EP4114205A1 - Beverage composition and method of forming the same - Google Patents

Abstract

The present invention provides a beverage composition comprising, at least 600mg polyphenols, and at least 1 g of fibre. A beverage composition according to one aspect of the present invention may be for use in a method of improving cognitive function in a subject in need thereof, a method of managing and/or treating type II diabetes, a method of reducing postprandial glycaemia, a method of promoting weight-loss and/or improving Body Mass Index in a subject in need thereof, a method of reducing the risk of cardiovascular disease and/or a method of preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin response.

Description

DESCRIPTION

Title of Invention

Beverage composition and method of forming the same

This application claims priority from GB2003327.0 filed on 06 March 2020, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention This disclosure relates to a beverage composition which provides a desirable satiety and a method of making the beverage composition. In particular, it relates to a beverage composition comprising polyphenols and fibre. The disclosure further relates to uses of the beverage composition in each of improving cognitive function, managing and/or treating type II diabetes, reducing postprandial glycaemia and promoting weight-loss.

Background

Consumers want to be provided with energy from the food and beverages they consume. However, as consumers are becoming more aware of their diet and nutrition, they are seeking out foods and beverages which are natural and healthy. When providing an energy source to meet the consumer’s need: for satiet and a temporary boost in mental and physical capacity, high giycaemic index meals and snacks are not always appropriate, since they do not generally provide a longrlasting effect. Caffeine is often used-in place of calorie based energy sources, but many consumers now prefer to use caffeine judiciously, due to their belief that consumption should be limited.

There is a range of solid, biscuit-based snack-foods already available on the market, and it is known from the research on these products that the provision of certain slowly-available calorie sources can provide for long-lasting energy, increased satiety and delayed onset of hunger. There is a general desire for the provision of such products, since they would be expected to provide a number of health benefits.

It is known that one way to achieve increased feelings of satiety is to provide sources of fibre not easily digested by the body. This delays gastric emptying and increases a feeling of fullness after consumption.

Description of Invention

Accordingly, it is desirable to provide alternative food products and, in particular, beverage compositions and method for making the same with beneficial health effects as described herein and/or to tackle at least some of the problems associated with the prior art or, at least, to provide a commercially viable alternative thereto.

In a first aspect there is provided a beverage composition comprising at least 600mg polyphenols and at least 1g of fibre.

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The present invention relates to a beverage composition. By a “beverage composition” is meant a composition suitable for human consumption as a beverage either immediately or after reconstitution or dilution with a beverage medium, typically water.

Accordingly, in one embodiment the beverage composition is ready-to-drink and has a volume of from 100 to 1000ml, preferably 100 to 750ml, preferably 150 to 500ml, preferably 200 to 300ml. That is, in a ready-to-drink beverage having a volume of from 100 to 1000ml there is provided at least 600mg polyphenols and at least 1¾ of fibre.

In an alternative embodiment the beverage composition is provided as a concentrate for forming a beverage on reconstitution with water. The final volume of the beverage after formation will be the same as the ready-to-drink composition discussed above. The format of the concentrate may be a concentrated liquid or gel formulation for dilution with a beverage medium. For example, a beverage may be formed after the concentrate is added to a receptacle and water added, or after the concentrate is added to a vessel containing water (e.g. added to a bottle and shaken). The concentrate may also be a powder, such as a freeze-dried or spray-dried powder, and reconstituted in the same manner. Given the challenges of suspending significant amounts of fibre, it is most preferred that the composition is a ready-to-drink formulation or liquid concentrate.

Polyphenols

The beverage composition comprises at least 600mg polyphenols. Polyphenols are micronutrients that can enter our diet through certain plant-based foods. Polyphenols are characterized by the presence of large multiples of phenol structural units. The number and characteristics of these phenol structures underlie the unique physical, chemical, and biological properties of particular members of the class. They are known to have antioxidants and potential health benefits. It is thought that polyphenols can improve the health of individuals with, or reduce the risk of the following: digestion issues, weight management difficulties, diabetes, neurodegenerative disease, and cardiovascular diseases. The level of polyphenols in a composition can be quantified with Folin Ciocalteu reagent using colorimetric assays; such assays are well known in the art.

Many foods in a healthy diet contain high levels of naturally occurring phenols, such as from fruits, vegetables, cereals, tea and coffee. Fruits like grapes, apple, pear, cherries and berries contains up to 200-300 mg polyphenols per 100 grams fresh weight. Typically a glass of red wine or a cup of tea or coffee contains about 100 mg polyphenols.

As will be appreciated, a dose of 600mg of polyphenols in a beverage of up to 11 and preferably smaller, is a large dose of polyphenols compared to that which can be achieved by consuming a natural polyphenol source. Blackcurrants typically contain about 250mg per 100g of fresh fruit, so to achieve the minimum required dose you would need to consume at least 250g of the fruit. Preferably the beverage composition comprises from 700 to 2000mg polyphenols, preferably 800 to 1200 mg polyphenols.

In one embodiment, concentrations of total polyphenols in a beverage composition may be 500 to 10,000 ppm, 600 to 8,000 ppm, 700 to 7,500 ppm, 800 to 7,000 ppm, 1 ,000 to 6,500 ppm, 1 ,200 to 6,000 ppm, 1 ,500 to 5,500 ppm, 1 ,800 to 5,200 ppm, 2,000 to 5,000 ppm, 2,250 to 4,750 ppm, 2,500 to 4,500 ppm, 600 to 6,000ppm, 800 to 6,000ppm, 1 ,200 to 4,000ppm, 2,000 to 3000ppm or 800 to 1 ,200ppm.

Polyphenols can be produced synthetically. However, preferably the beverage composition comprises a concentrated fruit extract as a source of the polyphenols. The use of a natural source provides a more nutritionally diverse selection of polyphenols and is considered to be healthier by the end consumer, permitting a clean label on the product (i.e. no artificial chemical additives). The fruit extract will typically consist essentially of the polyphenols.

Preferably the fruit extract is derived from one or more anthocyanin-rich fruits. Anthocyanin- rich fruits are characterised by having a red, purple, blue or black colour derived from the anthocyanin content. As used herein, an anthocyanin-rich fruits is one comprising at least 200mg polyphenols per 100g. Preferred anthocyanin-rich fruits are selected from blackcurrant, blackberries, blueberries and pomegranate, or a combination of two or more thereof. It is most preferred that the anthocyanin-rich fruits comprises or consist essentially of blackcurrants.

Preferably the polyphenols consist essentially of blackcurrant polyphenols. Blackcurrants are a rich source of polyphenols and can be readily treated and concentrated to obtain a rich source of polyphenols. The polyphenols are often present in the skins of the blackcurrant (and in other anthocyanin-rich fruits), so the polyphenols can desirably be obtained as a process by-product after the juice has been obtained from the fruit. That is, the polyphenols can be obtained as a useful additive from the materials which would otherwise be considered a waste product.

Preferably, the polyphenols in the fruit comprise a large proportion of anthocyanins. Preferably, the anthocyanin profile comprises (or consists essentially of) 4 anthocyanins: 3- O-glucosides and the 3-O-rutinosides of delphinidin and cyanidin. In one embodiment, the anthocyanin profile may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% of the 4 anthocyanins.

In one embodiment, fruit extract containing polyphenol can be obtained from a whole fruit, part of fruit and/or pomace of fruit. Part of fruit may be flesh, pulp, peel, flavedo and/or albedo.

Fibres

The beverage composition comprises at least 1g of fibre. The fibre present is also known as dietary fibre. Dietary fibre is the portion of plant-derived food that cannot be completely broken down by human digestive enzymes. It has two main components: 1 ) Soluble fibre - which dissolves in water - is readily fermented in the colon into gases and physiologically active by-products, such as short-chain fatty acid^'s produced- in' the colon by gut bacteria; it is viscous, may be called prebiotic fibre, and delays gastric emptying which, in humans, can result in an extended feeling of fullness. 2) Insoluble fibre - which does not dissolve in water - is inert to digestive enzymes in the upper gastrointestinal tract and provides bulking.

Dietary fibre consists of non-starch polysaccharides and other plant components such as cellulose, resistant starch, resistant dextrins, inulin, lignins, chitins, pectins, beta-glucans, and oligosaccharides. Preferably the beverage composition comprises from 1.25 to 3.5g of fibre, preferably from 1 .5 to 2g of fibre. The level of fibre in a composition can be quantified in accordance with AOAC 991 .43; such testing approaches are well known in the art.

As will be appreciated, 1g of fibre is a significant amount to be present in a beverage. A pulp-containing orange juice, which is a rich source of fibre, typically comprises around 0.2g/100ml of fibre. Therefore, particularly for the smaller volumes of beverage contemplated herein, it can be seen that the dosage of fibre is particularly high. Techniques for assessing fibre levels in foods are well known in the art. in one embodiment, concentrations of fibres in a beverage composition may be 0.5 to 20 g/L, 1.0 to 19 g/L, 1.5 to 18 g/L, 2.0 to 17 g/L, 2.5 to 16 g/L, 3.0 to 15 g/L, 3.5 to 14g/L, 4.0 to 13 g/L, 4.5 to 12 g/L, 5.0 to 11 g/L, 5.0 to 10 g/L, 1.0 to 10 g/L, 1.3 to 10 g/L, 2.0 to 6.7 g/L, or 3.3 to 5.0 g/L.

Preferably substantially all of the fibre is natural fibre. Preferably the fibre is provided as a cellular pulp (where pulp is defined as a mashed fruit mass). Preferably the composition comprises or consists essentially of citrus fibre as a source of the fibre. For citrus fibre the bulk of the fibre is provided by the cell walls of the pulp structure. Suitable citrus sources include lemon, lime, orange and grapefruit products and blends of two or more thereof.

Citrus fibre is particularly preferred because it can be provided in the desired amounts without providing an unpleasant effect on the flavour and without leading to a sticky or creamy mouthfeel. Consumers are familiar with the sensation of beverages containing citrus pulp. It is noted that some other fibre sources can be associated with an astringency and/or gelling of the beverage.

In one embodiment, fibre can be obtained from a whole fruit, part of fruit and/or pomace of fruit. Part of fruit may be flesh, pulp, skin, peel, flavedo and/or albedo. Preferably, fibre can be obtained from the pulp of the fruit.

In one embodiment, a beverage composition can contain a commercially available fibre. For example, Unipectin-100, Unipectin-300 and Unipectin-900 produced by Cargill Incorporated, and^' Citrus Pectin produced^ by Doehler can be used* for a beverage composition according to one embodiment of the present invention.

Calorie

Preferably the beverage composition comprises less than lOOKcal. As noted above, an orange juice typically comprises around 0.2g/100ml of fibre. However, this is also associated with approximately 8g of sugar per 100ml and about 45kcal per 100ml. Thus, the provision of at least 1g of fibre while having less than lOOkcal is quite distinct from any typical fruit juice beverage.

Alternatively, the beverage composition may comprise lOOKcal or less. In a preferred embodiment, the beverage composition comprises less than 95Kcal, less than 90Kcal, less than 85Kcal, less than 80Kcal, less than 75Kcal, less than 70Kcal, less than 65Kcal, less than 60Kcal, less than 55Kcal, less than 50Kcal, less than 45Kcal or less than 40Kcal. In a preferred embodiment, the beverage composition comprises less than 100Kcal/100ml, less than 95Kcal/100ml, less than 90Kcal/100ml, less than 85Kcal/100ml, less than 80Kcal/100ml, less than 75Kcal/100ml, less than 70Kcal/100ml, less than 65Kcal/100ml, less than 60Kcal/100ml, less than 55Kcal/100ml, less than 50Kcal/100ml, less than 45Kcal/100ml or less than 40Kcal/100ml. In other embodiment, the beverage composition comprises 10 to 100 Kcal/100ml, 15 to 95 Kcal/100ml, 20 to 90 Kcal/100ml, 25 to 85 Kcal/IOOml, 30 to 80 Kcal/IOOml, 35 to 75 Kcal/100ml, 40 to 70 Kcal/100ml, 5 to 50 Kcal/100ml, 6 to 45 Kcal/100ml, 7 to 40 Kcal/100ml, 8 to 35 Kcal/100ml, 9 to 30 Kcal/100ml, or 10 to 25 Kcal/100ml.

Preferably the beverage obtained from the beverage composition and, in particular when the beverage composition is a ready-to-drink composition, comprises less than 5g/100ml of sugar. The provision of sugar in the beverage assists with the reduced postprandial glycaemia benefits observed. However, it is preferred that the sugar is generally provided by an accompanying meal or snack. Accordingly, it is desirable that the beverage composition has a low sugar content.

Preferably the beverage composition consists essentially of the fibre (as provided as a citrus fibre extract), the polyphenols (as provided as a concentrated fruit extract), together with any added sweetener (including sugar) and flavourings (and water when provided in concentrate and ready-to-drink formats). Other conventional ingredients such as stabilisers and pH regulators may be presents in small amounts, as required.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 200ml. That is, the recommended serving size is 200ml. At doses with a smaller size the level of fibre can lead to an overly pulpy beverage.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 250ml. That is, the recommended serving size is 250ml.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 300ml. That is, the recommended serving size is 300ml.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 330ml. That is, the recommended serving size is 330ml. A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 500ml. That is, the recommended serving size is 500ml.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 750ml. That is, the recommended serving size is 750ml.

A most preferred beverage composition is a ready to drink beverage composition comprising at least 600mg polyphenols and at least 1g of fibre, such as 800 to 1200mg of polyphenols and 1 to 2g of fibre, and preferably having less than 45Kcal, provided as a dose having a volume of about 1000ml. That is, the recommended serving size is 1000ml. At large dose sizes the fibre may be harder to keep suspended and the dose itself may be larger than most consumers wish to consume.

A most preferred beverage composition is a ready to drink beverage composition comprising 600 to 6,000ppm of polyphenols and^; 1.0 to 10 g/L of fibre, preferably 1 ,200 to 4,000ppm of polyphenols or 800 to 1 ,200ppm of polyphenols and; 1.3 to 10 g/L of fibre, and preferably having 1 to 50 Kcal/100ml, preferably 1 to 30 Kcal/100ml. A serving size may be 50ml to 1 ,000ml, preferably 100ml to 800ml, more preferably 200ml to 500ml or 600ml to 750ml. A serving may contain at least 600 mg polyphenols, preferably700 to 2000mg polyphenols, more preferably 800 to 1200 mg polyphenols. More than one serving may be contained in one container. For example, when a serving size of the beverage composition is 200ml and one container contains 1 ,000ml of the beverage composition, five servings can be provided by the container. Alternatively, a serving size may be constituted by more than one containers. For example, when a serving size is 200ml, four 50ml bottles or two 100ml bottles may constitute the serving size. Generally, a consumer would expect to get the benefits of a beverage composition from one whole bottle of the drink, but sometimes the serving size (or portion size) required to get the benefit is different from the pack size. When a serving size is different from a pack size, then the serve size may be indicated, for example, on the label, in promotional material, or in accompanying information leaflets.

The present inventors have now discovered that the provision of a synergistic mixture of polyphenols and fibre can have a number of beneficial health effects as described herein. In particular, without wishing to be bound by theory, it is considered that the fibre enhances a feeling of fullness, while the polyphenols serve to decrease the rate of sugar uptake. In combination, these provide a delayed release of energy to the consumer. It is particularly surprising that these benefits can be achieved in the form of a beverage composition, since the level of solids (i.e. non-water components) achievable in such a beverage are so tightly constrained. That is, there is a limit to the amount of fibre and other additives that can be introduced into a beverage before it becomes distinctly unpalatable due to, for example, thickening. For example, beverages containing a large amount of fibre tend to become extremely thick almost like a solid, or have a gel-like, lumpy or pasty texture. Nonetheless, despite these constraints, the present inventors have been able to produce a desirable beverage having observable benefits.

The inventors investigated the inclusion of fibre alone to provide slow release energy, but the fibre was difficult to formulate at the required doses due to the thickness of the formulation and the associated cost. The inventors separately investigated the use of polyphenols to slow down the glucose entering the body. However, polyphenols were too expensive and tasted bitter at doses which affected glycaemia. In providing a blend of the two, the inventors were able to provide an effective composition which overcame these disadvantages associated with the individual components.

The inventors have provided a low calorie beverage with a violet purple colour. The pulp provides a full mouthfeel and the beverage is not too sweet, slightly acidic and has a pleasant fruity flavour. Advantageously, the beverage is obtained entirely from natural fruit ingredients.

The inclusion of large doses of polyphenols is expensive and leads to bitter formulations. Surprisingly, the inventors have found that effective doses of polyphenols do not need to be unduly large, particularly when based on anthocyanin-rich fruits. Indeed, a smaller dose of around 800mg was found to be as effective as a larger dose of about 1600mg.

The inventors looked at different doses of blackcurrant and sweet orange polyphenols. The significant effect of 800mg of blackcurrant was strongly supported by plasma GIP secretion, which is tightly coupled to the rate of glucose absorption in the intestine. The differences in early postprandial glucose concentrations had minor effects on insulin secretion.

Studies were conducted on soluble and insoluble dietary fibre from lemon and orange pomace sourced from Spain. This was to compare their characteristics and infer their biofunctionality. The object was to identify potent fractions of fibre to use and thereby reduce the quantity of fibre required. Lemon fibre was found to retain a suitable viscosity at concentrations higher than that of other considered fibre sources. Studies were then conducted on blackcurrant and pulp. It was found that BC and pulp, but not pulp only, produced a small, but statistically significant inhibitory effect in the rise in glucose, insulin, c-peptide and GIP in the first 30 mins postprandially, providing further support for previous findings suggesting that BC PP may inhibit carbohydrate digestion and intestinal glucose absorption. Reaction time and cognitive performance/working memory were improved by BC and pulp but not pulp, suggesting that BC PP may improve cognitive function. There were no differences in subjective scores of appetite nor food intake following the drinks indicating that BC + pulp is unlikely to modify energy intake at the next eating event. A lower dose of BC PP, equivalent to approximately 100 g fresh fruit, slows the rate of early postprandial glycaemia in healthy adults indicating slower intestinal absorption.

According to another aspect the beverage composition as described herein is provided for use in improving cognitive function in a subject in need thereof. There is also provided a method of improving cognitive function by administering the beverage composition described herein to a subject. Improved cognition could include promoting a temporary improvement in cognitive function, maintaining normal cognitive function, and reducing the risk of cognitive impairment in either the short-term or the long-term, for example, minimizing the likelihood of age-related decline in mental function, for example, a temporary improvement in a healthy subject, a maintenance of normal cognition in a healthy subject, an acute or chronic improvement in a subject with an existing cognitive impairment, or a reduction of risk of the development of abnormal cognitive function.

In one embodiment, use of the beverage composition according to the present invention in a non-therapeutic method of improving cognitive function in a subject is provided. As used herein, "a non-therapeutic method of improving cognitive function in a subject" includes promoting a temporary improvement in cognitive function, maintaining normal cognitive function, and reducing the risk of cognitive impairment in either the short-term or the long- term, for example, minimizing the likelihood of age-related decline in mental function, for example, a temporary improvement in a healthy subject, a maintenance of normal cognition in a healthy subject, maintaining or increasing alertness/feelings of wakefulness/mental performance in a healthy subject, and decreasing reaction time and feelings of sleepiness in a healthy subject. In another embodiment, a beverage composition for use in a therapeutic method of improving cognitive function in a subject in need thereof is provided. As used herein, "a therapeutic method of improving cognitive function in a subject in need thereof' includes an acute or chronic improvement in a subject with an existing cognitive impairment, a reduction of risk of the development of abnormal cognitive function, a reduction of risk of the development of Alzheimers, Dementia, mild cognitive impairment and mild cognitive decline and age-related cognitive decline. According to another aspect the beverage composition as described herein is provided for use in a method of managing and/or treating type II diabetes, including managing the symptoms of type II diabetes. Furthermore, the beverage composition is provided for the use in a method of reducing the risk of developing type II diabetes. There is also provided a method of managing and/or treating type II diabetes by administering the beverage composition described herein to a subject. Long term reduction of poor postprandial glycaemic control is associated with reduced risk of developing diabetes.

According to another aspect the beverage composition as described herein is provided for use in a method of reducing the risk of cardiovascular disease (CVD). There is also provided a method of reducing the risk of cardiovascular disease (CVD) by administering the beverage composition described herein to a subject.

In one embodiment, use of the beverage composition according to the present invention in a non-therapeutic method of maintaining a healthy heart and circulation in a subject is provided. In another embodiment, a beverage composition for treating, preventing or curing cardiovascular disease in a subject is provided.

According to another aspect the beverage composition as described herein is provided for use in a method of reducing postprandial glycaemia. There is also provided a method^; of reducing postprandial glycaemia by administering the beverage composition described herein to a subject.

In particular, without wishing to be bound by theory, the inventors found that a combination of fibre and polyphenols reduced post prandial glycaemia by both slowing transit and reducing glucose uptake, leading to a slow release of glucose into the blood, consistent with a number of beneficial health outcomes including improving mental energy, and reducing the likelihood of over-release of insulin which can have the undesirable effect of reducing blood glucose below optimal.

In one embodiment, use of the beverage composition according to the present invention in a non-therapeutic method of managing available energy (calorie source) in the blood and consumer perceived energy (defined as feelings of mental and physical vigor and ability to perform physically and mentally) as required for daily life in a subject is provided.

According to another aspect the beverage composition as described herein is provided for use in a method of preventing or reducing the risk of low blood glucose resulting from the glycaemia induced insulin response. There is also provided a method of reducing preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin response by administering the beverage composition described herein to a subject.

According to another aspect the beverage composition as described herein is provided for use in a method of promoting weight-loss and/or improving Body Mass Index (BMI) in a subject in need thereof. There is also provided a method of promoting weight-loss by administering the beverage composition described herein to a subject.

In one embodiment, use of the beverage composition according to the present invention in a non-therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject is provided. As used herein, "a non-therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject" includes smoothing of the blood glucose curve, sustained provision of energy into the blood, sustained feelings of energy, avoiding peaks and troughs in energy and mood, maintaining normal weight, and weight loss for minor aesthetic concerns that have not required GP intervention. In another embodiment, a beverage composition for use in a therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject in need thereof is provided. As used herein, "a therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject in need thereof' includes treatment of obesity, prevention of diseases relating to obesity, administration to a subject who has been required/advised by a medical practitioner to protect health or if the user is diagnosed by a medical practitioner as overweight or clinically obese.

For the above described uses, the use is in particular for a subject when consuming their standard diet. In these instances, the composition helps to limit a rise in blood-glucose levels after consumption of a meal or snack, particularly a carbohydrate rich meal, such as to less than 50% of a normal response (or of a subject not receiving the beverage composition), more preferably to less than 30% of a normal response.

According to another aspect there is provided method of forming a beverage composition as described herein, the method comprising mixing a polyphenol-rich fruit extract with a natural fibre ingredient. The sources of the extract and ingredient are discussed herein.

According to another aspect there is provided a beverage composition for being consumed with or before intake of carbohydrate comprising at least 600mg polyphenols, and at least 1g of fibre. There is also provided a method for administrating the beverage composition described herein to a subject with or before the subject's intake of carbohydrate. Since the beverage composition is consumed or administered before the intake of carbohydrate, the postprandial glycaemia may be reduced. In one embodiment, carbohydrate may be carbohydrate containing food, such as bread, cereal, rice, pasta, potato and sweets. In one embodiment, the beverage composition may be administered to a subject before the subject consumes carbohydrate or may be administered to a subject with carbohydrate. The beverage composition may be administered to a subject immediately before, 15 minutes, or 30 minutes before the subject consumes carbohydrate, these times are based on the length of time normally taken for liquids to pass through the digestive system.

According to another aspect the beverage composition as described herein is provided as a meal or snack replacement. As used herein, a meal or snack replacement means a substitute for a solid food meal or snack and is intended to provide the nutrition (both macro and micro-nutrient profiles) of a typical full meal /snack respectively.

According to another aspect the beverage composition as described herein is provided for improving cognitive function, for managing and/or treating type II diabetes, for reducing postprandial glycaemia, for promoting weight-loss and/or improving Body Mass Index, for reducing the risk of cardiovascular disease, and/or for preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin response. In one embodiment, polyphenols and fibre are contained as active ingredients. In one embodiment, the beverage composition as described herein may be used for subjects with a high blood glucose level, subjects who concern or start to concern blood» glucoseTevel, subjects who concern body fat, slightly obese subjects, subjects with a tendency of metabolic syndrome, or the like.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described herein, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about," it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Brief Description of Drawings

The invention will now be described in relation to the following non-limiting figures, in which Figures 1- 20 relate to Study 1.

Figure 1 shows the assessment of subjects in Study 1 discussed below.

Figure 2 shows Mean Glucose over Time by Treatment Intent to Treat Population

Figure 3 shows Mean Insulin over Time by Treatment Intent to Treat Population

Figure 4 shows Mean C-Peptide over Time by Treatment Intent to Treat Population

Figure 5 shows Mean GIP Over Time by Treatment Intent to Treat Population.

Figures 6 to 11 show Mean Ratings of Palatability in the Intent to Treat Population.

Figure 6 asked how much did you enjoy the beverage? (0 = Not at all to 100 = Extremely enjoyable).

Figure 7 asked how hard was it to consume the given amount of beverage? (0 = Not hard at all to 100 = Extremely hard). Figure 8 asked how did you find the taste of the beverage? (0 = Very bad to 100 = Very good). Figure 9 asked how bitter did you find the beverage? (0 = Not bitter at all to 100 =

Extremely bitter).

Figure 10 asked how did you find the texture of the beverage? (0 = Unpleasant to 100 = Pleasant).

Figure 11 asked did you experience any aftertaste? (0 = None to 100 = Strong).

Figures 12 to 20 show Mean Ratings of Mood and Satiety Over Time by Treatment in the Intent to Treat Population.

Figure 12 asked how happy do you feel? (0 = Not happy at all to 100 = Extremely happy). In this figure at 60 minutes the placebo is the lowest line and the fruit pulp is the highest. Figure 13 asked how alert do you feel (0 = Not alert at all to 100 —Extremely alert).

In this figure at 10 minutes the placebo is the middle line and> the fruit pulp is the lowest.

Figure 14 asked how hungry to do you feel? (0 = Not hungry at all to 100 = Extremely hungry). In this figure at 30 minutes the placebo is the lowest line and the fruit pulp is the middle line.

Figure 15 asked how full do you feel? (0 = Not full at all to 100 = Extremely full). In this figure at 30 minutes the placebo is the middle line and the fruit pulp is the highest.

Figure 16 asked how energised do you feel? (0 = Not at all to 100 = Extremely). In this figure at 60 minutes the placebo is the lowest line and the fruit pulp is the highest.

Figure 17 asked how would you rate your digestive comfort? (0 = Very uncomfortable to 100 = Very comfortable). In this figure at 60 minutes the placebo and fruit pulp are the highest and the mixture is the lowest. Figure 18 asked how thirsty do you feel? (0 = Not at all to 100 = Extremely). In this figure at 10 minutes the placebo is the lowest line and the fruit pulp is the middle.

Figure 19 asked how much do you think you can eat? (0 = Nothing at all to 100 = A lot). In this figure at 30 minutes the placebo is the lowest line and the fruit pulp is the middle.

Figure 20 asked how strong is your desire to eat? (0 = Not at all to 100 = Very strong). In this figure at 60 minutes the placebo is the lowest line and the fruit pulp is the highest.

In the above figures, error bars represent standard error.

Examples

The invention will now be described in relation to the following non-limiting examples. Study 1

A study was conducted to confirm the effects of the inventive composition on trial participants. In the following study the following abbreviations have been used:

Postprandial glycaemia refers to the transient rise in blood glucose levels that occurs after consuming a meal. Large fluctuations in blood glucose levels, experienced on a frequent basis, may impair the functioning of pancreatic beta cells, and thus elevate the risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (Blaak et al., 2012).

It has previously been shown that consuming a drink containing fruit polyphenols immediately before a meal, may reduce, or slow the rate of, the rise in blood glucose. Specifically, the consumption of drinks that contained blackcurrant and/or apple polyphenol extracts favourably modified postprandial carbohydrate absorption (Acosta et al., 2016).

Fruit polyphenols slow glucose absorption by inhibiting digestive enzyme activity, reducing sodium- glucose linked transporter 1 (SGLT-1) / glucose transporter 2 (GLUT-2) glucose transporter activity (Williamson, 2013), or potentially by binding to starch molecules (Shen et al., 2012). Importantly, other fruit components, namely soluble fibres, may also impact on carbohydrate digestion by slowing gastric emptying rates (Di Lorenzo et al., 1988), interacting with the intestinal mucus layer, and affecting starch hydrolysis by amylase (Grundy, 2016). It is not yet known whether the addition of both polyphenols and fibre to a drink has additive or synergistic effects on reducing postprandial glycaemia. Although limited, there is a growing body of evidence showing: beneficial acute effects of polyphenols in cognitive function which is of great interest in many work and academic environments where fast cognitive enhancement is wanted to perform a task or an exam (Bell et al., 2015).

The aim of this study was to investigate the effects of fruit polyphenol extracts combined with pulp (source of fibre), on postprandial outcomes following a mixed carbohydrate (starch and sucrose) test meal. The primary endpoint was glycaemic response, and additional secondary endpoints included cognitive performance testing, postprandial plasma insulin, glucose-dependent insulinotropic peptide (GIP) - a gut hormone and incretin that is tightly coupled to the rate of glucose absorption - and C-peptide (which is co-secreted with insulin following a meal and is a more specific indicator of the rate of insulin secretion). Perceived satiety, subjective mood feelings and ad libitum energy intake were also assessed. The study utilised a randomised, double-blind, cross-over design. Each subject consumed a placebo drink (no polyphenols or fibre), a pulp only drink (1.5 g fibre), and a BC extract combined with pulp drink (800 mg total polyphenols and 1.5 g fibre), at three separate study visits. Postprandial changes in plasma glucose, insulin, GIP and C- peptide concentrations were measured. It was hypothesised that intake of the BC and pulp drink reduces postprandial glycaemia compared to placebo, and the drink containing pulp alone was used to benchmark the effect of pectin fibre. 2 Study Objectives

2.1 Primary Objective

The primary endpoint is iAUC_{0-30 min} for plasma glucose, which is defined as the change in area under the curve from baseline plasma glucose concentration (0 mins) to the 30 minute sample.

2.2 Secondary Objectives

1. iAUC_{0-150 min} for plasma glucose concentration.

2. iAUC_{0-30 min} and iAUC_{0-150 min}for plasma insulin, C-peptide and GIP concentration.

3. Incremental C_MAX (iC_MAX) for plasma glucose, insulin, C-peptide and GIP concentrations, defined as the maximum change from baseline over 150-min sampling period.

4. T_MAX for plasma glucose, insulin, C-peptide and GIP, defined as the time to maximum concentration.

5. Plasma Concentration of glucose, insulin, C-peptide and GIP at each time point (T10, 20, 30, 45, 60, 75, 90, 120 and 150 min).

6. Responses to the VAS payability questions at T10 and T230 mins, and the mood and satiety questions at T-10, 10, 30, 60, 90, 120 and 150 mins, will be summarised using descriptive statistics.

7. Ad libitum energy intake at T215 min.

8. Cognitive testing scores at baseline (T -45) and endpoint (T 165). The Computerised Mental Performance Assessment System (COMPASS, Northumbria University, Newcastle Upon Tyne, UK), a software platform, was used to deliver the· cognitive tasks, This assessment has been described in Jackson 2012 paper. These were presented in the same order as outlined below with the cognitive domain described i brackets. Alt reaction times were measured in milliseconds and; accuracy was measured as a percentage of correct responses. a) Choice Reaction Time (attention/response inhibition). An arrow appeared on the screen pointing to the left or right. This task required that participants respond with pressing the ‘Z’ (left) or ‘M’ (right) key corresponding to the direction of the arrow. There was a randomly varying inter-stimulus interval of between 1 and 3 s for a total of fifty stimuli. Accuracy and mean reaction time were recorded. b) Rapid visual information processing (RVIP, cognitive performance/working memory): required that participants respond by pressing the ‘space bar’ on the laptop keyboard every time they detected three consecutive odd or even numbers in a sequence of rapidly (100 per min) presented single digits (1 to 9). Accuracy (percentage of target strings correctly detected), mean reaction time were for correct responses and number of false alarms were recorded. c) Simple Reaction Time (psychomotor speed/attention): required the participants to press the ‘space bar’ on the laptop keyboard as quickly as possible every time an arrow pointing upwards appeared on the screen. Stimuli were presented with an inter-stimulus duration that varied randomly between 1 and 3.5 seconds. Mean reaction time was recorded. d) Stroop Task (attention/response inhibition): a computerised version of the Stroop Task (Stroop 1992). A series of words describing colours (GREEN, BLUE, RED, YELLOW) were 5 randomly presented on at a time in different coloured text (e.g. RED was presented in yellow text, etc.)· Fifty stimuli were presented, where participants were required to click the colour box located on the right side of the screen that matched the colour of the text the word was presented in. Accuracy, mean reaction time and number of false alarms were recorded. e) Four-Choice Reaction Time (attention). A visual representation of the four direction arrow keys of a standard keyboard was presented on the screen. The arrows 'lit up’ at random on the screen and participants were requested to press the corresponding arrow key. In all, each arrow was the target stimulus twelve times, with a total of forty-eight stimuli. Accuracy and mean reaction time were recorded. f) Serial 3 subtractions (cognitive performance/working memory): participants were requested to subtract 3 consecutively from an original randomly generated number between 800 and 999 for the duration of the task (4 min), as quickly and accurately possible. Responses were cleared once the key ‘Enter’ was pressed and participants were only shown the first number on the screen after which they had to keep subtracting from memory. In the case of incorrect responses, subsequent responses are scored positively if they are correct in relation to the new number. Total and incorrect responses and errors were recorded.

3 Investigational Plan

3.1 Overall Study Design and Plan Description

A randomised, placebo-controlled, double-blind, cross-over study was conducted. Over three separate visits, all subjects will consume three drinks; the placebo, the pulp only and the BC + pulp drink. The BC + pulp drink contains a combination of BC extract providing 800 mg of total polyphenols (TP) and fruit pulp providing 15 g fibre. Subjects will be randomly allocated to the different treatment arms.

The minimum wash out period was a minimum of 4 days. Where possible, study visits were separated by no more than 21 days. In order to minimise the influences of cyclical reproductive hormones in female subjects, the female subjects only attended study visits during the middle two weeks of the menstrual cycle (weeks 2 and 3) and not the first (week 1) or the last week (week 4).

Standardised diet and exercise guidance were given prior to the visit (Source document). Subjects arrived on each study visit between 08.00 and 10.00 h, after a 12 h overnight fast, and having followed a low polyphenol/ low fibre diet for 48 hours, a low-fat diet for 24 hours, and having avoided caffeine and decaffeinated tea and coffee from noon the previous day. Subjects performed a computer-based cognitive battery test (approximately 30 mins) and were then cannulated in a forearm vein and two baseline fasting blood samples were taken (T-10 and -5 min). Following consumption of the test drink (T0 min), the high carbohydrate meal (starch and sucrose) was served (white bread with apricot jam). Postprandial blood samples were collected at T10, 20, 30, 45, 60, 75, 90, 120 and 150 min for plasma glucose analysis, serum insulin, c-peptide and GIP. The cannula was then removed after the T150 min, and the subject performed another 30-min cognitive battery test, followed by an ad libitum pasta meal (T215 min). Visual analogue scales (VAS) questionnaires on study meal palatability, satiety and other subjective feelings were completed at T-10, 10, 30, 60, 90, 120, 150 and 230 min. 3.2 Rationale of Study Design, including the Choice of Control Groups

A randomised, placebo-controlled, double-blind, study design is considered the gold standard for a human trial. The present study consisted of a full cross-over design. Early postprandial glucose concentrations (iAUC_0-30min) were chosen as the primary outcome as BC extract was expected to slow down the rate of glucose absorption. A 2.5-hour postprandial period was chosen to allow observation of the most relevant changes in postprandial glycaemia and insulinaemia and subsequent changes in C-peptide and GIP following a high-carbohydrate meal. A starch/sucrose test meal of white bread and apricot jam was administered, in line with previous similar studies conducted at KCL allowing cross-study comparisons.

3.3 Selection of Study Population

The study population comprised of healthy men and women aged 18 to 70 y from the general adult population. Subjects were recruited using advertisements (posters among KCL, internal email circulars, social media [Facebook, Twitter] and external advertising including newspaper advertisement) (Source document). Subjects that met the inclusion criteria following the pre- screening telephone questionnaire were invited to attend a screening visit that took place at the Metabolic Research Unit (MRU), Franklin-Wilkins Building, King's College London, at least 1 week before the first study visit. At this visit, informed written consent was given after which blood pressure and anthropometric data (height, weight, waist and hip circumferences, and body fat percentage) were measured, and a fasted blood sample was taken· and assessed for exclusion criteria. Subjects also had a practice run of the cognitive battery test. Subjects accepted onto the study according to the inclusion and exclusion criteria (see sections 3.3.1 and 3.3.2) completed: a 7-day food diary (Source document) where the. habitual: dietary intake was recorded:

3.3.1 Inclusion criteria

1. Consent

Demonstrated understanding of the study and willingness to participate as evidenced by voluntary written informed consent and has received a signed and dated copy of the informed consent form.

2. Age

Aged over 18 and under 70 years at date of enrolment.

3. Compliance

Understood and was willing, able and likely to comply with all study procedures and restrictions. a. The subject was willing to adhere to the Dietary Guidelines for Participants. b. The subject was willing to avoid strenuous exercise for 24h before each study visit. c. The subject was willing to avoid caffeine from midday the day before each study visit. d. For female subjects only: The subject provided an approximate date for the start of their menstrual cycle.

4. General Health

Good general and mental health with, in the opinion of the investigator a) No clinically significant and relevant abnormalities of medical history or physical examination. b) Absence of any condition that would impact on the subject’s safety or wellbeing or affect the individual’s ability to understand and follow study procedures and requirements.

3.3.2 Exclusion criteria

1. Intolerance / allergy / hypersensitivity

Those with a known or suspected intolerance or hypersensitivity to the study materials (or closely related compounds) or any other stated ingredient.

2. Blood donation a) Those who have donated blood within 3 months of the date of the screening visit. b) Those for whom participation in this study would result in having donated more than 1500 millilitres of blood in the previous 12 months.

3. Participation in Clinical trials a) Simultaneous participation in another clinical trial b) Previous participation in this trial

4. Smoking a) Those who were current smokers,, or used an electronic cigarette b) Reported giving up smoking within the last 6 months.

5. Weight Loss

Those who have experienced weight loss of over 3kg (7 lbs) in the preceding 2 months.

6. Alcohol consumption

Those who consumed over 28 Units/week (men) or over 21 Units/Week (women) of alcohol.

7. Concomitant Medication

Those who were taking medications that could interfere with the study such as alpha-glucosidase inhibitors (acarbose: Glucobay), insulin-sensitising drugs (metformin: Glucophage, Glucophage SR, Eucreas, Janume; thiazolidinediones: Actos, Competact), sulfonylureas (Daonil, Diamicron MR, Glibenese, Minodiab, Amaryl Tolbutamide), and lipid-lowering drugs (statins, nicotinic acid, colestyramine anhydrous, ezetimibe, fibrates). Other medications were reviewed by a medical representative from KCL on a case by case basis.

8. Nutritional Supplements

Nutritional supplements that could interfere with the study such as higher dose vitamins/minerals (>200% RNI), B vitamins, Vitamin C, calcium, copper, chromium, iodine, iron, magnesium, manganese, phosphorus, potassium and zinc. Subjects already taking vitamin or minerals at a dose around 100% or less up to 200% of the RNI, or evening primrose/algal/fish oil supplements were asked to maintain habitual intake patterns, ensuring that they take them every day and not sporadically. They were advised not to stop taking supplements or start taking new supplements during the course of the study. 9. Medical History

Those with a medical history of; a) Phenylketonuria b) Heart attack (myocardial infarction) or stroke c) Cardiovascular problems/angina/thrombosis d) Cancer within the last 5 years e) Diabetes (Type I or Type II) f) Stomach or inflammatory bowel disease g) Kidney problems h) Liver disease, adult jaundice or anaemia i) History of drug and/or alcohol addiction (>60 units/wk)

10. Female health a) Pregnancy: Subjects known to be pregnant or intending to become pregnant over the duration of the study b) Breast-feeding: Women who were breast-feeding or lactating at the time

Exclusion criteria assessed after taking measurements

• Those with an average blood pressure reading of ≥160/100 mmHg

• Those with body mass index < 18 or > 35 kg/m²

• Those from whom a fasted venous blood sample could: not be taken

• Those with Full Blood Counts and Liver Function test results outside of the normal range.

• Those with total cholesterol ≥ 7.5 mmol/L; fasting triacylglycerol concentrations ≥ 5.0 mmol/L

3.3.3 Subject restrictions 3.3.3.1 Lifestyle

Screening: Subjects were advised to avoid eating or drinking anything, except water, from after 8 pm the previous night.

Study: Subjects were asked to modify their diets to avoid high-fibre and high-polyphenol foods for 48 hours before their study visits. They were also asked to avoid fatty foods, oily fish and drinking alcohol, and refrain from taking part in any strenuous exercise for 24 hours before their study visits, and abstain from caffeine intake from noon the day before the visit. Subjects were provided with dietary guidelines to follow before their visits (Source document). Subjects were also requested to avoid eating or drinking anything, except water, from after 8.00 pm the day before each visit. On study visits, subjects were asked to stay seated, apart from walking the short distance between clinical rooms in the Metabolic Research Unit, for the duration of the visit (approximately 3 h). 3.3.3.2 Medications and treatments

Subjects were not allowed to take any of the medications prohibited in the exclusion criteria throughout the course of the study. If a subject was prescribed any such medications during the course of the study, they would be withdrawn from the study. 3.3.4 Removal of subjects from therapy or assessment

Subjects had the right to withdraw from the study at any time for any reason, and they were able to withdraw their data from the study up until the 31/03/2019.

The investigator had the right to withdraw subjects from the study in the event of intercurrent illness, adverse events (AEs) or product failure after a prescribed procedure, protocol deviations, administrative reasons or other reasons.

When a subject decided to withdraw, all efforts were made to complete and report the observations as thoroughly as possible. A complete final evaluation at the time of the subject’s withdrawal was made with an explanation of why the subject was withdrawing from the study.

3.4 Treatments 3.4.1 Treatments administered

Three different treatments, including a placebo, a fibre only, and a polyphenol and fibre containing drinks were supplied by the New Product Development team at LRS. The drinks were 200 ml and were taste and colour matched as far as possible. Table 2 shows the study drinks formulations Table 2. Study supplies formulations. 3.4.2 Identity of investigational product(s)

3.4.2.1 1dentity of study drinks

The BC polyphenol extract were supplied by BerryPharma, Iprona. The BC polyphenol extract contains Anthocyanins; expressed as Delphinidin-3-rutinoside (spectrophotometry by pH- Differential.) g / kg 20.0 - 28.0 and Polyphenols; expressed as Catechin (Folin Ciocalteaus) g / kg 25.0 - 55.0.

The fruit pulp was supplied by Citresa, Orangina Schweppes Suntory, Spain (Citricos Y Refrescantes)

3.4.2.1 1dentity of study breakfast

Subjects were given a mixed starch/sucrose meal containing approximately 100-g thick sliced white bread (Hovis, London, UK) with 32-g Hartley's smooth apricot jam (Hain Daniels Group, Leeds, UK), both high carbohydrate foods which are low in phenolics (<6-mg/100-g fresh weight). These are typical meal serving sizes and were well-tolerated by subjects. The total amount of carbohydrate provided in the study meal and study drink was approximately 75 g. Importantly, this is the same amount of carbohydrate used’ in 2 h oral glucose tolerance tests (75 g). The jam was refrigerated and labelled with date opened and Glu-MIX study label and was used within 6 weeks of opening. The bread was bought and frozen on day of purchase to prevent.mould growth onless busy weeks and used within 3 months. The bread was defrosted in time for breakfast. The bread packaging was labelled, with Glu-MIX study and date; Clean utensils and; sanitised· work surfaces; were used when preparing the test meal. Bread was weighed on a plate and then jam was spread evenly over the bread.

3.4.3 Treatment assignment

The randomisation schedule was generated according to a Williams square, for a 3 by 3 crossover study. Within the randomisation list, each subject is assigned placebo in one of the study periods, pus the pulp only and test (BC + pulp) drinks in the other study periods. Furthermore, each treatment will be followed by every other treatment an equal number of times.

3.4.4 Selection of doses in the study

The total PP dose of 800 mg has been selected based on the previous study results (HVS-007, GLU-FX) where 800 mg TP dose of BC was found to significantly reduce postprandial iAUC blood glucose in the first 30 min. A fibre concentration of 0.75 g / 100 ml was chosen as this exceeds the dose needed to fulfil the nutrition claim of ‘rich in fibre’ (3 g fibre/ 100 kcal). The pectin in the drinks will be from pulp.

3.4.5 Selection and timing of dose for each subject

All subjects received the three drinks as per the double-blind randomisation schedule. One drink was administered per study visit. Study visits were separated by minimum of 4 days, and where possible no more than 21 days, which was considered a sufficient wash out period for an acute design study.

The study drinks were provided in opaque brown glass bottles, with a black straw, to mask slight differences between treatment drinks for colour and flavour, respectively. The study drinks were stored frozen and were removed from the freezer and placed in a fridge, allowing 48 h to defrost, before the study visit. The study drinks were removed from the fridge approximately 10 min before provision to the subject. The researcher vortexed the beverage for approximately 1 min to ensure that the drink was fully dispersed. Immediately before providing the drink to the subject, the researcher further inverted the drink bottle 10 times. The drink was also shaken thoroughly by the researcher immediately before giving the drink to the subject. The lid to the bottle was removed and a black straw inserted into the bottle. Subjects were given up to 2 minutes to consume the test drink. Subjects were then given 7 minutes to consume the mixed carbohydrate breakfast. Subjects were asked to refrain from tampering with the study drinks bottles in any way.

3.4.6 Blinding The study was double blinded where only independent consultants were privy to the randomization schedule and treatment allocation. This blind remained in place throughout the study and it was not broken at study completion and analysis.

3.4.7 Treatment compliance

The researcher checked that the entire volume of beverage for each study test drink provided was consumed in the allocated time (2 min). Empty bottles were kept on site and the study monitor checked these at the end of the study to check for compliance.

3.5 Demographic, Efficacy and Safety Variables and Schedule of Study Events

3.5.1 Schedule of study events

A schedule of study events is presented in the following table. Details of the individual variables recorded are discussed in Sections 3.5.2 to 3.5.5 inclusive.

N cn>

3.5.2 Demographic and patient characteristics

All demographic and subject’s characteristics were entered in the CRF. The researcher recorded the following subject’s characteristics: year of birth, month of birth, age, gender and ethnic group. The following procedures were completed: height using a stadiometer; waist and hip circumference using a tape measure; body composition and weight by bio-electrical impedance using a TANITA BC-418 segmental body composition analyser; blood pressure using an automated blood pressure monitor (A&D Medical). Data was collected on subjects’ relevant medical or surgical history, including allergies or food intolerances. Habitual nutritional intake data was obtained from 7-day estimated food diaries. Buccal cells were collected at the screening visit using a buccal collection kit (Isohelix). A screening fasting venous blood sample was obtained to analyse glucose, full blood count, liver function test and full lipid profile.

3.5.3 Efficacy measurements and evaluations

The study would be considered a success if the primary efficacy endpoint was found to be significantly different for the pulp and BC drink compared to placebo. Thus, a significantly reduced peak postprandial glycaemia concentration (iAUC0-30min) for test treatment (BC and pulp) compared to placebo. The pulp only drink was used to benchmark the effect of fibre alone.

3.5.4 Safety measurements and evaluations

AEs were regarded as treatment emergent when they occurred after the first administration of one of the randomised treatments. All AEs with an onset date during the washout period between study periods were assigned to the treatment received in the previous period. All AEs that started after the last administration of treatment was assigned to the treatment taken in Period 3 or the last treatment period preceding· the end of the study. AEs with an onset date/time prior to the date/time of treatment administration in Period 1 were considered as non-treatment emergent.

If the intensity of a continuous AE worsened from one treatment period / baseline period to the following treatment period, then the AE was considered as emergent in the period in which it worsened.

3.5.4.1 Adverse events

An adverse event (AE) was any untoward medical occurrence in a subject following administration of an investigational product, which did not necessarily have a causal relationship with this treatment.

AEs were documented throughout the study. The investigator asked subjects the following question during each visit including any follow-up visits: “Have you felt unwell, experienced any symptoms or taken any medication (since your last visit) (today) (since your last dose) (since the last session).” AEs were recorded in the CRF by diagnosis and not by symptoms when possible (e.g., cold, seasonal allergies, etc. instead of runny nose). The investigator recorded the intensity of the AE and the relationship to the study treatment in the CRF. The gradings for intensity and relationship were as follows • Intensity:

- Mild - easily tolerated, causing minimal discomfort and not interfering with normal everyday activities.

- Moderate - sufficiently discomforting to interfere with normal everyday activities. - Severe - incapacitating and/or prevents normal everyday activities.

• Relationship to study treatment:

- Not related - The event was clearly related to other factors such as the subject’s clinical state, therapeutic interventions, or concomitant medications administered to the subject.

- Unlikely - The event was most likely produced by other factors such as the subject’s clinical state, therapeutic interventions, or concomitant medications administered to the subject; and did not follow a known response pattern to the trial intervention

- Possible - The event followed a reasonable temporal sequence from the time of drug administration; and/or followed a known response pattern to the trial drug; but could have been produced by other factors such as the subject’s clinical state, therapeutic interventions, or concomitant medications administered to the subject.

- Probable - The event followed a reasonable temporal sequence from the time of drug administration; and followed a known response pattern to the trial drug; and could not be reasonably explained by other factors such; as the subject’s clinical state, therapeutic interventions, or concomitant medications administered to the subject. - Highly Probable - The event followed a reasonable temporal sequence from the time of drug administration; and followed a known response pattern to the trial drug; and cannot be reasonably explained by other factors such as the subject’s clinical state, therapeutic interventions, or concomitant medications administered to the subject; and either occurred immediately following trial drug administration, or improved on stopping the drug, or reappeared on repeat exposure, or there was a positive reaction at the application site.

Additionally, AEs were categorized as serious or non-serious. A serious adverse event was any untoward medical occurrence that, at any dose a) Resulted in death. b) Was life-threatening. NOTE: The term 'life-threatening' in the definition of 'serious' refers to an event in which the subject was at risk of death at the time of the event. It does not refer to an event, which hypothetically might have caused death, if it were more severe. c) Required hospitalization or prolongation of existing hospitalization.

NOTE: In general, hospitalization signifies that the subject had been detained (usually involving at least an overnight stay) at the hospital or emergency ward for observation and/or treatment that would not have been appropriate in the physician’s office or out- patient setting. Complications that occurred during hospitalization were AEs. If a complication prolonged hospitalization or fulfilled any other serious criteria, the event was serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE was to be considered serious.

Hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline was not considered an AE. d) Resulted in disability/incapacity, or

NOTE: The term disability means a substantial disruption of a person’s ability to conduct normal life functions. This definition was not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhoea, influenza, and accidental trauma (e.g. sprained ankle) which may interfere or prevent everyday life functions but do not constitute a substantial disruption. e) Was a congenital anomaly/birth defect.

Medical or scientific judgement was exercised in deciding whether reporting was appropriate in other situations, such as important medical events that may not have been immediately life- threatening or resulted in death or hospitalization but ma have jeopardized the subject or may have required medical or surgical intervention to prevent one of the other outcomes listed in the above definition. These were also considered serious. Examples of such events are invasive or malignant cancers, intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization, or development of drug dependency or drug abuse.

3.5.4.2 Laboratory assessments

Abnormal results during the defined time period for AE reporting that the investigator considers clinically significant must be recorded as an AE or SAE. If the clinically significant abnormal lab was associated with a diagnosis, the diagnosis should be recorded on the CRF. 3.5.4.3 Vital Signs and other observations related to safety

Abnormal results during the defined time period for AE reporting that the investigator considers clinically significant must be recorded as an AE or SAE. If the clinically significant abnormal lab was associated with a diagnosis, the diagnosis should be recorded on the CRF.

3.5.6 Primary efficacy variable(s) iAUCo-30min 3.5.7 Drug concentration measurements

Not applicable (not a drug trial and none was administered)

3.5.8 Changes in the conduct of the study

No changes were made in the conduct of the study. 3.6 Data Quality Assurance

The study monitor reviewed the CRFs at the study site, in accordance with the monitoring plan, and collected the white copy for all the CRFs. Any queries were generated to the Investigator or designee enabling the errors to be addressed prior to review. The data manager then run the reports and listings on the CRF and raised queries for site clarification or correction. 3.7 Data Analysis Methods

3.7.1 Determination of sample size

Based on the previous study (HVS-007, GLU-FX), the plasma glucose iAUCO-30 mean response in the placebo and 800mg BC groups was 1.28 mmol/L and 1.05 mmol/L, respectively (i.e. a difference of 0.23 mmol/L). The within subject standard deviation was 0.29. To show a difference of 0.23 mmol/L or ~18% compared to placebo in the plasma glucose iAUCO-30, 36 subjects completing all 3 treatment periods will provide 90% power to detect differences between the pulp and BC polyphenol drink versus placebo at the two-sided 5% significance level.

In order to account for a ~5% attrition rate, 38 subjects were randomized into the study.

3.7.2 General considerations for data analysis 3.7.2.1 Interim analysis

No interim analysis was done in this study.

3.7.2.2 Handling of dropouts or missing data

Subjects who did not complete all four study periods; but had data from at least one of the study periods, were included in the analysis, where possible. This was achieved by including subject as a random effect in the analysis.

For the calculation of AUC, if one value is missing over the assessment period, the AUC simply used the values available. If there was more than 1 missing value or either the start or end values were missing, no AUC was calculated for that subject/period combination.

Blood plasma concentration values that were below the limit of quantification (LOQ) were set to ½ LOQ for statistical analysis. This is a widely used method that reduces the bias that would be associated with excluding LOQ values from analysis. The LOQ for each of the secondary metabolites was determined prior to sample analysis. 3.7.3 Study populations

The Safety population is defined as all subjects who are randomised and receive one of the study treatments. The Safety population was used to summarise treatment emergent adverse events.

The Intention to Treat (ITT) population is defined as all subjects who receive at least one of the study treatments and who have at least one post-baseline efficacy assessment. The ITT population was the primary population for the efficacy analysis.

The Per Protocol (PP) population is a subset of the ITT population. Subjects with a major protocol violation affecting the efficacy assessments were excluded from the PP population at the visit(s) affected by the violation. For the primary variable (Glucose iAUCo-30min), efficacy analysis on the PP population will be performed if there is more than a 10% difference in the number of subjects for the ITT and PP populations.

Descriptive statistics (number of subjects, mean, standard deviation, median, minimum and maximum for continuous variables, and frequency and percentage for categorical variables) are presented for demographic and baseline data. 3.7.4 Efficacy analysis and statistical methods

Statistical analysis was performed using SAS version 9.4.

For each of the analyses described below, the assumptions of Normality and homogeneity of variance was assessed, and where appropriate, data was transformed prior to analysis or a non- parametric analysis was performed (the Wilcoxon signed rank test). For all of the primary and secondary variables, in addition to the analyses described, the data is summarised using descriptive statistics and presented graphically. For parameters that are assessed at repeated time points, the data presentations include descriptive statistics over time, and line graphs of mean values over time.

For all variables measured at -10min and -5min, the baseline was calculated as the mean of the two values.

All efficacy variables were analysed under a null hypothesis of no difference between the test treatments and the placebo, against an alternative hypothesis of a difference between test treatments and placebo. For all variables measured at -10min and -5min, the baseline was calculated as the mean of the two values. 3.7.4.1 Primary efficacy parameters

The primary efficacy endpoint was iAUC_o-30min for plasma glucose, which is defined as the area under the change from baseline plasma glucose concentration versus time curve from zero to the T30 min sample time. The iAUC was calculated using the trapezoidal rule. In order to convert the results back to the original units of measurement (mmol/L), the iAUC was divided by the total duration (30 minutes). The iAUCo-30min was analysed using a linear mixed model. Terms in the model included treatment group and period as fixed effects, subject as a random effect, and subject-level and period-level baseline glucose as covariates.

The null and alternative hypotheses were: HO: There is no numerical difference in the comparison of all pairs (of all pairs including placebo) of treatments as measured by iAUCo-30min.

H1: There is a numerical difference between some pairs (of all pairs including placebo) of treatments as measured by iAUCo-30min.

From the above model, two-sided treatment comparison Dunnett’s tests were performed for all comparisons between placebo and test treatments. Treatment differences are presented with Dunnett’s adjusted 95% confidence intervals. The assumption of normality and homogeneity of variance was investigated. Violation of these assumptions were overcome by using suitable transformations or performing a non-parametric test.

3.7.4.2 Secondary efficacy parameters The secondary endpoints were:

• iAUCo-i50min for plasma glucose concentration.

• iAUCo-30min and iAUCo-isomin for plasma insulin, C-peptide (a more specific measure of insulin secretion) and GIP concentration.

• Incremental Cmax (iCmax) for glucose, insulin, C-peptide and GIP concentration, defined as the maximum change from baseline over 120-min sampling period.

• Tmax for plasma glucose, insulin, C-peptide and GIP concentration.

• Plasma concentration of glucose, insulin, C-peptide and GIP at each time point (T10, 20, 30, 45, 60, 75, 90, 120 and 150 mins).

• Responses to palatability questions at T10 mins and T230, using a 100mm VAS questionnaire. · Responses to mood and satiety questions at T-10, 10, 30, 60, 90, 120 and 150 mins, using a

100mm VAS questionnaire.

• Ad libitum energy intake, determined from the total amount of pasta consumed across the three servings at the end of each treatment period.

The iAUC, iCmax, and Tmax parameters were analysed using the same mixed models or non- parametric methodology as the primary endpoint. The concentrations of each parameter over time was summarised using descriptive statistics and plots.

The responses to the VAS questionnaires was summarised using descriptive statistics over time and by treatment group. Ad libitum energy intake and cognitive testing scores were analysed using the same linear mixed model or non-parametric methodology as the primary endpoint.

3.7.5 Safety parameters

Adverse events were coded by the medical representative of the sponsor, using a 2-tier hierarchy (Level 1 and Level 2 terms), which were akin to the ‘System Organ Class’ (Level 1) and ‘Preferred Term’ (Level 2) categorisation in the Medical Dictionary for Regulatory Activities (MedDRA). Frequencies and percentages of subjects with treatment emergent AEs are presented by treatment group, according to the Level 1 and Level 2 coded terms. Summaries of treatment emergent AEs, treatment related AEs and serious AEs are presented. No statistical comparisons with respect to the occurrence of AEs were made between treatment groups.

3.7.6 Changes in the Planned Analyses

The only change to the protocol was adding and swirling 20 ml of water to all drinks once the participant has finished it to ensure that most pulp would have been ingested. This change became effective from the 7th of August 2018.

4 Study Subjects

4.1 Disposition of Subjects

Fifty eight volunteers who met the initial eligibility assessed by questionnaire attended a screening visit at King’s College London, of whom 20 did not meet the inclusion criteria. Thirty- eight participants were randomized to treatment and 37 completed the study (see Figure 1).

4.2 Protocol Deviations

There were no protocol deviations.

4.3 Data Sets Analysed

The full dataset was analysed on the intention to treat population. 4.4 Demographic and Other Baseline Characteristics

4.4.1 Demographics

Demographics of the intention to treat population are presented in Table 3.

4.4.2 Baseline Characteristics

Baseline characteristics and baseline fasting plasma levels of the intention to treat population are presented in Table 4 and Table 5, respectively.

4.4.3 Current medical disorders and medical history

Medical history was recorded in the database, but no data tabulations were produced. 4.5 Concomitant Medications

Concomitant medications usage was recorded in the database, but no data tabulations were produced.

4.6 Compliance Subjects were compliant with the study protocol for the test drink and meal, except for two subjects on the BC + Pulp and Placebo groups, and three on the Pulp only group (Table 6). One subject spilled the drink to an estimated amount of 10-20 ml. Two subjects went slightly over the allocated time to eat the test meal.

5 Efficacy Results 5.1 Primary Efficacy Parameters

Glucose iAUCo-30min results are summarized in Tables 7.1 and 7.2. The BC + Pulp drink significantly inhibited the rate of increase in plasma glucose during the first 30 min of the postprandial period compared to both placebo and pulp only drink (iAUCo-30min mean difference [95% Cl] BC + pulp vs. placebo, -0.16 mmol/L [-0.27, -0.05]; and BC + pulp vs. pulp alone, -0.21 [-0.32, -0.10]). However, such an effect was not observed for the pulp only drink relative to placebo.

5.2 Secondary Efficacy Parameters

5.2.1 iAUCo.i5omin for plasma glucose concentration

Glucose iAUCo-i50min results are summarized in Tables 7.3 and 7.4. There was no drink effect for the change from baseline in plasma glucose during the 150 min postprandial period, as assessed by the iAUCo-isomin-

5.2.2 iAUCo-30min and iAUCo-isomin Plasma insulin, C-peptide and GIP concentrations

5.2.2.1 Insulin Insulin iAUCo-3omin and iAUCo-isomin results are summarized in Tables 8.1 and 8.2. The BC + pulp drink significantly inhibited the rate of increase in plasma insulin during the first 30 min of the postprandial period both compared to placebo and to pulp alone drinks (iAUCo-30min mean difference [95% Cl] BC + pulp vs. placebo, -2.67 mU/L [-4.70, -0.34]; BC + pulp vs. pulp alone, - 5.21 [-7.55, -3.02]). No significant drink effect was observed for the pulp only drink relative to placebo, although there was a trend for greater first phase insulin secretion following fruit pulp. There was no drink effect for the change from baseline in plasma insulin during the 150 min postprandial period, as assessed by the iAUCo-isomin.

5.2.2.2 C-peptide

C-peptide iAUCo-30min and iAUCo-isomin results are summarized in Tables 9.1 and 9.2. There was no significant difference in the change from baseline in plasma C-peptide between BC

+ pulp and placebo, either during the first 30 mins postprandially nor over the 150 min postprandial period, as assessed by the iAUCo-3omin and iAUCo-isomin, respectively. However, C-peptide secretion was significantly lower following BC + pulp compared with pulp alone during the first 30 mins. No significant drink effect was observed for the pulp only drink relative to placebo, although there was a trend for greater C-peptide secretion in the first 30 min following fruit pulp. 5.2.2.3 GIP

GIP iAUCo-30min and iAUCo-isomin results are summarized in Tables 10.1 and 10.2. Both the BC + pulp and pulp alone drinks significantly inhibited the rate of increase in plasma GIP during the first 30 min of the postprandial period relative to placebo (iAUCo-3omin mean difference [95% Cl] BC + pulp vs. placebo, -36.26 ng/L[(-49.23, -23.23]; Pulp vs. placebo, -11.22 [-22.31, -0.00]); and during the 150 min postprandial period (iAUCo-isomin mean difference BC + pulp vs. placebo [95% Cl] -28.62 ng/L [-47.99, -6.17]). No drink effect was observed for the iAUCo-isomin for the pulp only relative to placebo. The BC + pulp vs. pulp alone significantly attenuated the increase in GIP for both iAUCo-30min and iAUCo-isomin (mean difference [95% Cl] -29.06 [-41.69, -15.46] and -21.08 [- 41.29, -1.91], respectively).

5.2.3 iCmax for glucose, insulin, C-peptide, non-esterified fatty acids, and GIP, concentrations 5.2.3.1 Glucose

Glucose iCmax results are summarized in Tables 11.1 and 11.2. There was no drink effect for the maximum change in glucose from baseline during the 150 min postprandial period.

5.2.3.2 Insulin

Insulin iCmax results are summarized in Tables 12.1 and 12.2. There was-no drink effect for the maximum change in insulin from baseline during the 150 min postprandial period.

5.2.3.3 C-peptide

C-peptide iCmax results are summarized in Tables 13.1 and 13.2. There was no drink effect for the maximum change in C-peptide from baseline during the 150 min postprandial period.

5.2.3A GIP GIP iCmax results are summarized in Tables 15.1 and 15.2. There was no drink effect for the maximum change in GIP from baseline during the 120 min postprandial period, although there was a non-significant trend for a reduction following BC + pulp relative to placebo.

5.2.4 Tmax for plasma glucose, insulin, C-peptide, and GIP

5.2.4.1 Glucose Glucose iTmax results are summarized in Tables 15.1 and 15.2. There was no significant difference in the time to reach maximum concentration (T max) of glucose for the BC + pulp relative to placebo. The Tmax was significantly longer in the Polyphenol + Fruit Pulp compared to the Fruit Pulp alone drink (median (95%CI), 7.5 (7.50, 15.00)) and Tmax was significantly shorter in the Fruit Pulp drink compared to Placebo (median (95%CI), -7.5 (-12.50, 0.00)). 5.2A.2 Insulin

Insulin iTmax results are summarized in Tables 16.1 and 16.2. There was no drink effect for the time to reach maximum concentration in insulin. 5.2.4.3 C-peptide

C-peptide iTmax results are summarized in Tables 17.1 and 17.2. There was no drink effect for the time to reach maximum concentration in C-peptide.

5.2A.4 GIP GIP iTmax results are summarized in Tables 18.1 and 18.2. There was no drink effect for the time to reach maximum concentration in GIP.

5.2.5 Plasma concentration of glucose, insulin, C-peptide, and GIP over time

5.2.5.1 Glucose

The postprandial changes in plasma glucose after the test drinks and standard carbohydrate meals are shown in Figure 1. There were statistically significant lower concentrations of glucose following BC + pulp drink at 20 and 30 mins (change from baseline mean difference [95%CI] -0.27 [-0.47, - 0.07] and -0.27 [-0.52, -0.02], respectively) and following pulp only drink at 120 mins (change from baseline mean difference [95%CI] -0.32 [-0.62, -0.02]), relative to placebo. There were also lower glucose concentrations following BC + pulp compared to pulp alone at 10, 20 and 30 mins (change from baseline mean difference [95%CI] -0.11 [-0.20, -0.02], -0.37 [-0.57, -0.17] and -0.30 [-0.55, - 0.04], respectively).

5.2.5.2 Insulin

The postprandial changes in insulin after the test drinks and standard carbohydrate meals are shown in Figure 2. There were statistically significant lower concentrations of insulin following the BC + pulp drink at 30 min (change from baseline mean difference [95%CI] -8.25 [-13.16, -2.39]), and greater insulin concentrations following the pulp only at 20 mins (change from baseline mean difference [95%CI] -4.18 [0.69, 7.62]), relative to placebo. There were also lower insulin concentrations following BC + pulp compared to pulp alone at 10, 20 and 30 mins (change from baseline mean difference [95%CI] -2.31 [-4.23, -0.59], -7.14 [-11.12, -3.89] and -8.04 [-13.41 , - 4.60], respectively).

5.2.5.3 C-Peptide

The postprandial changes in C-peptide after the test drinks and standard carbohydrate meals are shown in Figure 3. There were no significant differences of C-peptide concentrations following the BC + pulp drink relative to placebo. At 10 mins c-peptide concentrations were significantly greater following pulp only compared to placebo (change from baseline mean difference [95%CI] 0.12 [0.02, 0.22]). The C-peptide concentrations were significantly lower following the BC + pulp drink compared to the pulp only drink at 10, 20, 30 and 45 mins (change from baseline mean difference [95%CI] -0.15 [-0.25, -0.07], -0.31 [-0.47, -0.17], -0.36 [-0.61, -0.15] and -0.26 [-0.46, -0.02], respectively). 5.2.5.4 GIP

The postprandial changes in GIP after the test drinks and standard carbohydrate meals are shown in Figure 4. There were statistically significant lower concentrations of GIP following the BC + pulp drink at 10, 20 and 30 mins (change from baseline mean difference [95%CI] -12.86 [-25.21 , -3.91], -61.96 [-85.35, -42.39], and -62.12 [-87.0, -37.86], respectively) and the pulp only drink at 30 mins (change from baseline mean difference [95%CI] -22.82 [-45.20, -0.67]), relative to placebo. There was also a significant lower GIP concentration following BC + pulp compared to pulp alone at 20 and 30 mins (change from baseline mean difference [95%CI] -55.67 [-74.69, -34.10] and -44.23 [- 67.79, -20.44], respectively). 5.2.6 Palatability

The mean ratings of palatability in the intention to treat population are presented in Figures 6 to 11. No significant differences were found in these ratings.

5.2.7 Mood and satiety

The mean ratings of mood and satiety in the intention to treat population are presented in Figures 12 to 20. No significant differences were found in these ratings.

5.2.8 Energy intake

The ad libitum energy intake results are summarized in Tables 19.1 and 19.2. There were no significant treatment effects on energy intake.

5.2.9 Cognitive Function The cognitive test results are summarized in Tables 20.1 to 20.6. The BC + pulp drink significantly reduced the amount of time to respond to the four choice reaction time task by 42 msec relative to placebo (mean difference [95% Cl] -41.89 [-97.18, -4.64]), which· is a measure of attention. The BC + pulp drink resulted in significantly fewer errors in the serial subtraction of 3’s (measure of working memory), relative to placebo and to pulp alone (mean difference [95% Cl] -1.0 [-2.0, 0.0] and -1.0 [-2.0, -0.], respectively). There were no significant treatment effects on simple reaction time, RVIP (rapid visual information processing), simple reaction time and Stroop task.

5.3 Drug Dose, Drug Concentration, and Relationships to Response

N/A

5.4 Tabulation of Individual Response Data Subject data listings were not produced.

5.5 Efficacy Conclusions

The primary efficacy analysis was based on the iAUCo.30 for postprandial glycaemia. Efficacy was reached with the BC + pulp drink where the rate of increase in plasma glucose was significantly inhibited. However, in absolute terms the difference is small and the relevance to glycaemic control is limited.

6 Safety Evaluation 6.1 Extent of Exposure

Each subject received a single dose of each test product. It is expected that no study participants will come to harm under the conditions of the study design. 6.2 Adverse Events (AEs)

6.2.1 Brief summary of adverse events

A total of 7 treatment emergent adverse events (TEAEs) were reported by 5 subjects (13.2%, Table 21.1). None of the AEs were considered treatment related. Six AEs were mild and one was moderate in intensity. None of the TEAEs were serious and all had resolved by the end of the study. One of the TEAEs (near faint) led to one subject withdrawal from the study.

6.2.2 Display of adverse events

Adverse events are presented in Tables 21.2 to 21.3.

6.2.3 Analysis of adverse events There were no statistical comparisons between groups.

6.2.4 Listing of adverse events by subject A full listing of AEs is presented in Table 21.2.

6.3 Deaths, Other Serious Adverse Events, and Other Significant Adverse Events

6.3.1 Listing of deaths, other serious adverse events and other significant adverse events No deaths, serious adverse events (SAEs) or other significant AEs were reported.

6.3.2 Narratives of deaths, other serious adverse events, and certain other significant adverse events

Not applicable.

6.3.3 Analysis and discussion of deaths, other serious adverse events, and other significant adverse events

Not applicable.

6.4 Pregnancy

No pregnancies occurred throughout the study in the female cohort.

6.5 Safety Conclusions No adverse events were considered treatment related, so this experiment reveals no evidence of safety concerns related to the treatments within the context applied and based on the measures made.

7 Discussion and Overall Conclusions 7.1 Discussion This study aimed to investigate the effects of fruit polyphenol extracts combined with orange pulp (a source of fibre: pectin), on postprandial outcomes following a mixed carbohydrate (starch and sucrose) test meal. The BC polyphenol extract in combination with orange pulp significantly inhibited the rate of increase in plasma glucose during the early postprandial period (0-30 min). Pulp alone failed to show a significant inhibitory effect on glucose in the early postprandial phase but lowered plasma glucose concentrations at the end of the sampling period (T+150 min).

BC + pulp showed a significant lowering effect on insulin secretion and GIP in the first 30 mins postprandially relative to placebo. Pulp alone showed a significant inhibitory effect on GIP but not insulin secretion. Neither of the test drinks had an inhibitory effect on C-peptide in the first 30 mins postprandially relative to placebo, although there was a trend for an increase following fruit pulp, resulting in a significant lowering of C-peptide concentration 0-30 min following BC + pulp relative to pulp alone. The BC + pulp significantly increased the attention (measured as response speed to the four reaction choice task) and working memory (measured as reduced number of errors) capacity in comparison to the placebo drink when measured 150-180 min following consumption of the test drinks.

The significant effect of the BC + pulp on glucose absorption was strongly supported by plasma GIP secretion, which is tightly coupled to the rate of glucose absorption in the intestine.

7.2 Conclusions The primary and secondary outcomes support the conclusion that only the BC + pulp drink consistently modified the early acute metabolic response to a mixed high carbohydrate meal, but that this was probably attributable to the addition of BC polyphenols (i.e. to the combination) as the glucose-lowering effects of the pulp alone drink were only apparent at the final sampling timepoint: 150 min. The improved parameters of attention and working memory following BC + pulp is a novel and intriguing finding, which merits further research using a study design where these outcomes are the primary endpoints. Future research should also investigate the chronic effects of daily consumption of BC polyphenol and fruit fibre-enriched drinks on cardiometabolic risk factors and cognitive function in populations at moderate risk of type 2 diabetes and cardiovascular disease. 8 References

1. Bell L, Lamport D.J, Butler L.T and Williams C.M. A Review of the Cognitive Effects Observed in Humans Following Acute Supplementation with Flavonoids, and Their Associated Mechanisms of Action. Nutrients 2015; 7, 10290-10306.

2. Blaak E, Antoine J, Benton D, Bjorck I, Bozzetto I, Bronus F, Diamant M, et al. Impact of postprandial glycaemia on health and prevention of disease. Obesity reviews 2012; 13, 923-

984.

3. Castro-Acosta M, Smith L, Miller R.J, McCarthy D.l, Farrimond J.A, Hall W.L. Drinks containing anthocyanin-rich blackcurrant extract decrease postprandial blood glucose, insulin and incretin concentrations. Journal of Nutritional Biochemistry 2016; 38, 154-161. 4. Cierello A, Esposito K, Piconi L, Ihnat MA, Thorpe JE, Testa R, Boemi M, Giugliano D.

Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients. Diabetes 2008; 57(5): 1349-54.

5. Grundy MML, Edwards CH, Mackie AR, Gidley MJ, Butterworth PJ, Ellis PR. Re-evaluation of the mechanisms of dietary fibre and implications for macronutrient bioaccessibility, digestion _: and postprandial metabolism. The British Journal of Nutrition 2016; 116(5): 816-833.

6. ICH Topic 6 Guideline for Good Clinical Practice CPMP/ICH/135/95 17^th July 1996. 7. Jackson PA, Deary ME, Reay JL, Scholey AB, Kennedy DO. No effect of 12 weeks’ supplementation with 1 g DHA-rich or EPA-rich fish oil on cognitive function or mood in healthy young adults aged 18-35 years. The British Journal of Nutrition 2012; 107: 1232-1243.

8. N6meth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY, Williamson G, Swallow DM, Kroon PA. Deglycosylation by small intestinal epithelial cell b-g!ucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans. European Journal of Nutrition 2003; 42: 29. doi:10.1007/s00394-003-0397-3

9. Neveu V, Perez-Jimenez J, Vos F, Crespy V, du Chaffaut L, Mennen L, Knox C, Eisner R, Cruz J, Wishart D, Scalbert A. Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database 2010; doi: 10.1093/database/bap024

10. Shen W1, Xu Y, Lu YH. Inhibitory effects of Citrus flavonoids on starch digestion and antihyperglycemic effects in HepG2 cells. Journal of Agricultural and Food Chemistry 2012; 60(38):9609-19.

11. Stroop JR (1992) Studies of interference in serial verbal reactions. (Reprinted from J Exp Psychol 18, 643-662, 1935). J Exp Psychol Gen 121 , 15-23.

12. Williamson G. Possible effects of dietary polyphenols on sugar absorption and digestion. Molecular Nutrition & Food Research 2013; 57(1):48-57.

13. World Medical Association Declaration of Helsinki, 48^th World Medical Assembly, Somerset West, Republic of South Africa, October 1996.

9 Tables, Figures and Graphs 9.1 Tables

Table 3 Summary of Demographic Characteristics Intent to Treat Population

^{: ~~ ~} Total (N=38)

Sex, n (%)

N 38

Male 16 (42.1)

Female 22 (57.9)

Age (years)

N 38

Mean (SD) 37.26 (15.147)

Median 31.70 Min, Max 19.8, 67.6

Ethnicity, n (%)

N 38

White 22 (57.9)

Mixed 5 (13.2)

Asian 2 (5.3)

Black 6 (15.8)

Chinese/East Asian 3 (7.9) Table 4 Summary of Baseline Measures Intent to Treat Population Total (N=38)

Height (cm)

N 38

Mean (SD) 169.8 (11.06)

Median 168.5 Min, Max 149, 192

Weight (kg)

N 38

Mean (SD) 72.42 (16.501)

Median 72.05 Min, Max 44.2, 108.5

BMI (kg/m²)

N 38

Mean (SD) 24.95 (4.459)

Median 24.35 Min, Max 18.4, 34.3

Total % body fat

N 38

Mean (SD) 26.81 (9.499)

Median 28.00 Min, Max 8.0, 43.8

Waist measurement (cm) N 38

Mean (SD) 88.16 (13.750)

Median 88.15 Min, Max 61.0, 116.6

Hip measurement (cm)

N 38

Mean (SD) 95.86 (11.186)

Median 96.10 Min, Max 71.5, 117

Waist: Hip ratio

N 38

Mean (SD) 0.919 (0.0877)

Median 0.935 Min, Max 0.73, 1.07)

Systolic BP (mmHg)

N 38

Mean (SD) 109.8 (14.97)

Median 113.5 Min, Max 75, 136

Diastolic BP (mmHg)

N 38 Mean (SD) 73.3 (9.48)

Median 73.0 Min, Max 56, 91

Table 5 Summary of Baseline Fasting Plasma Levels Intent to Treat Population

Total (N=38)

Glucose (mmol/L) N 38

Mean (SD) 4.93 (0.581) Median 5.00 Min, Max 3.3, 6.7

Total Cholesterol (mmol/L) N 38

Mean (SD) 4.80 (0.912)

Median 4.90 Min, Max 2.7, 6.4

TAG (mmol/L) N 38

Mean (SD) 0.94 (0.572) Median 0.75 Min, Max 0.3, 3.2

Table 6 Compliance with Test Drink and Meal Intent to Treat Population

BC Polyphenol + Fruit Fruit Pulp Placebo Pulp (N=37) (N=38)

(N=37)

Compliant with Test Drink and Meal, n (%)

N 37 37 38

Yes 35 (94.6) 34 (91.9) 36 (94.7)

No 2 (5.4) 3 (8.1) 2 (5.3)

Compliant means subject fully consumed test drink within 2 minutes and meal within 7 minutes.

Table 7.1 Summary of Glucose iAUCo-30min Intent to Treat Population.

BC Polyphenol + Fruit Pulp Fruit Pulp Placebo

(N=37) (N=37) (N=38) iAUC(0-30) (mmol/L) N 37 37 38

Mean (SD) 0.56 (0.329) 0.78 (0.354) 0.73 (0.366) Median 0.52 0.74 0.69 Min, Max 0.0, 1.4 0.1, 1.8 0.2, 1.6 BC Polyphenol + Fruit Pulp Fruit Pulp Placebo

Adj Mean (95% Cl) [1] 0.57 (0.45,0.68) 0.78 (0.66,0.89) 0.73 (0.62,0.84)

[1] Adj Mean represents the adjusted mean from a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates.

Table 7.2 Comparisons of Glucose iAUCo-₃₀min Intent to Treat Population.

Difference 95% Cl P-value iAUC(0-30) (mmol/L)

BC Polyphenol + Fruit Pulp vs -0.16 (-0.27, -0.05) 0.0038

Placebo

Fruit Pulp vs Placebo 0.05 (-0.06, 0.16) 0.3889

BC Polyphenol + Fruit Pulp vs Fruit -0.21 (-0.32, -0.10) 0.0003

Pulp

From a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

Table 7.3 Summary of Glucose iAUCo-isomin Intent to Treat Population.

BC Polyphenol + Fruit Pulp Fruit Pulp Placebo

(N=37) (N=37) (N=38) iAUC(0-150) (mmol/L) N 37 36 38

Mean (SD) 0.88 (0.851) 0.81 (0.885) 0.95 (0.897)

Median 0.85 0.67 0.91 Min, Max -0.8, 2.8 -0.6, 3.1 -0.6, 3.5

Adj Mean (95% Cl) [1] 0.90 (0.62,1.19) 0.83 (0.54,1.11) 0.95 (0.67,1.23)

[1] Adj Mean represents the adjusted mean from a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates.

Table 7.4 Comparisons of Glucose iAUCo-isomin Intent to Treat Population

Difference 95% Cl P-value iAUC(0-150) (mmol/L)

BC Polyphenol + Fruit Pulp vs -0.05 (-0.28, 0.18) 0.6756

Placebo

Fruit Pulp vs Placebo -0.12 (-0.36, 0.11) 0.3003

BC Polyphenol + Fruit Pulp vs Fruit 0.07 (-0.16, 0.31) 0.5336

Pulp

From a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

Table 7.5 Summary of Glucose iAUC Per Protocol Population.

BC Polyphenol + Fruit Fruit Pulp Placebo Pulp (N=36) (N=37) (N=36) iAUC(0-30) (mmol/L)

N 36 36 37

Mean (SD) 0.54 (0.317) 0.79 (0.357) 0.73 (0.370)

Median 0.52 0.74 0.67 Min, Max 0.0, 1.4 0 1, 1.8 0.2, 1.6

Adj Mean (95% Cl) [1] 0.55 (0.44,0.67) 0.78 (0.67,0.90) 0.72 (0.61 ,0.84) iAUC(0-150) (mmol/L)

N 36 35 37

Mean (SD) 0.89 (0.863) 0.81 (0.898) 0.97 (0.902)

Median 0.95 0.66 0.92 Min, Max -0.8, 2.8 -0.6, 3.1 -0.6, 3.5

Adj Mean (95% Cl) [1] 0.89 (0.61,1.18) 0.82 (0.53,1.11) 0.96 (0.67,1.24)

[1] Adj Mean represents the adjusted mean from a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates. Table 7.6 Comparisons of Glucose iAUC Per Protocol Population.

Difference 95% Cl P-value iAUC(0-30) (mmol/L)

BC Polyphenol + Fruit Pulp vs -0.17 (-0.28, -0.07) 0.0018

Placebo

Fruit Pulp vs Placebo 0.06 (-0.05, 0.17) 0.2566

BC Polyphenol + Fruit Pulp vs Fruit -0.23 (-0.34, -0.13) <.0001

Pulp iAUC(0-150) (mmol/L)

BC Polyphenol + Fruit Pulp vs -0.06 (-0.31, 0.18) 0.6008

Placebo

Fruit Pulp vs Placebo -0.14 (-0.38, 0.11) 0.2632

BC Polyphenol + Fruit Pulp vs Fruit 0.07 (-0.17, 0.32) 0.5437

Pulp

From a linear mixed model with fixed factors for treatment and period, and subject as a rando m effect, and subject baseline and period baseline as covariates.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

Table 8.1 Summary of Insulin Incremental AUC Intent to Treat Population

BC Polyphenol + Fruit Pulp Fruit Pulp Placebo

Mean (SD) 12.89 (7.180) 18.62 (9.803) 15.66 (8.287)

Median 12.53 16.76 ^{14 00} Min, Max 1.1, 29.6 5.4, 46.6 3.5, 45.6 iAUC(0-150) (mmol/L) N 37 36 38

Mean (SD) 32.65 (17.041) 33.44 (15.230) 32.48 (17.577)

Median 26.46 31.54 27.83 Min, Max 11.1, 87.4 10.6, 80.7 11.0, 80.6

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

P-value from a Wilcoxon signed ranks test.

Difference and Cl are based on the Hodges-Lehmann estimator:

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

P-value from a Wilcoxon signed rank test.

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher AUC.

P-value from a Wilcoxon signed rank test.

[1] Adj Mean represents the adjusted mean from a linear mixed model with fixed factors for treatment and period, and subject as a random effect, and subject baseline and period baseline as covariates.

Table 11.2 Summary of Change from Baseline in Glucose Cmax Intent to T reat Population yp p

From a linear mixed model with fixed factors for treatment and period, and subject as a random eff ect, and subject baseline and period baseline as covariates.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Cmax.

Table 12.2 Comparisons of Change from Baseline in Insulin Cmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Cmax.

P-value from a Wilcoxon signed rank test.

Table 13.1 Summary of Change from Baseline in C-Peptide Cmax Intent to Treat Population

Table 13.2 Comparisons of Change from Baseline in C-Peptide Cmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Cmax.

P-value from a Wilcoxon signed rank test.

Table 14.1 Summary of Change from Baseline in GIP Cmax Intent to Treat Population Table 14.2 Comparisons of Change from Baseline in GIP Cmax Intent to Treat Population Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Cmax.

P-value from a Wilcoxon signed rank test.

Table 15.1 Summary of Glucose Tmax Intent to Treat Population Table 15.2 Comparisons of Glucose Tmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Tmax.

P-value from a Wilcoxon signed rank test. Table 16.1 Summary of Insulin Tmax Intent to Treat Population

Table 16.2 Comparisons of Insulin Tmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Tmax.

P-value from a Wilcoxon signed rank test.

Table 17.1 Summary of C-Peptide Tmax Intent to Treat Population Table 17.2 Comparisons of C-Peptide Tmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Tmax.

P-value from a Wilcoxon signed rank test.

Table 18.1 Summary of GIP Tmax Intent to Treat Population Table 18.2 Comparisons of GIP Tmax Intent to Treat Population

Difference and Cl are based on the Hodges-Lehmann estimator.

Difference is first named drink minus second named drink such that a positive difference indicates the first drink has higher Tmax. P-value from a Wilcoxon signed rank test. Table 19.1 Summary of Ad Libitum Energy Intake Intent to Treat Population

[1] Adj Mean represents the adjusted mean from a linear mixed model with fixed factors for treatment and period, and subject as a random effect.

Table 19.2 Comparisons of Ad Libitum Energy Intake Intent to Treat Population

From a linear mixed model with fixed factors for treatment and period, and subject as a random effect.

Difference is first named drink minus second named drink such that a negative differen ce indicates more food was eaten after the second named drink.

Table 20.1 Comparisons of Choice Reaction Time Intent to Treat Population

Difference and Cl for change from baseline based on the Hodges-Lehmann estimator. Difference is first named drink minus second named drink.

P-value from a Wilcoxon signed rank test.

Table 20.2 Comparisons of RVIP Intent to Treat Population

Difference and Cl for change from baseline based on the Hodges-Lehmann estimator. Difference is first named drink minus second named drink. P-value from a Wilcoxon signed rank test.

Table 20.3 Comparisons of Simple Reaction Time Intent to Treat Population

Difference and Cl for change from baseline based on the Hodges-Lehmann estimator. Difference is first named drink minus second named drink.

P-value from a Wilcoxon signed rank test.

Table 20.4 Comparisons of Stroop Intent to Treat Population

Table 20.5 Comparisons of Four Choice Reaction Time Intent to Treat Population

Reaction Time Overall (msec)

Difference and Cl for change from baseline based on the Hodges-Lehmann estimator. Difference is first named drink minus second named drink. P-value from a Wilcoxon signed rank test.

Table 20.6 Comparisons of Serial subtraction Intent to Treat Population

Difference and Cl for change from baseline based on the Hodges-Lehmann estimator. Difference is first named drink minus second named drink.

P-value from a Wilcoxon signed rank test.

Table 21.1 Summary of Treatment Adverse Events nAE = Number of Events. N(%) = Number (%) of subjects.

Table 21.3 Summary of Treatment Emergent Adverse Events by Treatment Group Safety Population nAE = Number of Events. N (%) = Number (%) of subjects. Study 2

A study was performed to assess the effects of polyphenols alone. Full details of the study are not included, since the findings do not relate to the inclusion of fibre.

Elevated postprandial glycaemia is implicated in the development of type 2 diabetes mellitus (T2DM) and other chronic diseases, such as cardiovascular disease (Blaak et al., 2012). Purported mechanisms include increased oxidative stress, glycation of functional proteins, pancreatic beta cell dysfunction and vascular damage (Cierello et al., 2008). Thus, control of postprandial glycaemia is important in the prevention and management of metabolic diseases, including T2DM.

Emerging evidence suggests that the acute intake of fruit polyphenols may reduce postprandial glycaemia. Dr Wendy Hall’s research group has previously investigated the effects of drinks containing 800 mg or 1600 mg blackcurrant (BC) polyphenol extract on plasma glucose responses to a carbohydrate meal (Castro-Acosta et al., 2016). A 1600 mg BC extract drink (but not 800 mg drink) was found to inhibit average incremental area over baseline (AOB; TO to 30 min) of plasma glucose by a mean of -0.34 mmol/l.h. Moreover, this dose also reduced postprandial insulin, GIP and GLP-1 at AOB TO to 30 min, AOB TO to 120 min, and at T90 min, respectively. No effects on postprandial glycaemia were found with the 800 mg BC extract dose, however, the placebo drink contained tannins, which may have masked the effects of the BC extracts on postprandial glycaemia.

The primary proposed mechanisms of BC polyphenols on postprandial glycaemia are inhibition of digestive enzyme activity and sodium-glucose linked transporter 1 (SGLT-1) / glucose transporter 2 (GLUT-2) glucose transporters on intestinal cells (Williamson, 2013). Importantly, polyphenols from other fruits may impact glucose homeostasis via different mechanisms. For example, citrus polyphenols, such as those in sweet-orange (SO), only demonstrate a weak inhibition of digestive enzyme activity, however they may moderate carbohydrate digestion by binding to starch molecules (Shen et al., 2012). The effects of SO polyphenols on postprandial glycaemia and related metabolites are not yet known. BC and SO have distinct polyphenol profiles, with blackcurrants rich in anthocyanins, proanthocyanidins, and flavanols, and SO rich in flavanones, notably hesperidin (Neveu et al., 2010). Theoretically, combining fruit polyphenols with different structures and physiological properties, such as BC and SO extracts, may have synergistic effects on reducing postprandial glycaemia.

This research project investigated the acute effects of BC and SO polyphenol extracts, and their combination, on postprandial glycaemia following a mixed carbohydrate (starch and sucrose) meal. It was hypothesised that BC and SO polyphenol extracts alone will inhibit postprandial glycaemia, compared to placebo (no fruit polyphenols). Furthermore, it was hypothesised that a combination of BC and SO extracts would have a greater effect on postprandial glycaemia than either BC or SO polyphenol extracts alone. To test these hypotheses, we investigated the effects of drinks containing a low dose of BC extract (800 mg polyphenols), a high dose of BC extract (1600 mg polyphenols), a low dose of SO extract (800 mg), a mixture of low dose BC and SO extracts (800 mg / 800mg), or placebo (no polyphenols) on postprandial glycaemia, insulinaemia and plasma concentrations of gut hormones. A randomised, controlled, cross-over, single-meal, incomplete block, study design was used, with each subject consuming the placebo plus 3 out of 4 treatment drinks over the course of the study.

Most people of European origin are able to digest the milk sugar lactose, but this genetic trait is not so common in other worldwide populations. The characteristic of digesting lactose during adulthood is known as lactase persistence, yet the natural state is to lose this ability after weaning. In European populations, the activity of the lactase-phlorizin hydrolase (LPH) enzyme is maintained into adulthood by a single nucleotide polymorphism (SNP) in the upstream region of the gene. The derived allele (C-13910) is believed to be causative of LP in European adults.

Importantly, LPH activity is also linked to the deglycosylation of dietary flavonoids - a critical step in their absorption (Nemeth, K., et al., 2003). Blackcurrant extracts are rich in anthocyanin glycosides which are hydrolysed by LPH. Since the anthocyanins are hypothesised to inhibit postprandial glycaemia - at least in part, by their action in inhibiting digestive enzymes (amylase, disaccharidase) - a faster rate of hydrolysis to their aglycone form (anthocyanidins) might be expected to be associated with a reduction in the degree of inhibition of glucose absorption.

This study investigated whether there is an association between the well-characterised LP allele and postprandial glycaemic responses to a carbohydrate meal, preceded by the intake of a drink containing blackcurrant and/or orange polyphenol extracts relative to a placebo drink.

Study Objectives Primary Objective(s)

To determine the effect of blackcurrant and sweet orange polyphenol-rich extracts on the rate of glucose absorption. The primary endpoint is iAUC_{0-30 min}for plasma glucose, which is defined as the change in area under the curve from baseline plasma glucose concentration versus time from zero to the T30 min sample time.

Secondary Objective(s)

To determine the effects of blackcurrant and sweet orange polyphenol-rich extracts on related parameters of glucose absorption, including markers of glycaemic response, insulin and gut hormone secretion, and appetite, as follows:

1. iAUC_{0-120 min} for plasma glucose concentration.

2. iAUC_{0-30 min} and iAUC_{0-120 min} for plasma insulin, C-peptide, NEFA, GIP, PYY and GLP-1 concentration.

3. Incremental C_MAX (iC_MAX) for plasma glucose, insulin, C-peptide, NEFA, GIP, PYY and GLP-1 concentration, defined as the maximum change from baseline over 120-min sampling period.

4. T_MAX for plasma glucose, insulin, C-peptide, NEFA, GIP, PYY and GLP-1 , defined as the time to maximum concentration.

5. Plasma concentration of glucose, insulin, C-peptide, NEFA, GIP, PYY and GLP-1 at each time point (T10, 20, 30, 45, 60, 75, 90 and 120 min). 6. Responses to the VAS palatability questions at T10 mins, and the mood and satiety questions at T-10, 10, 30, 60, 90 and 120 mins, will be summarised using descriptive statistics.

Investigational Plan

Overall Study Design and Plan Description

A randomised, placebo-controlled, double-blind, cross-over study was conducted. Subjects were randomised to one of the four treatment arms in an incomplete-block design (Table 1). Over four separate visits, all subjects consumed four drinks; the placebo and three of the four test drinks. Low doses contained 800 mg of total polyphenols, and high doses contained 1600 mg of total polyphenols. The test drinks were the following a) low dose of BC polyphenols (L-BC), b) high dose of BC polyphenols (H-BC), c) low dose of SO polyphenols (L-SO) and d) high dose of blended polyphenols (H-Blend) containing a low dose of both BC and SO polyphenols.

Table 1 - Randomisation to treatment arms in incomplete block design

The minimum wash out period was 7 days and in order to minimise the influence of cyclical reproductive hormones in female subjects, the female subjects only attended study visits during the middle two weeks of the menstrual cycle (weeks 2 and 3).

Standardised diet and exercise guidance were given prior to the visit (Source document). Subjects arrived on each study visit between 08.00 and 10.00 h, after a 12 h overnight fast, and having consumed no caffeine since noon the previous day. They were then cannulated in a forearm vein and two baseline fasting blood samples were taken (T-10 and -5 min). Following consumption of the test drink (T0 min), the high carbohydrate meal (starch and sucrose) was served (white bread with apricot jam). Postprandial blood samples were collected at T10, 20, 30, 45, 60, 75, 90 and 120 min for plasma glucose analysis and serum insulin and plasma incretin and gut hormone analysis. The intervention ended once the T120 min sample was collected.

The cannula was then removed, and the subject was offered a light breakfast and hot drink.

Treatments

Treatments administered

Five different treatments, including a placebo and 4 active, polyphenol containing drinks were supplied by the New Product Development team, LRS. The drinks were 200 ml and were taste and colour matched as far as possible. Due to differences in the natural sugars present in the BC and SO extracts, different amounts of glucose, fructose and sucrose were added to provide the total sugar and extract values shown in Table 2. Table 2. Study supplies formulations. Sugars derived from the polyphenol containing extracts are indicated in parenthesis.

Drink 1 Drink 2 Drink 3 Drink 4 Drink 5 (High-BC) (Low-BC) (Low-SO) (High-Blend) (placebo) 1.6g Total 0.8g Total PP 0.8g Total PP 1.6g Total PP Og Total PP PP

BC extract 57.76 28.88 0.00 28.88 0.00 T. sugar (BC) 3.12 1.56 0.00 1.56 0.00 Glucose(BC) 1.29 0.64 0.00 0.64 0.00 Fructose(BC 1.59 0.79 0.00 0.79 0.00 )

Sucrose(BC) 0.18 0.09 0.00 0.09 0.00 SO extract 0.00 0.00 0.87 0.87 0.00 T. sugar (SO) 0.00 0.00 0.00 0.00 0.00 Total GLU 5.05 5.05 5.05 5.05 5.05

Total FRU 1.59 1.59 1.59 1.59 1.59

Total SUC 0.18 0.18 0.18 0.18 0.18

Total MALT 3.12 2.89 2.67 2.89 2.67

Total SUGAR 10.00 9,74 9.49 9.74 9.49 Discussion and Overall Conclusions

This study hypothesised that BC and SO polyphenol extracts alone would inhibit postprandial glycaemia compared to placebo and that a combination of BC and SO extracts would have a greater effect than either extract alone. The low dose of BC extract significantly inhibited the rate of increase in plasma glucose in the first 30 min postprandially, a period of time where plasma glucose concentrations are predominantly determined by the rate of intestinal absorption.

However, unexpectedly, the high dose of BC extract failed to show a significant inhibitory effect on glucose during this early postprandial period, and the H-Blend containing 800 mg of both extracts did not significantly modify glucose concentrations in the first 30 min. The SO extract did not show any significant effect. These results are puzzling, but they do confirm previous findings that blackcurrant polyphenols inhibit the glycaemic response in the early postprandial period, indicating a reduction in the rate of intestinal glucose absorption.

Regarding the secondary outcomes, both the low and high dose extracts showed a significant effect on insulin, C-peptide and GIP in the first 30 mins postprandially compared to placebo, strengthening the evidence that blackcurrant polyphenols modify the rate of delivery of glucose to the circulation. The high dose blend extract showed only a significant inhibitory effect on GIP, indicating a reduced rate of intestinal glucose absorption following this dose, and the low dose of SO extract showed no significant inhibitory effects in the first 30 min postprandially. None of the drinks had an inhibitory effect on NEFA, PYY and GLP-1 in the first 30 mins postprandially. Results were consistent with previous work showing that BC extract inhibits glucose absorption 0-30 min (Acosta Castro 2016). However, in the present study there was a lack of dose response effect, where only the low dose of BC extract showed a significant effect. The significant effect of the lower dose of BC on glucose absorption was strongly supported by plasma GIP secretion, which is tightly coupled to the rate of glucose absorption in the intestine. The primary and secondary outcomes support the conclusion that drinks containing blackcurrant polyphenol-rich extract slow down the rate of glucose absorption following a starch and sucrose containing meal, but that sweet orange-derived hesperidin has no perceptible effect.

References

14. Blaak E, Antoine J, Benton D, Bjorck I, Bozzetto I, Bronus F, Diamant M, et al. Impact of postprandial glycaemia on health and prevention of disease. Obesity reviews 2012; 13, 923- 984.

15. Castro-Acosta M, Smith L, Miller R.J, McCarthy D.l, Farrimond J.A, Hall W.L. Drinks containing anthocyanin-rich blackcurrant extract decrease postprandial blood glucose, insulin and incretin concentrations. Journal of Nutritional Biochemistry 2016; 38, 154-161.

16. Cierello A, Esposito K, Piconi L, Ihnat MA, Thorpe JE, Testa R, Boemi M, Giugliano D. Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients. Diabetes 2008; 57(5): 1349-54. 17. ICH Topic 6 Guideline for Good Clinical Practice CPMP/ICH/135/95 17^th July 1996.

18. Nemeth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY, Williamson G, Swallow DM, Kroon PA. Deglycosylation by small intestinal epithelial cell b-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans. European Journal of N utrition 2003; 42 : 29, doi : 10.1007/s00394-003-0397-3 19. Neveu V, Perez-Jimenez J, Vos F, Crespy V, du Chaffaut L, Mennen L, Knox C, Eisner R,

Cruz J, Wishart D, Scalbert A. Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database 2010; doi: 10.1093/database/bap024

20. Shen W1 , Xu Y, Lu YH. Inhibitory effects of Citrus flavonoids on starch digestion and antihyperglycemic effects in HepG2 cells. Journal of Agricultural and Food Chemistry 2012; 60(38):9609-19

21. Williamson G. Possible effects of dietary polyphenols on sugar absorption and digestion. Molecular Nutrition & Food Research 2013; 57(1):48-57.

22. World Medical Association Declaration of Helsinki, 48^th World Medical Assembly, Somerset West, Republic of South Africa, October 1996.

Unless otherwise stated, all percentages herein are by weight and all pressures are absolute, rather than gauge. Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the scope of the invention or of the appended claims.

Exemplary embodiments The present invention encompasses the following listing of exemplary embodiments:

1 . A beverage composition comprising: at least 600mg polyphenols; and at least 1g of fibre.

2. The beverage composition according to embodiment 1 , comprising from 700 to 2000mg polyphenols, preferably 800 to 1200 mg polyphenols. 3. The beverage composition according to embodiment 1 or embodiment 2, comprising a concentrated fruit extract as a source of the polyphenols.

4. The beverage composition according to embodiment 3, wherein the fruit extract is derived from one or more anthocyanin-rich fruits.

5. The beverage composition according to embodiment 4, wherein the anthocyanin-rich fruits are selected from blackcurrant, blackberries, blueberries and pomegranate, or a combination of two or more thereof. 6. The beverage composition according to any preceding embodiment, wherein the polyphenols consist essentially of blackcurrant polyphenols.

7. The beverage composition according to any preceding embodiment, comprising from 1.25 to 3.5g of fibre, preferably fro 15 to 2g of fibre.

8. The beverage composition according to any preceding embodiment, wherein substantially all of the fibre is natural fibre, preferably a cellular pulp.

9. The beverage composition according to any preceding embodiment, comprising citrus fibre as a source of the fibre.

10. The beverage composition according to any preceding embodiment, wherein the fibre consists essentially of citrus fibre. 11. The beverage composition according to any preceding embodiment, comprising less than

100Kcal.

12. The beverage composition according to any preceding embodiment, wherein the beverage composition is ready-to-drink and has a volume of from 100 to 1000ml, preferably 100 to 750ml, preferably 150 to 500ml, preferably 200 to 300ml.

13. The beverage composition according to embodiment 12, wherein the beverage comprises less than 5g/100ml of sugar. 14. The beverage composition according to any of embodiments 1 to 12, wherein the beverage composition is provided as a concentrate for forming a beverage on reconstitution with water.

15. The beverage composition according to any preceding embodiment for use a method of improving cognitive function in a subject in need thereof.

16. The beverage composition according to any of embodiments 1 to 14 for use in a method of managing and/or treating type II diabetes.

17. The beverage composition according to any of embodiments 1 to 14 for use in a method of reducing postprandial glycaemia.

18. The beverage composition according to any of embodiments 1 to 14 for use in a method of promoting weight-loss and/or improving Body Mass Index in a subject in need thereof.

19. The beverage composition according to any of embodiments 1 to 14 for use in a method of reducing the risk of cardiovascular disease.

20. The beverage composition according to any of embodiments 1 to 14 for use in a method of preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin* response.

21. A. method of forming a beverage composition according; to any of embodiments 1 to 1 , the method comprising mixing a polyphenol-rich fruit extract with a natural fibre ingredient.

22. A beverage composition for being consumed with or before intake of carbohydrate comprising: at least 600mg polyphenols; and at least 1g of fibre.

23. The beverage composition according to any of embodiments 1 to 13 for improving cognitive function.

24. The beverage composition according to any of embodiments 1 to 13 for managing and/or treating type II diabetes.

25. The beverage composition according to any of embodiments 1 to 13 for reducing postprandial glycaemia.

26. The beverage composition according to any of embodiments 1 to 13 for promoting weight-loss and/or improving Body Mass Index. 27. The beverage composition according to any of embodiments 1 to 13 for reducing the risk of cardiovascular disease.

28. The beverage composition according to any of embodiments 1 to 13 for preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin response.

29. The beverage composition according to any of embodiments 1 to 19, wherein a serving size of the beverage composition is 50ml to 1 ,000ml.

Claims

Claims:

1. A beverage composition comprising: at least 600mg polyphenols; and at least 1g of fibre, wherein the beverage composition comprises less than lOOKcal.

2. The beverage composition according to claim 1 , comprising from 700 to 2000mg polyphenols, preferably 800 to 1200 mg polyphenols.

3. The beverage composition according to claim 1 or claim 2, comprising a concentrated fruit extract as a source of the polyphenols.

4. The beverage composition according to claim 3, wherein the fruit extract is derived from one or more anthocyanin-rich fruits.

5. The beverage composition according to claim 4, wherein the anthocyanin-rich fruits are selected from blackcurrant, blackberries, blueberries and pomegranate, or a combination of two or more thereof.

6. The beverage composition^; according to any preceding claim, wherein-the polyphenols consist essentially of blackcurrant polyphenols.

7. The beverage composition according to any preceding claim, comprising from 1.25 to 3.5g of fibre, preferably from 1.5 to 2g of fibre.

8. The beverage composition according to any preceding claim, wherein substantially all of the fibre is natural fibre, preferably a cellular pulp.

9. The beverage composition according to any preceding claim, comprising citrus fibre as a source of the fibre.

10. The beverage composition according to any preceding claim, wherein the fibre consists essentially of citrus fibre.

11. The beverage composition according to any preceding claim, wherein the beverage composition is ready-to-drink and has a volume of from 100 to 1000ml, preferably 100 to 750ml, preferably 150 to 500ml, preferably 200 to 300ml.

12. The beverage composition according to claim 11 , wherein the beverage comprises less than 5g/100ml of sugar.

13. The beverage composition according to any of claims 1 to 10, wherein the beverage composition is provided as a concentrate for forming a beverage on reconstitution with water.

14. The beverage composition according to any preceding claim for use in a therapeutic method of improving cognitive function in a subject in need thereof.

15. The beverage composition according to any of claims 1 to 13 for use in a method of managing and/or treating type II diabetes.

16. The beverage composition according to any of claims 1 to 13 for use in a method of reducing postprandial glycaemia.

17. The beverage composition according to any of claims 1 to 13 for use in a therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject in need thereof.

18. The beverage composition according to any of claims 1 to 13 for use in a method of reducing the risk of cardiovascular disease.

19 The beverage composition according to any of claims 1 to 13 for use in a method of preventing or reducing the risk of low blood glucose resulting from the glycaemia-induced insulin response.

20. A method of forming a beverage composition according to any of claims 1 to 13, the method comprising mixing a polyphenol-rich fruit extract with a natural fibre ingredient.

21. Use of the beverage composition according to any one of claims 1 to 13 in a non- therapeutic method of improving cognitive function in a subject.

22. Use of the beverage composition according to any one of claims 1 to 13 in a non- therapeutic method of promoting weight-loss and/or improving Body Mass Index in a subject.