ammonia following consumption of cellulose at 32, 56 or 80 hours. It is difficult to test the increase in bacteria and/or ammonia in the colon in vivo. Instead, testing is conducted using in vitro models such as the model used in Example 8.

Optionally, at least 3.5 g or at least 7 g of the composition or bagasse is consumed. Optionally, the Rosebuira was increased over 3-fold (eg about 3.5-fold) 80 hours following consumption of 3.5 g of the composition. Optonally, the Roseburia was increased over 7-fold (eg about 7.5-fold) 80 hours following consumption of 7 g of the composition.

In some embodiments, the method is for the treatment of dysbiosis or diarrhoea

(infectious, travellers’ or antibiotic-associated diarrhoea).

In a further alternative fifth aspect, the present invention provides a method comprising:

- selecting a mammal in need of treatment for dysbiosis or diarrhoea (infectious, travellers’ or antibiotic-associated diarrhoea), or weight management; and

- administering at least 3 g of a composition according to the third or fourth aspects of the invention, their alternatives or their embodiments to that mammal wherein the method increases the quantity of probiotic bacteria in the colon of the mammal.

Optionally, 7 g of the composition is administered. Optionally, administration occurs at least once a day for 7 to 30 days. The composition may be in the form of a capsule (eg similar to probiotic capsules) or in the form of a food.

In a sixth aspect, the present invention provides a method of treating or preventing diabetes, obesity or hyperglycaemia comprising:

- selecting a mammal in need of said treatment or prevention; and

- administering to said mammal a composition according to the third or fourth aspects of the invention, their alternatives or their embodiments, wherein the composition decreased the bioavailability of the sucrose in the composition. In an alternative sixth aspect, the present invention provides a method comprising: - selecting a mammal in need of treatment for dysbiosis or diarrhoea (infectious, travellers’ or antibiotic-associated diarrhoea), or weight management; and

- administering at least 3 g of a composition according to the third or fourth aspects of the invention, their alternatives or their embodiments

wherein the method increases the quantity of probiotic bacteria in the colon of the mammal.

Optionally, 7 g of the composition is administered. Optionally, administration occurs at least once a day for 7 to 30 days. The composition may be in the form of a capsule (eg similar to probiotic capsules) or in the form of a food.

In a seventh aspect, the present invention provides a method of improving the prebiotic effect of a food comprising using the composition of the first aspect, third aspect (or its alternatives) or fourth aspect of the invention (or their embodiments) to prepare a food.

Preferably, the bagasse, composition and/or sugar compositions in all aspects of the invention are food grade.

In some embodiments of the first to seventh aspects of the invention, the method, composition, food or beverage promotes gut health. In an embodiment, promoting gut health comprises reducing or preventing one or more symptoms of a gut health associated condition selected from one or more of: irritable bowel syndrome,

inflammatory bowel disease, Crohn’s disease, colorectal cancer, gut leakiness, non- alcoholic fatty liver disease, metabolic syndrome, obesity, small intestinal bacterial overgrowth (SIBO), gastroenteritis, gut microbial dysbiosis, reduced gut microbial diversity, antibiotic treatment, post-surgery recovery, food intolerance, diarrhoea, gastritis, diverticulitis, flatulence, constipation, functional gut disorders and functional gastrointestinal and motility disorders.

In an embodiment, the functional gut disorder is selected from one or more of: functional abdominal bloating/distension, functional constipation, functional diarrhoea, unspecified functional bowel disorder, opioid-induced constipation, centrally mediated abdominal pain syndrome, narcotic bowel syndrome, opioid-induced hyperalgesia, functional pancreatic sphincter of oddi disorder, biliary pain, faecal incontinence, functional anorectal pain, and functional defecation disorders.

In an embodiment, the functional gastrointestinal and motility disorders is selected from one or more of: gastroesophageal reflux disease, intestinal dysmotility, intestinal pseudo-obstruction, small bowel bacterial overgrowth, constipation, outlet obstruction type constipation (pelvic floor dyssynergia), diarrhoea, faecal incontinence, hirschsprung's disease, gastroparesis and achalasia.

Promoting gut health optionally comprises promoting health of the gut microbiome in a subject. In an embodiment, promoting gut health of the gut microbiome comprises one or more of: increasing the level and/or activity of one or more beneficial bacteria, decreasing or maintaining the level and/or activity of one or more non-beneficial bacteria, increasing the resistance of the gut microbiome, increasing the resilience of the gut microbiome, and increasing the diversity of the gut microbiome. As used herein “resistance of the gut microbiome” refers to the insensitivity of the gut microbiome to a disturbance. As used herein“resilience of the gut microbiome” refers to the rate of the recovery of the gut microbiome after a disturbance (e.g. a disturbance may reduce the number or type of microorganism in the microbiome).

In an embodiment, the beneficial bacteria is selected from one or more or all of: lactic acid bacteria, Bifidobacteria, Bacteroidetes, Baciullus, Streptococcus, Escherichia, Enterococcus, Anaeropstipes, Bacillus, Odoribacter, Victivallis (phylum Lentisphaerae) and unclassified Lentisphaerae, unclassified Marinilabiaceae and Anaerophaga (family Marinilabiaceae), Anaerophaga, Cerasicoccus, Roseburia, and/or Shigella species.

In an embodiment, the lactic acid bacteria is selected from one or more of the genera selected from: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus,

Tetragenococcus, Vagococcus and Weissella. In an embodiment, the lactic acid bacteria is selected from one or more or all of: Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus brevis, Lactococus lactis, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus reuteri,

Pediococcus pentosaceus and Pedicoccus acidilacti.

In an embodiment, the Bifidobacteria is selected from one or more of: Bifidobacteria adolescentis, Bifidobacteria animalis, Bifidobacteria bifidum, Bifidobacteria breve, Bifidobacteria infantis, Bifidobacteria longum, and Bifidobacteria thermophilum.

In embodiment, the Baciullus is selected from one or more of: Baciullus cereus, Baciullus clausii, Baciullus coagulans, Baciullus licheniformis, Baciullus pumulis and Baciullus subtilis. In an embodiment, the Streptococcus is Streptococcus thermophiles. In embodiment, the Escherichia is beneficial strain of Escherichia coli.

In an embodiment, the Enterococcus is Enterociccus faecium.

In an embodiment, the non-beneficial bacteria is a pathogenic strain of bacteria.

In an embodiment, the non-beneficial bacteria is a pathogenic strain of bacteria selected from one or more of: Escherichia coli, Enterococcus, Helicobacter pylori, Clostridium, Vibrio cholerae, Bacteroides fragilis, Clostridium, Fusobacterium, Staphylococcus (e.g. pneumoniae), Legionella, Haemophilus, Pseudomonas, Prevotella, Salmonella, Campylobacter, and Shigella, Listeria.

In an embodiment, non-beneficial bacteria is a pathogenic strain of Escherichia coli.

In an embodiment, promoting gut health comprises modulating microbial diversity in the gastrointestinal tract of a subject. In an embodiment, modulating microbial diversity comprises increasing microbial diversity. This may occur, for example after a

disturbance which reduces the microbial diversity of the gastrointestinal tract.

The present invention has a number of specific forms. Additional embodiments of these forms are as discussed elsewhere in the specification. Preferred forms of the sugar compositions of the invention and the methods of their use include the following.

A food grade sugar composition comprising sucrose, at least 16 mg to 800 mg GAE polyphenol / 100 g carbohydrate and about 0 to 0.3% w/w ash, wherein the sugar is prebiotic.

A food grade sugar composition comprising sucrose, at least 16 mg to 800 mg GAE polyphenol / 100 g carbohydrate and about 0.05 to 0.3% w/w ash, wherein the sugar is prebiotic.

A prebiotic food grade very low glycaemic sugar comprising at least about 90% w/w or 95% w/w sucrose. Alternatively, the present invention provides an edible very low glycaemic prebiotic sugar comprising at least about 98% w/w sucrose or 99% w/w sucrose. The sugars of this embodiment optionally comprise: (i) about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g

carbohydrates, (ii) 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose, and/or (iii) a moisture content of 0.02% to 0.6%. A food grade solid very low glycaemic prebiotic sugar comprising at least about 90% or 95% w/w sucrose, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose.

A food grade solid very low glycaemic prebiotic sugar comprising at least about 98% or 99% w/w sucrose, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose.

A food grade very low glycaemic prebiotic sugar liquid or syrup comprising at least about 90% or 95% sucrose by solid weight, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % reducing sugars by solid weight, wherein the sugar is not more than 0.5% fructose by solid weight and not more than 1 % glucose by solid weight.

A food grade very low glycaemic prebiotic sugar liquid or syrup comprising at least about 98% or 99% sucrose by solid weight, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % reducing sugars by solid weight, wherein the sugar is not more than 0.5% fructose by solid weight and not more than 1 % glucose by solid weight.

Optionally, the above preferred very low glycaemic forms of the invention comprise about 45 mg GAE polyphenols/100 g carbohydrate to about 55 mg GAE

polyphenols/100 g carbohydrate.

Embodiments of the present invention provide food grade solids prebiotic sugars comprising at least about 95% w/w sucrose, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose. Embodiments of the present invention provide food grade prebiotic sugar liquids or syrups comprising at least about 95% sucrose by solid weight, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE

polyphenols/100 g carbohydrates and 0 to 1.5 % reducing sugars by solid weight, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose. Optionally, these prebiotic sugars are low glycaemic. Optionally, these prebiotic sugars comprise about 38 mg GAE polyphenols/100 g carbohydrates to about 70 mg GAE polyphenols/100 g carbohydrates or about 39 mg GAE polyphenols/100 g carbohydrates to about 60 mg GAE polyphenols/100 g carbohydrates.

Embodiments of the present invention provide food grade solid prebiotic crystalline sugars comprising at least about 95% w/w sucrose, about 37 mg GAE polyphenols/100 g carbohydrates to about 80 mg GAE polyphenols/100 g carbohydrates and 0 to 1.5 % w/w reducing sugars, wherein the sugar is not more than 0.5% w/w fructose and not more than 1 % w/w glucose and wherein the sugar has a first proportion of the polyphenols are entrained within the sucrose crystals and a second proportion of the polyphenols is distributed on the surfaces of the sucrose crystals. Optionally, about 70% to 95% of the polyphenols are on the outside of the sugar particles and about 5% to 30% of the polyphenols are the sucrose crystals are within the sucrose crystals.

Embodiments of the present invention provide a method for preparing a food grade solid prebiotic crystalline sugar comprising:

(i) washing sugar cane massecuite or an unrefined sugar including sucrose crystals, polyphenols and reducing sugars to remove an amount of polyphenols and an amount of reducing sugars from the massecuite and produce a first sugar; and

(ii) addition of an additive comprising polyphenols to the first sugar to prepare the second sugar;

wherein the first sugar comprises about 0 to 5% w/w or 0 to 1 %w/w reducing sugars and optionally about 0 to about 1 mg GAE polyphenols/100 g carbohydrate and the second sugar comprises about 37 mg GAE polyphenols/100 g carbohydrate to about 80 mg GAE polyphenols/100 g carbohydrate and 0 to 5% w/w or 0 to 1 %w/w % w/w reducing sugar. Optionally, the second sugar is about 45 mg GAE polyphenols/100 g carbohydrate to about 55 mg GAE polyphenols/100 g carbohydrate and very low glycaemic. Optionally, the first sugar is white refined sugar cane or beet sugar.

Embodiments of the present invention provide a method of increasing the quantity of probiotic bacteria in the colon of a mammal comprising consumption by the mammal of a prebiotic sugar composition of one of the above preferred embodiments. The method optionally promotes gut health and/or increases the quantity of one or more or all the following bacterial strains in the colon of the mammal: lactic acid bacteria,

Bifidobacteria, Bacteroidetes, Baciullus, Streptococcus, Escherichia, Enterococcus, Anaeropstipes, Bacillus, Odoribacter, Victivallis (phylum Lentisphaerae) and unclassified Lentisphaerae, unclassified Marinilabiaceae and Anaerophaga (family Marinilabiaceae), Anaerophaga, Cerasicoccus, Roseburia, and/or Shigella species.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1 shows a graph of Gl v polyphenol content in mg CE/100 g of sucrose sugars prepared by washing massecuite to various polyphenol contents, that is, an amount of endogenous polyphenols were retained and not exogenous polyphenols added. This figure shows sugars have low Gl at about 22-32 mg CE/100g polyphenols.

Figure 2 graphs moisture (% w/w or mg/100g), glucose (% w/w or mg/100g) and fructose (% w/w or mg/100g) content each separately against polyphenol content in sucrose sugars prepared by washing massecuite to various polyphenol contents.

Figures 1 and 2 show that beyond a point, when low Gl sugars are prepared by washing, more polyphenols are not better. Gl increases in sugars with polyphenol content above 32 mg CE/100 g. Without being bound by theory, it was thought that the increase in polyphenol content itself did not increase the Gl of the sugar. As the polyphenol content of the sugar increases above about 22-32 mg CE/100g polyphenols, the reducing sugar content of the sugar also increases and the sugar becomes hygroscopic so moisture content increases. The higher Gl of the reducing sugars such as glucose is then thought to overpower the Gl lowering polyphenols and raise the Gl of the sugar as a whole. The results in Figure 14 demonstrate that high polyphenol content can increase Gl in addition to increased glucose content.

Figure 3 graphs glucose (% w/w or mg/100g) and fructose (% w/w or mg/100g) content against polyphenol content in mg CE/100 g for sucrose sugars prepared by washing massecuite to various polyphenol contents. This figure also shows that Gl and reducing sugar content increases in sugars with polyphenol content above 32 mg CE/100g.

Figure 4 graphs the sucrose content (% w/w or mg/100g) and moisture levels (% w/w) against polyphenol content in mg CE/100 g for sucrose sugars prepared by washing massecuite to various polyphenol contents.

Figure 5 depicts the Triskelion Tiny-TIM system: a. gastric compartment; b. pyloric sphincter; c. small intestinal compartment; e. gastric secretion fluids; f. intestinal secretion fluids; g. pre-filter; h. dialysis membrane (hollow fiber); i. dialysate collection; k. pH electrode; I. pressure sensor; m. level sensor.

Figure 6 graphs the sucrose [mg] in the dialysate (bioaccessible fraction) and intestinal residue fraction at the end of the experiments and before brush border treatment (average ± sd, n=2) - see Example 7.

Figure 7 graphs the bioaccessible glucose+fructose [mg] measured over time in tiny- TIMcarbo dialysate samples treated with brush border enzymes (average ± sd, n=2) - see Example 7.

Figure 8 graphs the cumulative bioaccessible glucose+fructose [mg] measured over time in tiny-TIMcarbo dialysate samples treated with brush border enzymes (average ± sd, n=2) - see Example 7.

Figure 9 is a schematic representation of TIM-2. A. peristaltic compartments; B. pH- electrode; C. alkali pump; D. dialysis liquid circuit with hollow fibers; E. level-sensor; F. N2 gas inlet; G. sampling port; H. gas outlet; I. 'ileum effluent' container; J. temperature sensor.

Figure 10 graphs the cumulative ammonia production during the experimental period of 80h in TIM-2 for each test condition, expressed in mmol (mean ± stdev, n=3). * indicate p<0.05 (Tukey’s test).

Figure 11 is a multidimensional scaling (MDS) plot showing variation in samples from luminal content after 80h in TIM-2 under different test conditions (7g sugar cane bagasse, 3.5 g sugar cane bagasse, cellulose); n=3 per test condition; SCB: sugar cane bagasse - see Example 8.

Figure 12 is a heatmap depicting genera-level bacteria selected from the MDS plot (see figure 9); row normalized values were used (ratio of value per test condition / average of all test conditions per row) - see Example 8. Normalized values are as indicated in the legend. For instance, a value of 2 indicates an increased abundance of bacteria in the test condition relative to the average of all test conditions per row.

Figure 13 is a heatmap representing the shift in relative abundance of bacteria over time for sugar cane bagasse (SGB) test products compared to the shift over time for cellulose. - see Example 8. Shifts in relative abundance are as indicated in the legend, with darker shades indicated a greater shift. Figure 14 graphs the results of a study on the effect of polyphenol content on the Gl of sucrose in the form of traditional refined white sugar. With no polyphenol content the sugar had the Gl of sucrose (68). 15 mg CE/100 g polyphenols slightly lowered the Gl to about 66. 30 mg CE/100 g lowered the Gl to the low Gl of about 50 in accordance with the previous low Gl obtained in Figures 1 and 2. Surprisingly an increase to 60 mg CE/100 g polyphenols lowered the Gl to less than about 20, which is a dramatic and unexpected drop in Gl. Finally, an increase in the polyphenol content to 120 mg CE/100 g resulted in a surprising and steep increase in the Gl to above about 68, which is at about or higher than the original Gl of the sucrose and unexpectedly indicates that the Gl lowering effect of the polyphenols is negligible at that dose.

Figure 15 charts the results of a study on the effect of polyphenol content or polyphenol plus reducing sugar content on the Gl of sucrose in the form of traditional refined white sugar. 30, 60 and 120 mg CE/100g polyphenol content was tested and the results similar to those in Figure 3. Flowever, the Gl for 60 mg CE/100 g was shown to be about 15. Adding 0.6 % w/w reducing sugars (1 :1 glucose to fructose) to the 30 mg CE/100 g polyphenols and sucrose sugar raised the Gl from 53 to 70. Adding 0.6 % w/w reducing sugars (1 :1 glucose to fructose) to the 60 mg CE/100 g polyphenols and sucrose raised the Gl from 15 to 29. Adding 1.2% w/w reducing sugars (1 :1 glucose to fructose) to the 120 mg CE/100 g polyphenols and sucrose increased the Gl from 65 to 75. The presence of reducing sugar consistently increased the Gl.

Figure 16 graphs the Gl of several samples from Table 7 in Example 9.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example.

All of the patents and publications referred to herein are incorporated by reference in their entirety. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

The present invention is in no way limited to the methods and materials described.

The inventors of the present invention have developed a new prebiotic food. In preferred embodiments the prebiotic food is sugar cane bagasse, which has not previously been identified as having a prebiotic effect. Significant amounts of bagasse are produced during sugar production and are currently not used in food preparation. The identification of a new use for the product is of significance.

A further advance is the preparation of prebiotic sugars. As many popular foods, particularly foods with high sugar content, have a less than ideal impact on to the gastro-intestinal microbiome, the preparation of prebiotic sugars is a highly significant advance. The prebiotic sugars of the invention provide sugar substitutes that avoid one of the less desirable aspects of sugar and introduce a desirable prebiotic effect into sugars that will increase the health benefits of foods comprising the prebiotic sugars.

The sugars of the present invention are also of reduced bioavailability meaning that less of the calories in the sugar are absorbed by the mammal consuming the sugar. This is also a significant advance that improves the health profile of sugar containing foods including by minimising the risk of diabetes and obesity from frequent consumption of said foods.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

The term“amorphous” refers to a solid that is largely amorphous, that is, largely without crystalline structure. For example, the solid could be 80% or more amorphous, 90% or more amorphous, 95% or more amorphous or about 100% amorphous.

The term“bagasse” refers to the fibrous matter that remains after sugarcane or sorghum stalks or sugar beets are crushed to extract sugar cane juice. Bagasse is currently used as a biofuel and in the manufacture of pulp, paper and building materials. Optionally, the bagasse of the invention is sugarcane bagasse or sugar beet bagasse. Optionally, the bagasse of the invention is sugarcane bagasse or sorghum bagasse. Optionally, the bagasse of the invention is sugarcane bagasse.

Typically, bagasse can include:

Cellulose 45-55 percent

Hemicellulose 20-25 percent

Lignin 18-24 percent

Ash 1-4 percent

Waxes <1 percent

The terms“sugar cane bagasse” or“sugar cane fibre” refer to bagasse sourced from sugar cane.

The term“sugar” refers to a solid that contains one or more low molecular weight sugars such as sucrose, glucose, fructose or galactose.

The term“reducing sugar” refers to any sugar that is capable of acting as a reducing agent. Generally, reducing sugars have a free aldehyde or free ketone group. Glucose, galactose, fructose, lactose and maltose are reducing sugars. Sucrose is not a reducing sugar.

The term“phytochemical” refers generally to biologically active compounds that occur naturally in plants.

The term“polyphenol” refers to chemical compounds that have more than one phenol group. There are many naturally occurring polyphenols and many are phytochemicals. Flavonoids are a class of polyphenols. Polyphenols including flavonoids naturally occur in sugar cane. In the context of the present invention the polyphenols that naturally occur in sugar cane are most relevant. Polyphenols in food are micronutrients that are of interest because of the role they are currently thought to have in prevention of degenerative diseases such as cancer, cardiovascular disease or diabetes.

The term“refined white sugar” refers to fully processed food grade white sugar that is essentially sucrose with minimal reducing sugar content and minimal phytochemicals such as polyphenols or flavonoids.

The term“massecuite” refers to a dense suspension of sugar crystals in the mother liquor of sugar syrup. This is the suspension that remains after concentration of the sugar juice into a syrup by evaporation, crystallisation of the sugar and removal of molasses. The massecuite is the product that is washed in a centrifuge to prepare bulk sugar crystals.

The term“dysbiosis” refers to a gut microbiome that is imbalanced or disrupted. This imbalance in the microbiome can be associated with a disease, precede a disease or occur as the result of a disease. The imbalance, for example, could be a gain or loss of members of the microbiome community or changes in relative abundance of members of the microbiome community. Dysbiosis is associated with gut health associated conditions including irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, colorectal cancer, gut leakiness, non-alcoholic fatty liver disease, metabolic syndrome, obesity, small intestinal bacterial overgrowth (SIBO), gastroenteritis, gut microbial dysbiosis, reduced gut microbial diversity, antibiotic treatment, post-surgery recovery, food intolerance, diarrhoea, gastritis, diverticulitis, flatulence, constipation, functional gut disorders and functional gastrointestinal and motility disorders.

The term“low glycaemic” refers to a food with a glucose based Gl of 55 or less.

The term“very low glycaemic” refers to a food with a glucose-based Gl of less than half the upper limit of low Gl (ie the Gl is in the bottom half of the low Gl range).

The term“cane juice” or“sugar cane juice” refers to the syrup extracted from pressed and/or crushed peeled sugar cane. Ideally sugar cane juice is at least 60 Brix.

The terms“efficacious” or“effective amount” refer to an amount that is biologically effective. In this context, one example is an effective amount of polyphenols in the sugar particles to achieve a low Gl sugar, ie, a sugar that causes a low increase in blood sugar levels once consumed such that an insulin response is avoided.

The term“hi-maize” or“high amylose maize starch” refers to a resistant starch, ie a high molecular weight carbohydrate starch that resists digestion and behaves more like a fibre. Hi-maize is generally made from high amylose corn. There are 2 main structural components of starch; amylose - a linear polymer of glucose residues bound via a-D-

(1.4)-glycosidic linkages and amylopectin - a highly branched molecule comprising a-D-

(1.4)-linked glucopyranose units with a-D-(1 ,6)-glycosidic branch points. Branch points typically occur between chain lengths of 20 to 25 glucose units, and account for approximately 5% of the glycosidic linkages. Normal maize starch typically consists of approximately 25 to 30% amylose and 75 to 80% amylopectin. High amylose maize starch contains 55 to >90% amylose. The structure for amylose is (with an average degree of polymerisation of 500):

The structure for amylopectin is (with an average degree of polymerisation of 2 million):

The term“inulin” refers to one or more digestive resistant high molecular weight polysaccharides having terminal glucosyl moieties and a repetitive fructosyl moiety linked by b(2,1 ) bonds. Generally, inulin has 2 to 60 degrees of polymerisation. The molecular weight varies but can be for example about 400 g/mol, about 522 g/mol, about 3,800 g/mol, about 4,800 g/mol or about 5,500 g/mol. Where there the degree of polymerisation is 10 or less the polysaccharide is sometimes referred to as a

fructooligosaccharide. The term inulin has been used for all degrees of polymerisation in this specification. Inulin has the following structure:

One option is to use Orafti Inulin with a molecular weight of 522.453 g/mol. The term“dextrin” refers to a dietary fibre that is a D-glucose polymer with a-1 ,4 or a- 1 ,6 glycosidic bonds. Dextrin can be cyclic ie a cyclodextrin. Examples include amylodextrin and maltodextrin. Maltodextrin is typically a mixture of chains that vary from 3 to 17 glucose units long. The molecular weight can be for example 9,000 to 155,000 g/mol.

The term“digestive resistant dextrin derivatives” refers to a dextrin modified to resist digestion. Examples include polydextrose, resistant glucan and resistant maltodextrin. Fibersol-2 is a commercial product from Archer Daniels Midland Company that is digestion resistant maltodextrin. An example structure is:

The term“beet fibre” or“sugar beet fibre” refers to fibre from the sugar beet plant. The fibre may be sourced from the waste stream produced from extraction of sugar from sugarbeets. Sugar beet fibre contains hemicelluloses (22-32 %), pectins (22-29 %), cellulose (19-28 %), protein (5 %), ash (3 %) and moisture (7 %). Both soluble and insoluble polysaccharides are present in a roughly 2:1 ratio. The lignin content is low and considerably less than the lignosulphate content of sugar cane fibre.

The term“lignin” refers to cross-linked phenolic polymers with molecular masses in excess of 10,000 Da.

The term“hemicellulose” refers to a branched polysaccharide of about 500-3,000 sugar units. Examples include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. Sugar monomers in hemicellulose can include glucose, xylose, mannose, galactose, rhamnose, and arabinose. The term“cellulose” refers to a linear polysaccharide consisting of a linear chain of several hundred to many thousands of b(1 4) linked D-glucose units. Cellulose is an insoluble fibre. It is a major component of plant cell walls.

Probiotics

The term“probiotic” refers to live microorganisms which when present in an adequate amount in a host confers a health benefit to the host (such as a mammal subject).

Probiotics are microorganisms, in particular, beneficial bacteria existing in the gastro- intestinal tract of mammals. The probiotics in the colon are of particular interest.

Probiotics include lactic acid bacteria, Bifidobacteria, Bacteroidetes, Baciullus,

Streptococcus, Escherichia, Enterococcus, Anaeropstipes, Bacillus, Odoribacter, Victivallis (phylum Lentisphaerae) and unclassified Lentisphaerae, unclassified

Mariniiabiaceae and Anaerophaga (family Marinilabiaceae), Anaerophaga,

Cerasicoccus, Roseburia, and/or Shigella species.

The increase in certain probiotics increases butyrate production, acetate production, short chain fatty acid (SCFA), branched chain fatty acid (BCFA) or ammonia production.

SCFA constitute the major bacterial end products formed from fermentation of indigestible foods components. Preferably, the composition of the invention increases production of SCFA.

The importance of butyrate for host health is diverse. Butyrate is the main fuel for colonocytes and contributes to the conservation of the mucus barrier through the up- regulation of mucinencoding gene expression, assisting therefore, in maintaining a healthy epithelial layer in the colon. It has anti-inflammatory effects and induces apoptosis in in vitro cultured colon cancer cells and thus, might have anticarcinogenic effects.

Low ratios of acetate to propionate have hypolipidemic effects on the host. Acetate stimulates the synthesis of lipids, while propionate counteracts de novo lipogenesis from acetate demonstrating the importance of the SCFA ratios.

SCFA are essential to maintain a lower pH in the colon, which is a selective mechanism to favour the growth of beneficial bacteria over pathogens.

Prebiotics

The term“prebiotic” refers to something that is a nutrient for probiotics and/or its consumption results in changes in the composition and/or activity of the gastrointestinal microbiota conferring benefits upon the health of the host (e.g. gut health). This can be achieved by increasing the number and/or activity of probiotics in the colon of a mammal. Prebiotics can be soluble and insoluble and include inulin, lactulose, fructo- oligosaccharide (FOS), b-galacto-oligosaccharides (GOS) and Trans- galactooligosaccharides (TOS). The sugars of the invention are a new type of prebiotic.

Feeding our gut microbiota with dietary fibres is important because the fermentation of such substrates can stimulate and/or maintain the population of beneficial

microorganisms (eg Bifidobacterium), and stimulate the production of bacterial metabolites (eg SCFA).

Prebiotics are classified into short chain oligosaccharides (SCFA) consisting of 3-9 monosaccharide units and long chain polysaccharides, which have more than 10 units.

A substance can be tested for prebiotic effect using the in vitro TIM-2 test in accordance with the sugar cane bagasse testing in Example 8. The increase in SCFA, BCFA and/or ammonia following consumption of a substance can also be tested as described in Example 8.

Glycaemic response (GR)

GR refers to the changes in blood glucose after consuming a carbohydrate-containing food. Both the Gl of a food and the GL of an amount of a food are indicative of the glycaemic response expected when food is consumed.

Gl

The glycaemic index is a system for classifying carbohydrate-containing foods according to the relative change in blood glucose level in a person over two hours after consuming that a food with a certain amount of available carbohydrate (usually 50 g). The area under the two hour blood glucose response curve (AUC) is divided by the AUC of a glucose standard, where both the standard and the test food must contain an equal amount of available carbohydrate. An average Gl is usually calculated from data collected from 10 subjects. Prior to a test the person would typically have undergone a twelve hour fast. The glycaemic index provides a measure of how fast a food raises blood-glucose levels inside the body. Each carbohydrate containing food has a Gl. The amount of food consumed is not relevant to the Gl. A higher Gl means a food increases blood-glucose levels faster. The Gl scale is from 1 to 100. The most commonly used version of the scale is based on glucose. 100 on the glucose Gl scale is the increase in blood-glucose levels caused by consuming 50 grams of glucose. High Gl products have a Gl of 70 or more. Medium Gl products have a Gl of 55 to 69. Low Gl products have a Gl of 54 or less. These are foods that cause slow rises in blood-sugar.

Those skilled in the art understand how to conduct Gl testing, for example, using internationally recognised Gl methodology (see the Joint FAO/WHO Report), which has been validated by results obtained from small experimental studies and large multi- centre research trials (see Wolever et al 2003).

GL

Glycaemic load is an estimate of how much an amount of a food will raise a person’s blood glucose level after consumption. Whereas glycaemic index is defined for each type of food, glycaemic load is calculated for an amount of a food. Glycaemic load estimates the impact of carbohydrate consumption by accounting for the glycaemic index (estimate of speed of effect on blood glucose) and the amount of carbohydrate that is consumed. High Gl foods can be low GL. For instance, watermelon has a high Gl, but a typical serving of watermelon does not contain much carbohydrate, so the glycaemic load of eating it is low.

One unit of glycaemic load approximates the effect of consuming one gram of glucose. The GL is calculated by multiplying the grams of available carbohydrate in the food by the food’s Gl and then dividing by 100. For one serving of a food, a GL greater than 20 is high, a GL of 11-19 is medium, and a GL of 10 or less is low.

Cane juice or syrup

Cane juice and syrup contains all the naturally occurring macronutrients, micronutrients and phytochemicals normally removed in white refined sugar, which is 99.9% sucrose.

Molasses

Is a viscous by-product of sugar preparation, which is separated from the crystallised sugar. The molasses may be separated from the sugar at several stages of sugar processing. Molasses contains the same compounds as cane juice but is a more highly concentrated source of phytochemicals. Measuring proportions of surface and entrained polyphenols in solid crystalline sugars of the invention

The proportion of polyphenols on the surface of a solid crystalline sugar and the proportion of polyphenols entrained within the sugar crystal can be measured by washing the sugar with cold (refrigerated) water for 15 to 30 seconds. This time frame has shown to be sufficient to remove the surface polyphenols on sugars with no entrained polyphenols. However, the skilled person will be able to adjust the method for sugars with thick additive layers that need a longer wash to remove the surface polyphenols. The skilled person will also be able to determine when the wash needs to cease because a longer wash will result in dissolution of the sugar crystals into the water. After the wash, the wash water and sugar crystals are separated and the amounts of polyphenols in each quantified to determine the proportion of surface and entrained polyphenols.

The Gl, sucrose, polyphenol, glucose, fructose and moisture content of a sugar prepared from a controlled wash of sugar cane massecuite can all be defined, as set out in the Examples.

There is a“sweet spot” in the extent to which sugar is refined where Gl remains low. Too much washing removed the majority of polyphenol content and increased the Gl. Too little washing resulted in a higher reducing sugar content, which is thought to overpower the Gl lowering effect of the polyphenols and increase the Gl of the sugar.

The low Gl sweet spot was demonstrated by graphing the results of the sugars in Table 3 below. This graph demonstrates that at least 22 mg CE polyphenols/100 g sucrose needs to be retained during sugar processing to produce a low Gl sugar. If additional polyphenols are present but reducing sugars are too high then Gl lowering effect is removed. Respraying molasses back onto refined white and less refined raw sugars to produce a brown sugar may therefore not be an effective strategy to reduce Gl. Table 1 provides data on several sugars prepared by a controlled wash of sugar cane massecuite.

Table 1 - Example sugars

Figure 1 shows a graph of Gl v polyphenol content of these sugars. This figure shows sugars have low Gl at about 22-32 mg CE polyphenols/1 OOg carbohydrate.

Preparation of a sugar of the invention

A sugar according to the invention can be prepared from either sugar cane or sugar beet, from refined white sugar or a sugar prepared in accordance with Example 2 (ie a starting sugar also referred to as a“first sugar” in the summary of invention). Most starting sugars require the addition of further polyphenols to result in a sugar according to this invention. Beet sugar does not contain polyphenols and neither does refined white sugar contain more than trace amounts of polyphenols. However, polyphenols can be added to either to prepare a sugar according to the invention. Sugars prepared by controlled washing of sugar cane massecuite can be prepared with the desired polyphenol content directly but are expected to then contain too much reducing sugar for a low Gl and the reducing sugar content will also likely result in a sugar with unacceptable hygroscopicity. For example, if the starting sugar is prepared using the controlled washing method of Example 2 or as described in patent publication numbers WO 2018/018090 and/or WO 2018/018089 to produce a sugar of 20 to 45 mg CE polyphenols/100 g carbohydrate and suitable reducing sugar content, then the sugar still requires additional polyphenols.

The further polyphenols may be added to the sugar in a powdered or liquid form. One option is to spray the liquid or powdered polyphenols onto the sugar. The process for adding the polyphenol additive onto the sugar can be completed as described in Singaporean patent application no SG 10201806479U or international patent application number PCT/SG2019/050377. Any reducing sugars may be added with or separately to the polyphenols. Alternatively, the reducing sugars may be in the starting sugar.

It is preferred that the polyphenols added to the sugar are polyphenols that, even if not sourced from sugar cane, are present in sugar cane. The polyphenols can be sourced from sugar cane, for example, from a sugar processing waste stream and may be in the form of a sugar cane extract. In some embodiments, the additive is a liquid containing 1000 mg CE polyphenols/100 g carbohydrate and about 11 % solids (for example sugars) in water. 0 to 20% sugar is preferred in the additive. Where the sugar is prepared from sugar cane, the massecuite contains polyphenols. A proportion of the polyphenols in the massecuite are entrained within the sucrose crystals in the massecuite. Massecuite also contains a proportion of polyphenols that are not entrained in the sucrose crystals and the proportion of polyphenols not entrained in the sucrose crystals is generally significantly greater than the proportion of

polyphenols entrained within the sucrose crystals. The exact proportions can vary considerably based on variations in the process used to prepare the massecuite and variations in the sugar cane from which the massecuite is prepared. As an example, the quantity of polyphenols not entrained within the sucrose crystals could be tens to hundreds of times more than the amount of polyphenols entrained within the sucrose crystals. Optionally, the polyphenols entrained in the sucrose crystals in the massecuite are retained during processing of the massecuite and remain in the sugar particles. Optionally, an amount of the polyphenols not entrained within the sucrose crystals is retained during processing of the massecuite and remains on the surface of the sugar particles. In other words, a proportion of the polyphenols in the sugar particles can be endogenous to the sugar cane from which the sugar particles are prepared. The endogenous polyphenols may be separated from and then reintroduced to the sugar particles but remain with the bulk sucrose from which the sugar particles are seeded throughout processing and remain with the sugar particles through the washing process that follows seeding. Alternatively, the polyphenols are retained during processing of the massecuite and remain in the sugar composition because washing of the massecuite was ceased before removal of all of the polyphenols. A consequence of this process is that polyphenols entrained within the sucrose crystals remain within the sucrose crystals from the formation of those crystals and continue to remain within the sucrose crystals within the finished product. Optionally, the polyphenols remain in the sugar particles because washing of the massecuite was ceased before removal of all the polyphenols from the sugar particles (ie washing was ceased before the sugar particles became white). In some embodiments, washing of sugar cane massecuite is ceased when the sugar particles have been washed to contain suitable levels of reducing sugars (ie 0 to 1 % w/w). The polyphenol content is then determined and, if needed, additional polyphenols added to achieve the desired about 46 mg CE polyphenols/100 g carbohydrate to about 100 mg CE polyphenols/100 g carbohydrate. Alternatively, sugar cane can be refined until there is minimal polyphenol or reducing sugar content and the polyphenol content added to the sugar, for example, by a respraying process.

Alternatively, the sugar can be prepared from beet sugar. In this embodiment, the beet sugar is processed to ensure suitable reducing sugar levels and suitable polyphenol content added (as polyphenols are not endogenous to beet sugar).

The sugar particles of the present invention can be prepared to food grade quality by methods known to skilled person including using equipment that has covers to prevent external contamination of the sugar particles, for example by bird droppings, the use of magnets to remove iron shavings and other metals and other methods used to prepare food grade sugar.

Sugar particles of the present invention may optionally include additives or extracts such as added flavours, for example maple syrup flavour, colours or additives/extracts to produce additional health, taste, colour or nutritional benefits. Methods for including these additives are known to those skilled in the art.

Sugar particles of the invention may optionally be cocrystallised or agglomerated. Methods for performing these processed are known to those skilled in the art.

References

International patent application no. PCT/AU2017/050782 titled“Sugar Composition”.

Joint FAO/WHO Report. Carbohydrates in Human Nutrition. FAO Food and Nutrition. Paper 66. Rome: FAO, 1998.

Singaporean patent application no. SG 10201807121 Q, titled“Sugar composition”.

Singaporean patent application no SG 10201800837 U, titled“Amorphous sugar”.

Wolever TMS et al. (2003) Determination of the glycemic index values of foods: an interlaboratory study. European Journal of Clinical Nutrition, 57:475-482.

Kim, Dae-Ok, et al, Antioxidant capacity of phenolic phytochemicals from various cultivars of plums Food Chemistry (2003) 81 , 321 -26.

A copy of each of these is incorporated into this specification by reference. Examples

Example 1 - Preparation of sugar samples for Gl testing

Sugar 1 was prepared at a sugar mill by processing sugar cane to massecuite. The massecuite was washed until it had 22-32mg/100g polyphenol content. One method of achieving sugar with 22-32mg/1 OOg polyphenol content is to wash the massecuite in batches and wash each batch a different length of time. The polyphenol content of each washed batch can be analysed as set out in Example 2. The batch with the appropriate polyphenol content can then be selected. It will be understood by the person skilled in the art that each time massecuite is prepared its components vary. Therefore, there is no single set of wash conditions, eg time, spin and water flow, that will always result in a sugar with the desired polyphenol content. The appropriate wash time will vary depending on the components in the massecuite that is being washed.

Table 1 - Sugar samples

Example 2 - analysis of polyphenol content in sugar

40g of sugar sample was accurately weighed into a 100ml volumetric flask.

Approximately 40ml of distilled water was added and the flask agitated until the sugar was fully dissolved after which the solution was made up to final volume with distilled water. The polyphenol analysis was based on the Folin-Ciocalteu method (Singleton 1965) adapted from the work of Kim et a/ (2003). In brief, a 50 pL aliquot of

appropriately diluted raw sugar solution was added to a test tube followed by 650 pl_ pf distilled water. A 50 mI_ aliquot of Folin-Ciocalteu reagent was added to the mixture and shaken. After 5 minutes, 500 mI_ of 7% Na2C03 solution was added with mixing. The absorbance at 750nm was recorded after 90 minutes at room temperature. A standard curve was constructed using standard solutions of catechin (0-250 mg/L). Sample results were expressed as milligrams of catechin equivalent (CE) per 100g raw sugar. The absorbance of each sample sugar was determined and the quantity of polyphenols in that sugar determined from the standard curve.

Where the sugar is a less refined sugar prepared by a limited wash, an alternative method for analysis of the polyphenol content is to measure the amount of tricin in a sample using near-infra red spectroscopy (NIR). In these circumstances, the amount of tricin is proportional to the total polyphenols. Further information on this method is available in Australian Provisional Patent Application No 2016902957 filed on 27 July 2016 with the title“Process for sugar production”.

Example 3 - analysis of the reducing sugar content in sugar

There are several qualitative tests that can be used to determine reducing sugar content in a sugar product. Copper (II) ions in either aqueous sodium citrate or in aqueous sodium tartrate can be reacted with the sugar. The reducing sugars convert the copper(ll) to copper(l), which forms a copper(l) oxide precipitate that can be quantified.

An alternative is to react 3,5-dinitrosalicylic acid with the sugar. The reducing sugars will react with this reagent to form 3-amino-5-nitrosalicylic acid. The quantity of 3-amino-5- nitrosalicylic acid can be measured with spectrophotometry and the results used to quantify the amount of reducing sugar present in the sugar product.

Example 4 - Gl testing

The Gl testing was conducted using internationally recognised Gl methodology (see the Joint FAO/WFIO Report), which has been validated by results obtained from small experimental studies and large multi-centre research trials (see Wolever et al 2003).

The experimental procedures used in this study were in accordance with international standards for conducting ethical research with humans approved by the Fluman

Research Ethics Committee of Sydney University. Experimental procedures

Using standard methodology to determine a food’s Gl value, a portion of the food containing between 10 and 50 grams of available carbohydrate is fed to 10 healthy people the morning after they have fasted for 10-12 hours overnight. A fasting blood sample is first obtained from each person and then the food is consumed, after which additional blood samples are obtained at regular intervals during the next two hours. In this way, it’s possible to measure the total increase in blood sugar produced by that food over a two-hour period. The two-hour blood glucose (glycaemic) response for this test food is then compared to the two-hour blood glucose response produced by the same amount of carbohydrate in the form of pure glucose sugar (the reference food: Gl value of glucose = 100%). Therefore, Gl values for foods and drinks are relative measures (ie they indicate how high blood sugar levels rise after eating a particular food compared to the very high blood sugar response produced by the same amount of carbohydrate in the form of glucose sugar). Equal-carbohydrate portions of test foods and the reference food are used in Gl experiments, because carbohydrate is the main component in food that causes the blood’s glucose level to rise.

The night before each test session, the subjects ate a regular low-fat evening meal based on a carbohydrate-rich food, other than legumes, and then fasted for at least 10 hours overnight. The subjects were also required to avoid alcohol and unusual levels of food intake and physical activity for the whole day before each test session.

Measurement of the subjects’ blood glucose responses

For each subject, the concentration of glucose in each of the eight whole blood samples collected from them during each test session was analysed in duplicate using a

HemoCue ^® B-glucose photometric analyser employing a glucose dehydrogenase / mutarotase enzymatic assay (HemoCue AB, Angelholm, Sweden). Each blood sample was collected into a plastic HemoCue ^® cuvette containing the enzymes and reagents for the blood glucose assay and then placed into the HemoCue analyser while the enzymatic reaction took place. Therefore, each blood sample was analysed

immediately after it was collected.

For each of the 10 subjects, a two-hour blood glucose response curve was constructed for each of their test sessions using the average blood glucose concentrations for each of their eight blood samples. The two fasting blood samples were averaged to provide one baseline glucose concentration. The area under each two-hour blood glucose response curve (AUC) was then calculated in order to obtain a single number, which indicates the total increase in blood glucose during the two-hour test period in that subject as a result of ingesting that food. A glycaemic index (Gl) value for each test sugar was then calculated for each subject by dividing their two-hour blood glucose AUC value for the test food by their average two-hour blood glucose AUC value for the reference food and multiplying by 100 to obtain a percentage score.

l value (%) = Blood glucose AUC value for the test food x 100

Average AUC value for the equal carbohydrate portion of the reference food

Due to differences in body weight and metabolism, blood glucose responses to the same food or drink can vary between different people. The use of the reference food to calculate Gl values reduces the variation between the subjects’ blood glucose results to the same food arising from these natural differences. Therefore, the Gl value for the same food varies less between the subjects than their glucose AUC values for this food.

Table 2 - Gl for samples prepared in example 1

Example 5 - Relationship between Gl and polyphenol, glucose, fructose and moisture content

Increasing glucose and fructose in low Gl sugar can affect the Gl of sugar. In many unrefined sugars, as sucrose content decreases reducing sugar content increases. The increase in reducing sugars can increase the Gl of the unrefined sugar. This effect is counterintuitive and unexpected. Most consumers understand that less refined products are healthier or better for you. However, that is not necessarily the case for unrefined sugar. The healthiest sugars minimise reducing sugar content without refining out all the polyphenols responsible for the low Gl. There is a“sweet spot” in the extent to which sugar is refined where Gl remains low. The inventors of the present invention have researched less refined sugars by varying the extent of the massecuite washing. Too much washing removed the majority of polyphenol content and increased the Gl. Too little washing resulted in a higher reducing sugar content, which is thought to overpower the Gl lowering effect of the polyphenols and increase the Gl of the sugar.

The low Gl sweet spot was demonstrated by graphing the results of the sugars in Table 3 below. This graph demonstrates that at least 22mg CE/100mg sucrose needs to be retained during sugar processing to produce a low Gl sugar. If additional polyphenols are present but reducing sugars are too high then Gl effect is removed. Respraying molasses back onto refined white and less refined raw sugars to produce a brown sugar may therefore not be an effective strategy to reduce Gl.

Table 3 - Example sugars

Figure 1 shows a graph of Gl v polyphenol content of these sugars. This figure shows sugars have low Gl at about 22-32 mg CE/100g polyphenols. Figure 2 graphs moisture, glucose and fructose content each separately against polyphenol content. Figure 3 graphs glucose and fructose content against polyphenol content for these example sugars. Figures 2 and 3 illustrate why Gl is higher in sugars with higher polyphenol content (ie sugars that would otherwise be expected to remain low Gl). As the

polyphenol content increases above about 22-32 mg CE/100g polyphenols, the reducing sugar content of the sugar increases, the sugar becomes hygroscopic so moisture content increases and the higher Gl of the glucose and fructose begin to raise the Gl of the sugar as a whole despite the Gl lowering polyphenols.

Example 6 - Washing of massecuite to desired polyphenol content

Ten massecuite samples were prepared at two different sugar mills designated“Mill 1” and“Mill 2”. The polyphenol content of each sample was determined (see Example 2). The massecuite samples were washed until they were the depth of colour that is associated with the desired polyphenol content (ie roughly 500 to 2000 ICUMSA) and the polyphenol content measured. The results are in Table 4 below. The skilled person will understand that if the polyphenol content remains too high after the wash, a second wash is possible. The results for each sample are below. The polyphenol content of several of the samples below is too low. Those samples would have to be discarded. It is usual for some sugars prepared at a sugar mill to not meet specifications for various reasons.

Table 4 - Example sugars

Example 7 - Testing for sugar bioavailability

Sugar bioavailability testing was conducted in the Triskelion in vitro gastro-lntestinal Model (tiny-TIM), which simulates the successive dynamic processes in the stomach and the small intestine.

Test product #1 Less refined sugar: Sugar 1 of Example 1 (prepared by controlled wash of massecuite)

Name: Less refined sugar

Available carbohydrate content: ~99 g /100 g dry product

Intake in tiny-TIM: 6.25 g available carbohydrates

Test product #2 Reference product

Name: refined cane sugar (Rohrzucker, from Nordzucker, purchased in Germany) Available carbohydrate content: 100%

Intake in tiny-TIM: 6.25 g available carbohydrates

Meal matrix

A standardized amount of the test product (standardization: related to amount of available carbohydrates) was mixed with artificial saliva and drinking water. After a short pre-incubation at 30 °C, the mixture was put into the stomach compartment of the model together with the gastric residue containing gastric acid and enzymes (amylase, lipase, pepsin).

Test system: tiny-TIM

Information on the Triskelion tiny-TIM system is available at:

The tiny-TIM system consists of a gastric compartment and one small intestinal compartment connected by peristaltic valves (Figure 5). This model mimics the intraluminal pH, enzyme activity, bile salt concentrations, peristaltic movements, and gastrointestinal transit of the contents. The set-points for gastrointestinal simulation are controlled and monitored by specific computer programs. Released and dissolved food components are removed from the intestinal lumen by semipermeable membrane units connected to the small intestinal compartment. This allows the assessment of the so- called bio-accessible fraction, i.e. the fraction of the compound which is potentially available for small intestinal absorption.

The experiments in tiny-TIM were performed under simulation of the average physiological conditions of the Gl tract of human adults after the intake of a liquid product. The simulated parameters are among others: body temperature of 37°C; the swallowing of saliva with amylase; the pH curve in the stomach compartment in relation to the secretion of gastric acid; concentrations of pepsin and gastric-lipase in the stomach; the kinetics of gastric emptying; the secretion of pancreatic enzymes, including amylase in the intestinal compartment; secretion of bile; and secretion of bicarbonate to control intestinal pH.

The gastric half time was set to 30 minutes for liquids. The gastric pH decreased gradually in time from approximately 5.0 to 3.5 within 30 minutes and further to 2.0 within the next hour.

Prior to the performance of each experiment the secretion fluids (e.g. gastric juice with enzymes, electrolytes, bile, and pancreatic juice) was freshly prepared, the pH electrodes calibrated, and semipermeable membrane (hollow fiber) unit installed.

Sampling

The gastric content with the test product were gradually delivered into the small intestine via the 'pyloric sphincter' (Figure 5, item B). The carbohydrates were (partly) digested, depending on their digestibility. The digested and dissolved oligosaccharides are dialyzed continuously from the small intestinal compartment via a semi-permeable hollow fiber membrane systems.

During 170 minutes of digestion the dialysate (= bioaccessible) fractions were collected (Figure 5, item I). Samples were taken every 10 minutes using the fraction collector. Sodium azide (0.05% w/v) is added to each sample, to prevent any microbial growth during overnight incubation with brush border enzymes.

In total 17 samples were taken per tiny-TIM run.

Untreated dialysate samples were collected and stored for sucrose analysis.

Analysis on glucose and fructose using the brush border technique After pre-treatment of the tiny-TIM samples with the brush border enzyme mix, all samples were analyzed on free glucose and fructose using the glucose-fructose assay (R-Biopharm).

Obtained glucose and fructose concentrations were corrected for the dialyzed volume from the tiny-TIM system to obtain the absolute amount of glucose and fructose per sampling time point.

Sucrose analysis before BB treatment

Untreated dialysate samples were analyzed on sucrose content.

Samples are dissolved in diluted hydrochloric acid, and after stirring for 1 h in order to dissolve the sugars. In the resulting solution the content of the sugars were analyzed using high performance anion exchange chromatography (HPAEC).

Calculation

The bioaccessible carbohydrate fraction was calculated by multiplying the total dialysate volume (V) with the analyzed glucose+fructose concentration (C) (Equation 1 ).

Equation 1 : Bioaccessible fraction [mg] = C [mglmL] x V [mL]

The results of the duplicate tiny-TIM runs are presented as average ± sd.

Results

The bioaccessible amount of sucrose and glucose+fructose was measured during and after the tiny-TIM experiments. While bioaccessibility (fraction which is potentially available for small intestinal absorption in vivo) of sucrose was measured in the dialysate samples before brush border enzyme treatment, bioaccessibility of

glucose+fructose was measured after brush border enzyme treatment. The difference between the two measurements can give insight in mechanism underlying the glucose handling in the intestine for the test products.

Bioaccessibilitv of sucrose

Two test products were studied using tiny-TIMcarbo technology. Less refined sugar and white refined cane sugar (reference).

Figure 6 shows the amount of sucrose measured in the dialysate (=bioaccessible fraction) collected at the end of each experiment and before brush border treatment. Similar amounts of sucrose were taken up in the dialysate for both test products (6891.8 ± 70.6 mg refined cane sugar; 6938.3 ± 186.4 mg for Less refined sugar, average ± sd). The residual (intestinal) fraction contained comparable low amounts of sucrose for both test products (42.3 ± 3.1 mg refined sugar vs. 27.7 ± 12.8 mg Less refined sugar).

Bioaccessibilitv of qlucose+fructose after brush border treatment

Disaccharides such as sucrose, which became bioaccessible, were measured in dialysate fractions over time. After subsequent brush border enzyme treatment, sucrose is enzymatically converted into glucose and fructose. The measured glucose and fructose measurement is shown in Figure 7.

The pattern of bioaccessible glucose+fructose over time for the less refined sugar follows a similar curve compared with the reference sugar. The total amount, however, is less for the less refined sugar compared to the reference (Figure 8). The difference between the two is strongest between 30 min and 80 min after the start of the

experiment.

The recovered amount of sucrose in tiny-TIM, i.e. residual fraction and dialysate, for the test products was 6934.1 ± 67.5 mg (Refined cane sugar) and 6965.9 ± 173.7 mg (Less refined sugar). This recovered amount of sucrose was used to calculate the available glucose+fructose after brush border treatment as % of recovery (sucrose).

At the end of the experiment 96.0 ± 4.5% glucose+fructose of recovered sucrose (average ± sd, n=2) was bioaccessible from the reference test product, while 77.5 ± 5.2% glucose+fructose of input (average ± sd, n=2) was bioaccessible from the less refined sugar sugar. The less refined sugar is about 20% less bioavailable in the stomach and small intestine than refined white sugar.

Discussion and conclusions

The test products were tested in tiny-TIM using TIMcarbo technology. During passage of the test products through the lumen in TIM simulating the upper gastrointestinal tract, the carbohydrates were digested. If complex carbohydrates (ie carbohydrates comprising more than two saccharide units) were present, then they would have been digested to mono-, di- and oligosaccharides. The disaccharides (sucrose) and monosaccharides were dialyzed from the lumen and subsequently treated with brush border enzymes containing sucrase.

Less refined sugar contains polyphenols from sugar cane. The hypothesis is that phenols inhibit the digestion of sugar (saccharose/sucrose, a disaccharide) and hence absorption of monosaccharides (e.g. glucose). The digestion of disaccharides (saccharose/sucrose) is driven by brush border enzymes. The effect of the test product containing phenols on the brush border enzymes and hence digestion of saccharose was studied and compared with a reference product (refined sugar). It is part of the hypothesis that phenols bound in the test product are released into the dialysate.

The test product Less refined sugar showed a lower amount of glucose+fructose becoming bioaccessible after brush border treatment in the dialysate, compared to the reference test product. The amount of sucrose in the dialysate fraction was comparable between the two test products indicating a difference with Less refined sugar on the sucrose digestion with brush border enzymes, possibly via phenols.

The digestion resistant portion of less refined sugar is expected to proceed to the colon, where they would have a prebiotic effect, in particular increasing Prevotella spp. that are associated with weight loss.

Example 8

The prebiotic effect of bagasse was tested using the Triskelion TNO Intestinal Model 2. Two concentrations of Sugarcane bagasse (3.5% and 7%) were compared to a negative cellulose control.

The effect of the test product on ammonia was assessed. In addition, microbial composition can be assessed by 16S rDNA amplicon sequencing (V4 region).

Assessment of these parameters, i.e. growth and activity of (human) colonic bacteria, was done using an in vitro model which simulates colon conditions and more

importantly which is predictive for the human situation.

Test products

Test product #1

• Name: Sugar cane Bagasse

• Description: Composed of hemicellulose (~20-25%), cellulose (~45-55%),

Lignin (-18-24%)

• Batch number: Bagasse Nl 2018

• Form: Brown powder

• Storage conditions: ambient temperature (15-25°C), dry condition

• Expiry date: February 2019 • Supplier: Nutrition Innovation

• Amount in TIM-2: 7 g and 3.5 g

Test product #2 Negative Control

• Name: Cellulose

• Description: Cellulose, microcrystalline, extra pure, CAS: 9004-34-6

• Batch number: A0381663

• Form: powder, particle size 90 pm

• Storage conditions: ambient temperature (15-25°C)

• Expiry date: 30 November 2022

• Supplier: ACROS organics

• Amount in TIM-2: 7 g

SIEM and dialysis liquid

SIEM (standardized ileal effluent medium) simulates material passing the ileocecal valve in humans, or in other words material reaching the colon. The SIEM in the TIM-2 experiments contained the major non-digestible carbohydrates (pectin, xylan, arabinogalactan, amylopectin, starch) found in a normal western diet as well as protein (bactopepton, casein), some ox-bile, Tween 80 as well as vitamins and minerals.

SIEM does not require pre-digestion and was added to the system at a speed of 2.5 ml/hour. The speed of the dialysis liquid was 1.5 ml/min.

Test system: TIM-2

The TNO intestinal model TIM-2 (Figure 9) is a dynamic in vitro model of the proximal large intestine. The conditions in the proximal large intestine are mostly determined by the composition of the microbiota. The TIM-2 system (Figure 9) was inoculated with a dense and highly metabolic active colon microbiota of human origin. In the system the following standardized conditions are simulated: body temperature; pH in the lumen of the proximal colon (pH 5.8); anaerobiosis; delivery of a predigested substrate from the ‘ileum’ (SIEM); mixing and transport of the intestinal contents; absorption of water and absorption of metabolic products (via dialysis).

The content of the system was kept under strict anaerobic conditions (flushing with nitrogen). Fermentation products, metabolites and other low molecular weight compounds was continuously removed from the lumen via dialysis through a semipermeable membrane system inside the colon compartment.

During the experiment, the intestinal contents were mixed continuously by the peristaltic movements of the TIM-2 system. The pH was kept at pH 5.8 or above by automatic titration with 2 M NaOH (C in Figure 9). The pH was automatically adjusted minute by minute. The consumption of NaOH was monitored.

Prior to the performance of each experiment the secretion fluids and dialysis solutions were prepared freshly, the pH electrodes calibrated, new membrane units installed and the system was inoculated (one day before the start of the test period) with a

standardized microbiota of human origin.

Microbiota

The microbiota was prepared using fecal donations from a group of 4 healthy volunteers (2 male, 2 females, age 37.3 ± 6.4 years; BMI 23.0 ± 2.6 kg/m ² ). Individuals provided signed informed consent prior to participation, were non-smokers and had not used antibiotics, prebiotics, probiotics or laxatives 1 month before the donation. Fecal donations were pooled and afterwards mixed with dialysate in 1 :1 ratio (including 10% glycerol). Aliquots (35 ml_) from the microbiota obtained after pooling the fecal samples were snap-frozen in liquid nitrogen and stored in a freezer at <-72 °C. Before being introduced into the system, the inoculum was thawed by 1 h immersion in a 37 °C water bath.

Conduct of the study and sampling

The test products were studied in triplicate experiments. The TIM-2 experiments last for 80 hours, i.e. three days.

Set-up of experiments:

1 - Test product #1 (7 g/day)

2- Test product #1 (3.5 g/day)

3- Test product #2 cellulose (microcrystalline, by Acros Organics) as negative control

The TIM-2 units was flushed with nitrogen prior to inoculation and during the entire experiment.

At the start of the adaptation period, the TIM-2 system was inoculated with

approximately 30 ml of the standardized microbiota and 80 ml dialysis fluid. The microbiota was allowed to adapt to the model conditions and SIEM for 16 h. After the adaptation period the 80 h test period started, in which test product were added to TIM- 2 in a daily dose.

Administration of test product

During the test period, the test product were added to the system in a daily dose.

The test product were added to the system at the indicated dose in addition to the SIEM, which were added throughout the entire test period. The SIEM was added without carbohydrates, i.e. the test products replace the carbohydrates standard included in the SIEM during the experimental period. Since the test product are not digested in the small intestine, pre-digestion was performed.

Sampling

Samples were taken from the dialysis fluid (D in Figure 9) and from the lumen (G in Figure 9). Metabolites like short chain fatty acids (SCFA), branched-chain fatty acids (BCFA), and ammonia produced in TIM-2 are continuously removed from the lumen by a semipermeable membrane unit (D in Figure 9). This dialysate represents the amount available for absorption from the colon in vivo. The dialysate was collected after 8h and every 24 hours: t=0h, t=8h, t=32h, t=56h, t=80h after start of the test period. Volumes were measured and samples were taken from the dialysate (1 ml per analysis, 1 ml samples for back-up.)

Luminal samples (in 1 ml aliquots) were collected via the sampling port (G in Figure 9) at the same time points (after 8h and every 24 hours) in order to determine the metabolite concentration in the lumen.

In order to simulate the transit of the chime from proximal to distal colon, 25 ml (minus the ml used for sampling) of the lumen was removed after 8h and every 24 hours.

Sample storage

The samples were snap frozen in liquid nitrogen and stored at <-72°C until analysis. Backup samples were taken of every time point and stored at <-72°C until 1 month after submission of the final report. Number of sam to be on SCFA, BCFA, ammonia

The procedure described in Example 8 results in 3 x 3 (experiments in triplicate) x 5 (sample time points) x 2 (types of samples - lumen and dialysate) (90) samples in total (lumen and dialysate), analyzed on SCFA, BCFA, and ammonia.

Metabolites

The dialysate and lumen fractions of TIM-2 were analyzed with gas chromatography for SCFA, (butyrate, acetate, and propionate) and BCFA (iso-butyric acid and iso-valeric acid). In addition, the samples were analyzed enzymatically for ammonia. The SCFA, BCFA and ammonia values from t=0h were used to set the values of these metabolites in the TIM-2 system artificially to zero. Results are expressed as cumulative production over time.

For SCFA/BCFA, samples were prepared and analysed. Briefly, samples were centrifuged (12,000 rpm at 4 °C for 10 min). A mixture of formic acid (20%), methanol, and 2-ethyl butyric acid (internal standard, 2 mg/ml in methanol) was added to the supernatant. Samples were measured by gas-chromatography. A 3 pi sample with a split ratio of 75.0 was injected on a GC-column (ZB-5FIT inferno, ID 0.52 mm, film thickness 0.10 urn; Zebron; phenomenex, USA) in a Shimadzu GC-2014 gas

chromatograph. Standard curves were obtained by injecting calibrated quantities of a blend of volatile fatty acids (the measured SCFA and BCFA) and amounts were calculated from the graph obtained correlating peak height and time measured (all reagents from Sigma-Aldrich with the exception of formic acid which was from Merck).

Samples for ammonia analysis were centrifuged as described above. Ammonia was determined based on the Berthelot reaction in which ammonia first reacts with alkaline phenol and then with sodium hypochlorite to form indophenol blue. In the currently used method phenol was replaced by salicylic acid. The absorbance of the indophenol blue is directly proportional to the original ammonia concentration and is measured at 660 nm. The measurement is automated on a Cobas Mira plus auto analyzer (Roche, Almere, The Netherlands). Concentrations in the samples was determined via comparison with a series of standard solutions with known concentrations. 16S rDNA amplicon sequencing

The bacterial population in the TIM-2 samples were analyzed using Next Generation sequencing. DNA from TIM-2 lumen samples was isolated and purified for universal 16S rDNA amplification targeting the V4 region with universal primers.

Subsequently these amplicons were further processed for amplicon sequencing on the MiSeq system. The sequencing results from the MiSeq system were processed in a dedicated data processing pipeline. To this purpose, the Mothur pipeline is implemented in combination with the Silva database which provides comprehensive, quality checked and regularly updated databases of aligned small 16S rRNA sequences

(https://www.arb-silva.de/).

In brief, processing of the sequencing data using the Mothur pipeline is a computer- based script that comprises in summary: quality checks, normalization and identification of the sequences at taxon level 6 (genus level) by using the Silva data base (a database with 16S rRNA sequences linked to taxonomic information). Based on frequencies of the presence of specific sequences, the bacterial community composition can be described. This yields a community profile which is presented in a bar plot indicating the relative abundance of the various bacterial genera present. The bar plots give an overview on all bacterial genera present and identified, with exception of nonclassified bacterial genera, which are indicated as unknown genus but with a lower level identification (taxon or family for instance).

Data analysis

The metabolite concentration as measured in the different samples (lumen and dialysate over time) was multiplied by the volume of the respective sample. For the dialysates, the total volume of each sample was measured. For the lumen samples, a theoretical volume (same for all TIM-2 units and at every sampling time) was used to calculate the absolute amount of metabolites.

The SCFA, BCFA, and ammonia values from t=0h were used to set the values of these metabolites in the TIM-2 system artificially to zero.

The graphs in the report depict the total (cumulative) produced amount (luminal and dialysate samples) over time. Metabolite data are presented as mean ± standard deviation.

ANOVA (one-way) was performed on metabolite analysis in combination multiple comparison (Tukey’s) test using GraphPad Prism 7. In case of significant differences between treatments groups, the different groups were mentioned in the text and indicated in the graphics.

Microbiota composition was analysed using MDS plots (multidimensional scaling) performed in R. The relative abundances were expressed in bar plots and in heatmaps. For the latter, the change in bacterial abundance per sugar cane bagasse test product was expressed as ratio of t80h/t0h and compared to the ratio of t80h/t0h for cellulose.

Equation 1 :

80, ¾

relative abundance baetermm for for sugar cane bagasse fold change o¾

relative abundance

Results

Pilot

To test the behaviour of the test product and the mode of administration in the TIM-2 system, a pilot experiment was conducted with 10 g and 7 g of test product #1.

During the experiment with 10 g, a blockage of the system was observed. The test product seemed to be hardly degraded in the lumen. Due to bulking, 10g product could not be administered in daily shots during the entire test period. For 7 g, the daily shots could be administered to the system throughout the test period.

To test a concentration dependent effect, it was decided to use 3.5 g in addition to 7 g sugar cane bagasse.

Moreover, due to little change in pH seen with 7 g compared to cellulose during the first hours, it was decided to increase the experimental period by 8h to increase time for adaptation of the microbiota to the test product substrate. This extra sample time point resulted in 5 sample time points instead of 4.

Ammonia

Ammonia is a metabolite produced by microbial fermentation of nitrogen containing molecules (such as protein, peptides, peptone and urea). The cumulative (total) amount of ammonia was measured for each of the test conditions as shown in Figure 10 (individual data are shown in Table 5).

Table 5 - Ammonia (mM) measured in TIM-2 samples after adding the test products sugar cane bagasse (3.5g and 7g) and cellulose for all TIM-2 runs

The production of ammonia was highest for 3.5 g (125.3 ± 1.7 mmol), followed by 7 g sugar cane bagasse (122.3 ± 3.4 mmol) and cellulose (119.3 ± 1.7 mmol). ANOVA indicated significantly different ammonia values between the treatment groups. A Tukey’s posthoc test indicated furthermore a significant difference between 3.5 g and cellulose after 32h of experiment, between 3.5 g and cellulose as well as between 7 g and 3.5 g after 56h and between 3.5 g and cellulose after 80h.

Microbiota composition

To test the effect of the test products on the microbiota composition, 16S sequencing was performed in TIM-2 luminal samples after 80 h of experimental period.

Bacteroides are the most abundant bacteria present from all test conditions in TIM-2 after 80h. Bacteroides are known for their capability of degrading complex

carbohydrates. The changes in relative abundances of bacteria for sugar cane bagasse compared to cellulose were calculated as fold changes and presented in Figure 13 (see equation 1 ).

A fold change above 2.0 indicates a higher relative abundance for the sugar cane compared to cellulose after 80h in TIM-2. On the other hand, a fold change below 0.5 indicates lower relative abundance for sugar cane compared to cellulose and fold changes between 0.5 and 2.0 indicate that there is no significant change. Figure 11 shows the variation between luminal TIM-2 samples based on microbiota composition.

The separation of samples on the x-axis indicates that the 7 g sugar cane bagasse bacterial population distribution is most different from the cellulose bacterial population distribution. The variation between the samples is explained by 53.6%. Whereas on the y-axis, the samples from 3.5 g sugar cane bagasse bacterial population distribution are different from the bacterial population distributions of cellulose and 7 g sugar cane bagasse. This variation on the y-axis is explained by 22.5%. It should be noted that y- axis has a different scaling compared to the x-axis which means that samples from 3.5 g sugar cane bagasse are actually closer to 7 g sugar cane bagasse and cellulose in variation. The arrows indicate genera of bacteria with the highest abundance towards the samples they are pointing. Escherichia/Shigella, unclassified Porphyromonadaceae, Anaeropstipes and Bacillus are pointing towards 7 g sugar cane bagasse. Kurthia, Odoribacter, Victivallis and Salmonella are pointing towards 3.5 g sugar cane bagasse and Anaerophaga towards cellulose.

Figure 11 depicts bacteria that have the highest contribution to the variation between the respective test conditions/samples. In addition, the heatmap (figure 12) shows that Escherichia/Shigella showed an increase for only one out of the three TIM-2 runs with 7 g sugar cane bagasse. Bacillus, Anaerostipes and unclassified Porphyromonadaceae show an increase on sugar cane bagasse compared to cellulose, while Anaerophaga and unclassified Marinilabiaceae were more abundant for cellulose compared to sugar cane bagasse.

Discussion and conclusions

To the best of the applicant’s knowledge knowledge, to date, there are no studies examining the effect of sugar cane bagasse on human microbiota composition or activity. Therefore metabolites produced and changes in microbiota composition were studied in TIM-2.

Increased amounts of propionate in colon has been linked to prevention of weight gain in overweight adult humans as well as satiety. Propionate has furthermore cholesterol- lowering effects. Species of Roseburia have been reported to produce propionate. In line with the observation of increased propionate, Roseburia was dose-dependently increased in abundance with sugar cane bagasse (Appendix Table A 7: 7.4 fold increase for 7 g and 3.5 fold for 3.5 g vs. cellulose). It can hence be hypothesized that sugar cane bagasse has a positive effect on weight management.

Changes in microbiota composition

Most of the bacteria that were different in abundance with sugar cane bagasse were associated with SCFA metabolism. Anaeropstipes was seen increased in abundance with 7 gram sugar cane bagasse. Anaerostipes produces butyrate. In addition, Bacillus was seen higher in abundance with 7 gram sugar cane bagasse. Some Bacillus species can be used as probiotics for improvement of human health. Odoribacter, which was specifically higher in experiments with 3.5 gram sugar cane bagasse, has been linked to higher SCFA production and hence suggested to be beneficial to the host organism. Similarly, Victivallis (phylum Lentisphaerae) and unclassified Lentisphaerae were found increased in abundance for 3.5 gram sugar cane bagasse. Victivallis produces acetate from a variety of sugars and is a strict anaerobic bacterium found in human feces. This is in line with increased level acetate found for 3.5 g. Specific for cellulose, unclassified Marinilabiaceae and Anaerophaga (family Marinilabiaceae) were increased in

abundance. In industrial fermentation processes Anaerophaga has been associated with cellulose fermentation.

For cellulose, and the sugar cane bagasse test products, changes in microbiota composition were observed. Some bacteria were overlapping between the test products for 1 or 2 runs, while some were specifically increased only with one test product. The overlap between bacteria is expected since the test products consist of similar carbohydrate compounds. Sugar cane bagasse consists of cellulose, hemicellulose and lignin.

Most of the bacterial changes seen with sugar cane bagasse have been associated with health benefits.

Example 9 - Effect of polyphenols on Gl of sugar

The effect of polyphenol content on the Gl of sugar was studied. Traditional white sugar ie essentially sucrose was used as a control. Sugars with varied quantities of polyphenols were prepared by adding various amounts of polyphenol content to traditional white sugar.

Table 6 shows the results of testing of an in vitro Glycemic Index Speed Test (GIST) on the sugars prepared. The method involved in vitro digestion and analysis using Bruker BBFO 400MFIz NMR Spectroscopy. The testing was conducted by the Singapore Polytechnic Food Innovation & Resource Centre, who have demonstrated a strong correlation between the results of their in vitro method and traditional in vivo Gl testing. The results of the GIST testing is also graphed in Figure 14.

Table 6 - sugar polyphenol content v Gl

While the Gl of fructose is 19, the Gl of glucose is 100 out of 100. We therefore expect that the as glucose increases in less refined sugars the glycemic response also concurrently increases.

A second set of sugars were prepared in which reducing sugars (1 :1 glucose to fructose) were added to some of the white refined sugar plus polyphenol sugars. The Gl of these sugars was also tested using the GIST method and the results are in Table 7.

Table 7 - Effect of polyphenol and reducing sugar content on Gl

The Gl of several samples from Table 7 are graphed in Figure 15.