WO2012055577A1

WO2012055577A1 - An edible product

Info

Publication number: WO2012055577A1
Application number: PCT/EP2011/005599
Authority: WO
Inventors: Maria Christiana Peter Geraedts; Frederik Jan Troost; Wilhelmus Hermanus Maria Saris
Original assignee: Maria Christiana Peter Geraedts; Frederik Jan Troost; Wilhelmus Hermanus Maria Saris
Priority date: 2010-10-25
Filing date: 2011-10-24
Publication date: 2012-05-03
Also published as: GB2484929A; GB201018030D0

Abstract

An edible product comprising: an intact plant protein; a sugar in an amount greater than 2 times the amount of intact plant protein by weight or a sugar substitute; and a delivery vehicle. The delivery vehicle at least partially protects the plant protein from hydrolysis. The edible product may be used to induce or increase satiety in an individual

Description

An Edible Product

Field of the Invention The present invention relates to an edible product and, in particular, an edible product comprising a plant protein. The invention also relates to a method of inducing or increasing satiety in an individual and also to a method for treating clinical or nonclinical obesity. Background of the Invention

Over the last few decades, the food consumption of individuals and, more specifically, the caloric intake of individuals have increased significantly. This development has occurred in most Western countries and also in many other countries worldwide. Such a change in diet has given risen to an increase in the number of overweight individuals and individuals who are obese, either in a clinical sense (i.e. requiring medical attention) or in a non-clinical sense (i.e. for cosmetic reasons the individual would prefer to lose weight). As a consequence, there has been considerable interest in identifying products that reduce or alleviate feelings of hunger, that is to say products that induce or increase satiety in an individual, so that individuals can more easily regulate their consumption of food and keep food consumption within healthy limits.

It is, of course, known that, generally speaking, the feeling of hunger in an individual recedes and satiety increases, after consuming food. The identification of products that induce or increase satiety in an individual has involved a greater understanding of the biochemical and physiological of food components in influencing feelings of hunger and satiety in individuals. In particular, it has been found that nutrient induced gut-to-brain signalling plays a major role in the control of the digestive function, appetite, and energy intake [1]. These effects are mediated by a number of interrelated factors, including the release of signalling peptides from enteroendocrine cells, such as cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1). Most of these hormones are secreted upon food intake, and contribute to the termination of the meal. Since overweight individuals and obesity have become a major health problem [2], several types of diets have focused on favourable macronutrient compositions in order to stimulate the release of these satiety hormones [3-8]. Of all diets tested, high-protein diets seem to have the largest effects on reducing food intake [9, 10]. The effects of several protein hydrolysates on the release of CCK from the enteroendocrine STC-1 cell line have been determined [11]. It has been shown that all hydrolysates were able to induce elevated levels of CCK, but there were no differences between the hydrolysates. It has also been demonstrated that intact proteins are the most potent in stimulating CCK and GLP-1 release versus hydrolysates and specific peptides. Pea hydrolysate has been reported as most effective in suppressing hunger and stimulating satiety when compared with whole milk protein [12] It has been reported that some proteins (alpha-lactalbumin, gelatin, gelatin + TRP) are more satiating than other proteins (casein, soy, whey, whey-GMP) [41].

WO2009/053487 discloses that intact pea protein is particularly effective in increasing release of GLP-1 and is therefore believed to be effective in inducing or increasing satiety in humans. It is also reported therein that intact pea protein is suitable for reducing appetite and/or inducing or increasing satiety when brought into contact with receptors in the duodenum. WO2009/053487 also discloses the provision of intact pea protein or intact wheat protein incorporated into a delivery vehicle so as to avoid hydrolysis of the protein in the stomach thereby ensuring that intact protein reaches the duodenum of an individual consuming the protein.

Over the past few years, not only food intake, but also consumption of soft drinks has increased [13]. This high consumption of sugar-sweetened beverages has been linked with increased energy intake and obesity [14]. It has been demonstrated that overweight subjects who consume large amounts of caloric-sweetened beverages increase energy intake, body weight, fat mass, and blood pressure after a 10 week intervention, whereas this is not observed in a similar group receiving artificial sweeteners [15]. It has been suggested that intake of caloric sweetened beverages is linked to obesity, related to the potential mediating role of energy intake, e.g. that intake of caloric sweetened beverages brings less satiation, causing a higher amount of calories consumed at a given meal and thereby a higher daily energy intake [16, 17]. It has also been suggested that the intake of caloric sweetened beverages fails to trigger physiological satiety mechanisms, providing imprecise and incomplete energy compensation [18]. WO2004/105505 relates to food products comprising a satiety agent, in particular, a satiety agent that is in an encapsulated form where the encapsulant material has a certain degree of cross-linking. However, this document does not identify specific satiety agents, or combinations of satiety agents, which are particularly effective in inducing or increasing satiety in individuals. For example, there is a general explanation that the satiety agent may comprise a protein or a carbohydrate but specific proteins or carbohydrates are not identified as satiety agents. The document discloses the components of the encapsulant material but the encapsulant material is not, itself, encapsulated.

WO2004/016720 relates to the coating of polyunsaturated fatty acid-containing particles and of liquid pharmaceutical-containing particles. It discloses an example of PUFA oil-coated isolated soy protein particles which were coated with a layer of sucrose. However, the sucrose constituted a very small (2.8%) component of the finished product and was provided in order to form a barrier against the effects of oxidation of the particles and to improve the handling characteristics of the particles. The PUFA oil-coated soy protein particles were used as a solid feed material but the effect of the solid sucrose coating was not tested, in particular, the satiety inducing effect of the coating was not studied and was not discussed.

Accordingly, there remains a need to develop new products that induce or increase satiety in individuals. There is also a need for methods to treat or alleviate both clinical and non-clinical obesity. Summary of the Invention

According to one aspect of the present invention there is provided an edible product comprising:

i) intact plant protein

ii) a sugar or sugar substitute; and

iii) a delivery vehicle for at least partially protecting the plant protein from hydrolysis, such as in the upper Gl tract.

It is preferred that the sugar is not an inactive water soluble sugar. In particular, it is preferred that the sugar is not lactose, mannitol or tretalose. It is also preferred that the plant protein is an isolated plant protein. In this context, the term "isolated" means that the plant protein is separated from other components in its naturally occurring environment such as starch and fibers.

It is preferred that the sugar is provided in an amount greater than 2 times, preferably 2.5, 3, 4 or 5 times, the amount of plant protein, by weight.

In some embodiments, the sugar or sugar substitute constitutes at least 5% or at least 10% of the total edible product, by weight.

Preferably, the delivery vehicle includes 0%, less than 1% or less than 5% total sugar or sugar substitute, by weight, and/or 0%, less than 1% or less than 5% total plant protein by weight.

Conveniently, the delivery vehicle comprises an enteric coating of at least the plant protein.

Preferably, the product comprises at least 5g of the plant protein, more preferably at least 10g of the plant protein.

Advantageously, the product comprises between 2mg and 100mg of the sugar substitute. Alternatively, the product comprises at least 5g of sugar, preferably at least 10g, 15g or 20g of sugar.

Conveniently, the product further comprises a delivery vehicle for at least partially protecting the sugar or sugar substitute from hydrolysis

According to another aspect of the present invention there is provided a method of inducing or increasing satiety in an individual comprising the steps of:

i) delivering an intact plant protein to the duodenum of the individual; and ii) delivering a sugar or sugar substitute to the duodenum of the individual. Preferably, the method is non-therapeutic, such as forming part of a weight management programme.

According to a further aspect of the present invention there is provided a method of treating obesity in or controlling weight of an individual comprising the steps of:

i) delivering an intact plant protein to the duodenum of the individual; and ii) delivering a sugar or sugar substitute to the duodenum of the individual.

It is to be appreciated that the sugar or sugar substitute is provided in these methods for its effect in inducing or increasing satiety and/or treating obesity.

Advantageously, the method is a cosmetic method.

It is preferred that the sugar is provided in an amount greater than 2 times, preferably, 2.5, 3, 4 or 5 times, the amount of plant protein, by weight.

Conveniently, the individual has a body mass index of greater than 25, preferably greater than 30. Preferably, the intact plant protein comprises or consists of wheat protein, soy protein and/or pea protein.

In particular, the intact plant protein may be a proteinaceous extract of a plant of the subfamily Faboideae and the tribe Vicieae such as the genus Pisum.

Advantageously, the intact plant protein comprises or consists of a proteinaceous extract of a plant of the species Pisum sativum.

Preferably, the sugar substitute is a high intensity sweetener or a sugar alcohol.

Advantageously, the high intensity sweetener is a naturally occurring or non-naturally occurring high intensity sweetener.

Conveniently, the naturally occurring high intensity sweetener comprises a protein, preferably Thaumatin, Brazzein or Monellin. Alternatively, the naturally occurring high intensity sweetener comprises Stevia, a Steviol glycoside, Mogroside V (Lo Han Guo ), Monatin or Glycyrrhizin. Advantageously the Steviol glycoside is stevioside, steviol, Rebaudioside A , Rebaudioside B, Rebaudioside C, Rebaudioside D or Rebaudioside E.

Conveniently, the non-naturally occurring high intensity sweetener comprises a peptide, preferably aspartame (or a salt of aspartame-acesulfame), neotame, advantame or alitame.

Alternatively, the non-naturally occurring high intensity sweetener comprises a halosugar, preferably sucralose. Alternatively, the non-naturally occurring high intensity sweetener comprises Acesulfame-K, Saccharin (or sodium, potassium or calcium salts thereof), Cyclamate (that is to say, the sodium salt of cyclamic acid although the high intensity sweetener may alternatively be cyclamic acid or its calcium salt) or neohesperidin DC. Preferably, the sugar comprises sucrose, glucose or fructose.

Advantageously, the sugar alcohol comprises xylitol, sorbitol, maltitol, erythritol, isomalt or lactitol. Alternatively, the sugar substitute comprises a precursor of a sugar or a sugar substitute. The precursor preferably comprises maltodextrin.

Conveniently, the method comprises administering to the individual an edible product in accordance with the invention.

Definitions

As used herein, the term "delivery vehicle" means a physical product that protects the plant protein from hydrolysis in the stomach of an individual. Such protection may be only partial protection but a greater amount of the plant protein passes through the stomach of an individual without hydrolysis in the presence of the delivery vehicle compared with the absence of the delivery vehicle. In general terms, the delivery vehicle provides a physical barrier between the plant protein and acidic and/or proteolytic environments.

As used herein the term "edible product" means a product that can be safely eaten by humans when consumed in physiological amounts. It includes products that comprise ingredients that are generally regarded as safe and ingredients that have specifically received regulatory approval. Edible products include foods such as cereal products and energy bars as well as beverages such as protein shakes.

As used herein the term "plant protein" means a proteinaceous extract from a plant. That is to say a composition obtained from a plant that primarily (e.g. at least 90% by weight) comprises proteins from the plant. The proteins may be a mixture of plant proteins or may be a single plant protein.

As used herein the term "sugar" means any edible crystalline carbohydrate having a sweet flavour, for example sucrose, glucose and fructose.

As used herein, the term "sugar substitute" means a natural or synthetic food additive (aside from a sugar) that has a sweet flavour. The term "sugar substitute" also includes precursors of a sugar or one of the aforesaid food additives which are digested under physiological conditions into products having a sweet flavour.

Figures

Figure 1 is a graphical representation of hormone release from STC-1 cells after 2h exposure to several sweeteners, pea protein, and combinations of pea protein with the sweeteners. STC-1 cells were exposed to different sweeteners, pea protein, and a combination of pea protein with sweeteners for 2 hours. After the incubation period, CCK (Figure 1A) and GLP-1 (Figure 1 B) levels were measured in the supernatant. Results are expressed as mean ± SEM. * indicates a result significantly different from the negative control, p<0.05). Figure 2 is a graphical representation of hormone release from human duodenal biopsies in Ussing chambers after 2h exposure to sucrose, sucralose, pea protein, and combinations of pea protein with the sucralose or sucrose. Duodenal biopsies of lean and obese subjects were exposed to sucrose, sucralose, pea protein, or a combination of pea protein with sucrose or sucralose. The levels of CCK (Figure 2A) and GLP-1 (Figure 2B) were measured in the supernatant of basolateral side of the biopsies in the Ussing Chambers after being exposed to proteins for 2h to the apical side. Results are expressed as mean ± SEM. * indicates results significantly different from negative control, p<0.05). $ indicates results significantly different from lean subjects

Detailed Description

In general terms, the present invention provides an edible product comprising plant protein; a sugar or sugar substitute (which may collectively be referred to as "sweeteners"); and a delivery vehicle for at least partially protecting the plant protein from hydrolysis.

Plant Protein The plant protein may be obtained from any plant. The plant protein is distinguished from naturally occurring sources of plant protein (e.g. raw plants) in that the plant protein is isolated from the other components of plants (e.g. starch and fibers) with which it is associated in its naturally occurring form. Thus, in some embodiments, the plant protein comprises at least 50%, 60%, 70%, 80%, 90%, 95% or 99% by weight protein.

The plant protein may be a single type of protein (i.e. a protein of a single amino acid sequence) but is more preferably a mixture of different proteins. The proteins may be obtained from a single plant species or variety or may be a mixture of protein extracts from different plant species or varieties.

It is preferred that the plant protein is obtained from a plant that is generally regarded as edible. Exemplary plants are pea plants, wheat, soy and potato. Examples of products comprising potato protein include Satietrol™ and Slendeista®. It is important for the efficacy of the present invention that the plant protein, or at least a significant proportion of the plant protein, is intact after being consumed by an individual and passing through the stomach of the individual. The term "intact plant protein" in this context is to be understood to mean non-hydrolysed pea protein. This means that the protein bonds in the intact plant protein fraction should be intact, i.e. a degree of hydrolysis (DH) of less than 5% or even 0%. The Degree of Hydrolysis (DH) may be determined using a rapid OPA test (Nielsen, P.M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 2001 , 66, 642-646).

In the context of the present invention, the term intact plant protein is therefore to be interpreted to mean a preparation comprising at least 1 % non-hydrolysed plant protein, preferably more than 10%, more preferably over 20%, 30%, 40% or 50%, even more preferably over 60%, 70%, 80% or 90%, such as more than 92%, 94%, 96%, 97%, 98% or 99%. The invention therefore relates to an edible product as described above wherein the delivery vehicle comprises between 1 % and 100% intact plant protein as a fraction of the total protein content of the edible product. Intact plant protein may be obtained from commercial sources or freshly isolated from plants. Pea Protein

A preferred plant protein is pea protein. It is particularly preferred that the pea protein is an extract of a plant of the species Pisum sativum such as dried yellow or gold peas. In principle, any of the available varieties of the species Pisum sativum may be used. Pitsum sativum is of the tribe Vicieae and the subfamily Faboideae.

The amino acid profile of the pea protein is set out in Table 1.

Table 1 g/kg %/prot

Dry matter 867.0

Prot (N*6.25) 212.0

LYS 15.1 7.5

MET 2.1 1.0

CYS 3.2 1.6

MET+CYS 5.3 2.6

THR 7.8 3.9

TRP 1.9 0.9

ILE 8.7 4.3

ARG 18.7 9.3

PHE 10.0 5.0

HIS. 5.3 2.6

LEU 15.1 7.5

TYR 7.0 3.5

Phe+Tyr - 8.4

VAL 9.8 4.9

ALA 9.3 4.6

ASP 24.8 12.3

GLU 35.4 17.5

GLY 9.3 4.6

PRO 8.5 4.2

SER 10.0 5.0

Sum AA 202.0 100.0

CVB, 2000. [Feedstuff-tables. Chemical composition, digestibility, and feeding value of feedstuffs.] Central Bureau for Animal Nutrition, Lelystad, the Netherlands.

The pea protein may be obtained from commercial sources (such as Nutralys® F pea protein and Nutralys® S pea protein of Roquette, France) or freshly isolated from peas. A typical process for obtaining pea protein is to harvest peas which are then dried and milled in order to produce a pea flour. The flour is then hydrated and starch and fibres are separated out and removed. The proteins are then flocculated and the pea protein is then purified and carefully dried in an atomiser.

Thus in embodiments of the present invention, the pea protein is isolated from the components with which it is associated in nature.

The pea protein is a particularly preferred plant protein for use in the present invention because it is readily available, is relatively inexpensive and does not have a strong flavour.

Sugar

The sugar provided in embodiments of the invention may be any edible crystalline carbohydrate. Exemplary sugars are: sucrose, lactose, fructose, glucose and mixtures thereof. In preferred embodiments, the sugar is sucrose since it has been found that the combination of sucrose and pea protein is particularly effective in raising GLP-1 levels in individuals which is indicative of increasing or inducing satiety in individuals. However, in some embodiments, the sugar is a sugar other than sucrose. It is generally preferred that the sugar is not an inactive water soluble sugar such as lactose, mannitol or tretalose.

Sugar Substitute

As an alternative to sugar, embodiments of the present invention may instead comprise a sugar substitute or a mixture of one or more sugars and one or more sugar substitutes. The advantage of providing a sugar substitute is that in many cases the sugar substitute has little or no food energy thus the edible product not only increases or induces satiety in the individual but also does not give rise to significant caloric intake when consumed by the individual.

The sugar substitute may be any natural or synthetic food additive (aside from a sugar) that has a sweet flavour. One category of sugar substitute is sugar alcohols. Examples of sugar alcohols are xylitol, sorbitol, maltitol erythritol, isomalt and lactitol. The other category of sugar substitutes is the high intensity sweeteners which include both naturally occurring and non-naturally occurring high intensity sweeteners. The naturally occurring high intensity sweetener may be a protein such as Thaumatin, Brazzein or Monellin. Other suitable naturally occurring high intensity sweeteners include Stevia , a Steviol glycoside (such as stevioside, steviol, Rebaudioside A , Rebaudioside B, Rebaudioside C, Rebaudioside D and Rebaudioside E), ogroside V (Lo Han Guo ), Monatin and Glycyrrhizin.

Alternatively, the high intensity sweetener may be non-naturally occurring such as a peptide (e.g. aspartame, neotame, advantame or alitame) or a halosugar (e.g. sucralose). Other exemplary non-naturally occurring high intensity sweeteners include Acesulfame-K, Saccharin (including sodium, potassium and calcium salts thereof), Cyclamate (that is to say the sodium salt of cyclamic acid, although cyclamic acid, itself, or the calcium salt thereof are other examples) and neohesperidin DC.

Another exemplary high intensity sweetener is a salt of aspartame acesulfame.

It is particularly preferred that sucralose is provided as part of the edible product since it has been found that the combination of sucralose and plant protein (more specifically pea protein) is particularly effective in raising levels of CCK and GLP-1 in individuals which is indicative of increasing or inducing satiety in individuals. Furthermore, sucralose is not hydrolyzed in the stomach after consumption and therefore does not require the provision of a delivery vehicle in order to deliver it to the duodenum where it is believed to have its satiety inducing and/or increasing effect in conjunction with the plant protein. Another preferred sugar substitute is Acesulfame K which has also been found to be very effective in raising GLP-1 levels in individuals when administered in combination with plant protein (more specifically pea protein).

In further embodiments, the sugar substitute may comprise a precursor that is digested into a food additive having a sweet flavour in the stomach and/or duodenum of an individual. Thus in these embodiments, the sugar substitute, itself, may not have a sweet flavour but its digested product does have a sweet flavour. The digested product may be a natural or synthetic food additive, as described above, or may be a sugar. An example of such a sugar substitute is maltodextrin which is a polysaccharide consisting of D-glucose units and which is easily digestible to give a relatively constant delivery of glucose. Delivery Vehicle

It has been reported in WO2009/053487 that, while CCK and GLP-1 release is elevated after exposing duodenal tissue to intact pea protein this satiating effect is observed to a lesser extent when pea protein hydrolysates are used. Accordingly, it is necessary for an edible product of the present invention to comprise plant protein in a delivery vehicle that protects the plant protein from hydrolysis in the first part of the gastrointestinal tract, in particular the stomach. Furthermore, certain sugars and sugar substitutes are also be subject to hydrolysis in the stomach and may therefore be comprised in a delivery vehicle. For example, aspartame is normally broken down into its constituent amino acids (phenylalanine and aspartic acid) in the stomach and thus, in some embodiments in which the sugar substitute is aspartame, the aspartame is incorporated in a delivery vehicle. It is believed that, without a delivery vehicle, about 90% of plant protein is hydrolyzed (depending on the food matrix) in the human stomach after consumption. Therefore, the delivery vehicle must protect the plant protein from hydrolysis to the extent that greater than 10% of the plant protein by weight consumed by an individual remains intact on reaching the duodenum of the individual. It is preferred that greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the plant protein by weight consumed by an individual remains intact on reaching the duodenum of the individual.

The plant protein and optionally the sugar or sugar substitute, can be delivered intact to the human duodenum in several different ways. Suitable delivery vehicles include capsules, tablets or particles such as micropellets, microparticles or capsulated microparticles. Such delivery vehicles increase the resistance of the intact plant protein and optionally the sugar or sugar substitute (referred to hereafter as "the active components") against hydrolysis. The delivery vehicle may encapsulate, include, encompass and /or contain the active components.

The delivery vehicle is a vehicle that is suitable for enteric delivery, i.e. it should be suitable to be swallowed by the individual and it should be capable of passing through the gastrointestinal tract of the subject without getting blocked. Such a vehicle is often referred to as a gastrointestinal delivery vehicle. In all cases, the intact active components in the vehicle need to overcome the acidic environment of the stomach. One particularly advantageous way to achieve that goal is to provide the active components in a core with an outer enteric coating. Thus in some embodiments, the delivery vehicle comprises an enteric coating. Suitable enteric coatings include pH-triggered coatings, pressure-sensitive coatings or time- released coatings. Such coatings are disclosed in Bodmeier, R., H. G. Chen, and O. Paeratakul, A novel approach to the oral delivery of microparticles. Pharm Res, 1989. 6(5): p. 413-7; Dhaliwal, S., et al., Mucoadhesive microspheres for gastroretentive delivery of acyclovir: in vitro and in vivo evaluation. Aaps J, 2008. 10(2): p. 322-30; lchikawa, H. and Y. Fukumori, [Design of nanohydrogel-incorporated microcapsules for appropriate controlled-release of peptide drugs]. Yakugaku Zasshi, 2007. 127(5): p. 813-23; Mustata, G. and S. M. Dinh, Approaches to oral drug delivery for challenging molecules. Crit Rev Ther Drug Carrier Syst, 2006. 23(2): p. 111-35; Clear, N.J., et al., Evaluation of the Intelisite capsule to deliver theophylline and frusemide tablets to the small intestine and colon. Eur J Pharm Sci, 2001. 13(4): p. 375-84; and Malik, D. K., et al., Recent advances in protein and peptide drug delivery systems. Curr Drug DeNv, 2007. 4(2): p. 141 -51 each of which is incorporated herein by reference. For duodenal delivery of relatively large amounts of the active components, a pH- sensitive coating (e.g. a polymer coating) used on particles such as micropellets or microparticles is highly suitable. Tablets or capsules are also feasible. Particles are preferred however because they are easier to mix with foodstuff and large amounts of active component may be administered in the form of particles whereas the swallowing of large amounts of capsules is often considered problematic and troublesome. Moreover, the contact area of particles may be more advantageous resulting in a slower release of the active components. The use of compositions comprising the active components encapsulated into particles, such as micropellets or microparticles is thus preferred.

In some embodiments, there is provided an edible product as described above wherein the delivery vehicle is a particle. In another embodiment, there is provided an edible product as described above wherein the particles are micropellets or microparticles. In one embodiment of the present invention, an orally administrable particle containing the active components is formed by encapsulating the active components with an enteric coating. As used herein the term "enteric coating", is used to mean a material such as a polymer material or materials which encases the core consisting of the active components. As such, the polymeric enteric coating material does not usually contain any of the active components. It is preferred that a substantial amount or the entire enteric polymer coating material is dissolved before the active components are released from the delivery vehicle, so as to achieve delayed dissolution of the active component core.

A suitable pH-sensitive polymer is one which dissolves with intestinal juices at the higher pH levels (such as pH greater than 4.5), such as found within the small intestine and therefore permit release of the pharmacologically active substance in the regions of the small intestine and not in the upper portion of the Gl tract, such as the stomach. The polymer coating material may be selected such that the active components are released when the delivery vehicle reaches the small intestine or a region in which the pH is higher, such as more than pH 4.5. Preferred coatings are pH-sensitive materials, which remain intact in the lower pH environs of the stomach, but which disintegrate or dissolve at the pH commonly found in the small intestine of the patient. A very suitable enteric polymer coating material begins to dissolve in an aqueous solution at pH between about 4.5 to about 5.5. The pH-solubility behaviour of the enteric polymers are usually such that significant dissolution of the enteric polymer coating does not occur until the delivery vehicle has emptied from the stomach. The pH of the small intestine gradually increases from about 4.5 to about 6.5 in the duodenal bulb to about 7.2 in the distal portions of the small intestine (ileum). In order to provide predictable dissolution corresponding to the small intestine transit time of about 3 hours and permit reproducible release therein, the coating may begin to dissolve within the pH range of the duodenum and continue to dissolve at the pH range within the small intestine. Therefore, the amount of enteric polymer coating may be such that it is substantially dissolved during the approximate three hour transit time within the small intestine. There are means available in the art to form particles such as micropellets or microparticles from protein preparations.

An efficient way to produce such particles has been described in US6,224,910, which is incorporated herein by reference. The active components may accordingly be dispersed in an aqueous solution. The aqueous solution may also be sprayed onto nonpareils. Nonpareils are small, usually round particles of pharmaceutically inert materials. Generally, nonpareils that are formed from the combination of sucrose and starch are preferred. One such brand is Nupareils which is sold by Ingredient Technology Corporation. The preferred size is 500-595pm although sizes between 37 m and 4.76mm may be equally suited, depending on the specific intended use of the eventual particles, micropellets or microparticles.

Alternatively, particles such as micropellets, microparticles or microspheres (beads) may also be formed by any other conventional means, with or without the addition of filler substances. This allows for the formation of beads with a high load of active components. The active components should be capable of becoming somewhat sticky upon moistening or otherwise they should be mixed with minute amounts of suitable binders and optionally disintegrants. Hence, the delivery vehicle may also include one or more disintegrants or swelling agents in any practical amount. Conventionally, amounts within the range from about 1 % to about 4% by weight of the composition are preferred. Preferred disintegrants or swelling agents are sodium starch glycolate marketed under the trademark EXPLOTAB (Edward Mendell Co.), Ac-Di-Sol (cross- linked sodium carboxymethylcellulose) (FMC Corp), croscarmellose sodium, corn starch, or cross linked polyvinylpyrrolidone. A major portion of the active component blend may be wet massed extruded and spheronized as is conventionally performed in the art of bead formation whereas a minor portion of the blend may be used for dusting to prevent adhesion and sticking of the beads. One or more binders may be present in the core in any practical amounts. Conventionally, amounts within the range of from about 0 to about 10% are preferred, even more preferred are amounts of about 1 % by weight of the composition. Sodium carboxymethylcellulose is a preferred binder most suitable for use herein. Examples of other binders which may be used include Avicel. TM. PH101 , Avicel.TM. RC 591 , Avicel.TM. CL-61 1 , (FMC Corp), Methocel.TM. E-5 (Dow Corp.), Starch 1500 (Colorcon, Ltd.), Hydroxypropyl Methylcellulose (HPMC) (Shin-Etsu Chemical Co., Ltd.), Polyvinylpyrrolidone, Potassium Alginate and Sodium Alginate.

Another component which can be added to the active components is a stabilizing agent. Stabilizing agents provide physical protection for the active components, in particular the plant protein. Exemplary stabilizing agents include sugars such as lactose, mannitol and trehalose although these are to be distinguished from the sugar or sugar substitute that confers satiety. These act to protect the intact plant protein during the coating process. One advantageous way to form orally administrable particles such as micropellets or microparticles or microcapsules for use in the present invention is the following. An aqueous solution of the active components and the optional stabilizing agent is formed. The aqueous solution may include generally from about 0.5 to about 20% by weight of the intact plant protein with about 4 - 8% being preferred, from about 0.005% and 0.05% sugar or sugar substitute and from about 1 % to about 10% by weight of the stabilizing agent with about 5% being preferred. However, in some embodiments the delivery vehicle itself is substantially free of sugar, sugar substitute and/or plant protein. For example, the delivery vehicle may include 0%, less than 5% or less than 10%, by weight of any or all of these components. If the protein solution is to be sprayed on a nonpareil and has a low viscosity, it may be desirable to add 1- 0% of polyvinylpyrrolidone to bind the active components to the nonpareil.

The nonpareils may be coated with an amount of the aqueous active component solution to provide a coating such as for instance of 1 -10% plant protein by weight on a solids basis. Glatt brand powder coater granulators such as the GPCG-1 , GPCG-5, or GPCG-60 fluid bed coaters are suitable for use in this application. Coating conditions and times vary depending on the apparatus and coating viscosity. However, generally coating steps are best conducted at less than 50^° C and preferably less than 37^°C to avoid denaturing the pea protein. Subsequently the particles are coated with a water emulsion of a polymer which upon solidification is acid resistant. This protects the active components as they pass through the stomach and releases them into the small intestines where they can act to induce satiety. The particles or active component coated nonpareils are dried and subsequently coated with an acid stable polymer (enteric coating). Generally, the coating is applied in the same manner as the active components and with the same equipment. The coating composition is preferably a water based emulsion polymer. The preferred coating is an ethylacrylate methacrylic acid copolymer sold under the trademark Eudragit L 30D manufactured by Rhom Pharma. This has a molecular weight of about 250,000 and is generally applied as a 30% aqueous solution. An alternative coating is hydroxypropylmethyl cellulose acetate succinate.

Although Eudragit is the preferred coating polymer, the invention is not limited in this respect and other enteric coating polymers known in the art, such as hydroxypropyl methylcellulose phthalate HP50 (HPMCP- HP50) (USP/NF 220824), HP55 (HPMCP- HP55) (USP/NF type 200731 ) and HP55S available from Shin Etsu Chemical, Coateric.TM. (polyvinyl acetate phthalate) (Colorcon Ltd.), Sureteric.TM. (polyvinyl acetate phthalate) (Colorcon, Ltd.), or Aquateric.T . (cellulose acetate phthalate) (FMC Corp.) and the like may be employed instead.

In some embodiments, the coating composition is combined with a plasticizer to improve the continuity of the coating. Such plasticizers include triethyl citrate (Citroflex- 2), diethyl phthalate, triacetin, tributyl sebecate, and polyethylene glycol. Optionally an anti-adherent (anti-agglomerant) which is advantageously a hydrophobic material such as talc, magnesium stearate or fumed silica, with talc being referred, is applied after coating the beadlet or pellet. Thethylcitrate (TEC) sold by Morfley Inc. is a particularly preferred plasticizer. This can form about 1 -30% of the coating composition. Although plasticizers can be liquid, they are not considered to be solvents since they lodge within the coating altering its physical characteristics. It is preferred that the plasticizer does not act to dissolve the active components.

Talc (such as at 3.0% of coating composition) can also be added to prevent sticking between the particles if desired. Also, an antifoaming agent (such as for instance 0.0025% of coating composition) such as sorbitan sesquioleate (Nikko Chemicals Company Limited) or silicone may be added.

In some embodiments, particles comprising the active components and optional the stabilizing agents, are dried and are then coated with the enteric coating as previously described. The coating solution may be about 30% polymer, 0-30% plasticizer, 0 to 3% talc and 0 to 0.0025% antifoaming agent and water. It is desirable that there are no organic solvents including alcohols or even glycols present in the coating composition. The presence of these solvents during coating application can denature the intact plant protein. The coating is conducted in the same equipment used to coat the nonpareils with intact protein. The temperature for this coating should be at an optimum to ensure proper coating and as little as possible denaturation of the intact plant protein i.e. a temperature between about 30°C and 50^°C is preferred.

In preferred embodiments, the coating is made from components which naturally exist in food products. For example, the coating may be a shell made from a combination of shellac, carbohydrates and fatty acids. A particular commercial example of such a coating is Liposphere™ which can be used to make ultra fine shell coatings on powered ingredients and can be engineered to allow constant release of the active components at the neutral pH of the small intestine.

Particles comprising the active components may have any size distribution. Usually the size distribution is determined by the intended use. Preferred is a minimum size of 0.01 mm or more such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 0.08, 0.09, or 0.1 mm whereas the maximum diameter is determined by the ability of an individual to swallow the particles. A maximum diameter of 5 mm is preferred; however, less than 4, 3, or 2 mm, such as less than 1 mm is more preferred. Microparticles usually range in size between 1 and 100 μηι. Micropellets consist of agglomerates of particles or microparticles and can have any size that is practically useful. It is also to be noted that in some embodiments, the plant protein is provided in a first delivery vehicle and the sugar or sugar substitute is provided in a second delivery vehicle. For example, in one embodiment the plant protein is formed into a first set of enterically coated particles and the sugar or sugar substitute is formed into a second set of coated particles and the first and second sets of particles are mixed together.

The incorporation of sensitive proteins and the like into particles in order to protect the proteins from hydrolysis is known in the art. The term "a delivery vehicle for at least partially protecting the plant protein from hydrolysis" means that the vehicle such as particles are capable of increasing the resistance of the plant protein against hydrolysis such as enzymatic hydrolysis, e.g. by trypsin, chymotrypsin or pepsin or by acid hydrolysis under conditions comparable to a human stomach. Artificially, in a laboratory environment, a suitable test for determining the resistance of plant protein against hydrolysis is the incubation of the plant protein at a pH of approximately 1.5 as can be achieved by using more than 0.5N HCL, such as 1 N, 2N, or 4N for 10 minutes or more, such as 20 minutes, 30 minutes or 1 hour and then determining the degree of hydrolysis according to the method mentioned above. Increasing the resistance against hydrolysis in this context means an increase in the fraction of intact plant protein versus the fraction of hydrolysed plant protein when the plant protein is exposed to hydrolyzing conditions as outlined above. Such an increase should be measurable by determining the DH according to the methods as described above. Preferably, the increase should be 10 % or more, such as 20% 40%, 60%, 80 or more than 90%. An increase of resistance of 100% would mean that the amount of intact plant protein which is protected against hydrolysis is double the amount of intact plant protein which is not protected against hydrolysis.

Edible Product

The plant protein, sugar or sugar substitute and delivery vehicle (hereinafter referred to as "the prepared components") together form all or part of an edible product such as any conventional food or feedstuff. The prepared components may be mixed with drinks, such as fruit or dairy drinks, for example yoghurt, milk, buttermilk, cream, pudding. Alternatively, the prepared components may be incorporated in more solid food such as bread, cake, pastry, cheese, chocolate, butter, sweets (candy), muesli or chocolate bars (candy bars).

In other embodiments, the edible product is a food supplement. The term "food supplement" means any food component which provides specific nutritional or medicinal components and does not provide the full energy value required (i.e. generally less than 2000 or 2500 kcal/day) for an individual and includes food supplements in the form of a powder or medicament, as well as health products, such as health drinks. An ingredient that can be added to food before consumption or a preparation that can be consumed as such is also encompassed within the term.

In embodiments where the edible product is a beverage or food product, the prepared components can be combined with any common food ingredient. The term "beverage" is meant to include cordials and syrups, as well as formulations of a dry powder to be dissolved in water or another liquid component for the preparation of instant drinks such as juices, soups, yoghurt and other dairy stuff. In one specific embodiment, the prepared components are formulated as a dry powder for mixing by a consumer with water or milk in order to produce a protein shake. Other optional components of the dry powder include flavourings, and other proteins such as whey protein, casein protein, soy protein, egg-white protein, hemp seed and mixtures thereof.

In another specific embodiment, the prepared components are formulated as part of a food bar. Other optional components of the food bar are: flavourings, chocolate, cereals (such as rolled oats), nuts, honey, fruit, rice or mixtures thereof.

The prepared components may be placed in gel capsules for oral administration.

The preferred dosage range of the plant protein is between 0.1 and 1 g / kg bodyweight per day, with a range between 0.2 and 0.3 g/kg/day being preferred and 0.25 g/kg/day being particularly preferred. However, dosages outside this range are possible with 10g/kg/day being an effective upper limit. Other exemplary dosages of the plant protein are between 0.5 and 5 g/kg bodyweight per day preferably between 0.8 and 2 g/kg bodyweight per day, such as 0.9, 1.0, 1.2, 1.4, 1.6, andl .8 g/kg/day. Accordingly, the plant protein is typically provided in an amount of between 5g and 30g in the edible product, more preferably between 10g and 20g and more preferably between 13g and 17g, such as 15g.

The dosage ranges of the sugar differ from those of the sugar substitute since sugar substitutes typically are much sweeter than sugar.

Sugar is typically provided in an amount at least 2 times greater than the amount of the plant protein, by weight. Thus the edible product may comprise between 5g and 30g sugar, for example, or between 10g and 20g sugar. However, in some embodiments, the edible product comprises 2.5, 3, 4 or 5 times greater amounts of sugar than plant protein, by weight. In some embodiments, the edible product comprises at least 5% or at least 10% sugar and/or sugar substitute, by weight.

The dosage range of the sugar substitute may be between 50 and 500mg per kg of total edible product, preferably between 100 and 300mg/kg such as 200mg/kg. Alternatively, the dosage range of the sugar or sugar substitute may be between 50 and 500mg per I of total edible product in the case of a drink, preferably between 100 and 300mg/l such as 200mg/l. Since the edible product typically has a mass of between 40 and 200g, exemplary proportions (weight by weight) of the plant protein in the edible product are between 1 % and 75%, for example at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 75% plant protein or even at least 80% or 90% in some cases, or less than 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 75% plant protein. Exemplary amounts of sugar in the edible product are between 20% and 90% (weight by weight) for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Exemplary amounts of sugar substitute in the edible product are between 2mg and 100mg. Exemplary proportions (weight by weight) of the sugar substitute are between 0.005% and 0.05% of the total edible product.

Medicament

In some embodiments there is provided a medicament comprising intact plant protein and a sugar or sugar substitute. In these embodiments, the provision of a delivery vehicle for protecting the plant protein from hydrolysis is not essential since the medicament may be applied directly to the duodenum of an individual (i.e. bypassing the stomach). The invention therefore relates to a composition as described herein for use as a medicament. In embodiments where a medicament is provided, intact plant protein and sugar or sugar substitute is administered alone or in a mixture with a pharmaceutically acceptable adjuvant, carrier, diluent or excipient, in suitable pharmaceutical formulations. Examples of said formulations, and suitable carriers, diluents and excipients are described in "Remington's Pharmaceutical Sciences Handbook", Mack Pub. Co., N.Y. U.S.A. For example, for oral administration tablets, capsules, syrups, and the like may be provided, whereas for parental administration suitable formulations are sterile solutions or suspensions in acceptable liquids, implants, etc. The exact dosages will depend on several factors such as type and seriousness of the pathological conditions to be treated, patient's weight and sex, etc. but are in principle the same as described above in relation to the edible product. In some embodiments, the intact plant protein and the sugar or sugar substitute are administered separately, either simultaneously or sequentially. Uses of the product

The products described above, that is to say the edible product or medicament, are used as will now be described. In some embodiments, the edible product is eaten or drunk by an individual in order to induce or increase satiety in the individual. More specifically, when feelings of hunger arise, the individual consumes the edible product. The plant protein (and optionally the sugar or sugar substitute) are protected by the delivery vehicle and so pass through the stomach to the duodenum of the individual where they are released. In embodiments where the sugar or sugar substitute is not incorporated into the delivery vehicle, the sugar or sugar substitute is such that it in any case passes through the stomach of the individual without being broken down, or at least, with a significant proportion remaining intact. When the combination of the intact plant protein and the sugar or sugar substitute comes into contact with the duodenum, levels of CCK and/ or GLP-1 increase in the bloodstream of the individual with the effect that feelings of hunger are reduced or eliminated in the individual, that is to say that satiety is induced or increased in the individual.

The edible product is typically consumed once per day but may be consumed two, three or more times per day in some embodiments such as before meals. What is important is that the individual receives the dosage of plant protein and sugar or sugar substitute as set out above. The edible product may be eaten as part of a meal or may be eaten as a snack between or before meals. In alternative embodiments, the medicament described above is provided to an individual suffering from obesity. The obesity may be clinical in the sense of being a pathology or may be non-clinical in the sense of being unrelated to the health of the individual and being instead a cosmetic matter, such that the treatment merely controls the weight of the individual. The medicament may be consumed by the individual and, if so, is typically formulated as a syrup or as one or more pills or capsules. Alternatively, the medicament may be administered directly to the duodenum, for example, in conjunction with an endoscope.

The effect of the medicament is as described above in relation to the edible product except that after continued use, the repeated inducing or increasing of satiety in the patient results in the patient consuming less food (i.e. fewer calories per day) and thereby prevents weight gain or even causes weight loss in the patient thus treating or ameliorating obesity in the patient. In principle, the medicament may also be used for the therapy or prophylaxis of other conditions that are mediated by GLP-1 and/or CCK, such as diabetes, in particular type 2 diabetes.

It is particularly preferred that the methods described above are applied in relation to individuals who are overweight (i.e. have a BMI of greater than 25) or who are obese (i.e. have a BMI of greater than 30) since there is experimental evidence that indicates that the products of the invention have greater efficacy against such individuals.

Examples Example 1

This example tested the effect of sugars and sugar substitutes (hereafter "sweeteners") with and without pea protein on release of satiety hormones CCK and GLP-1 in vitro. MATERIAL AND METHODS

Test products

All sweeteners were dissolved in 300ml of HBSS (200ml as volume for one coffee or tea consumption, and 100ml as basal gastric juice volume). All sweeteners were dissolved accordingly to match the sweetness of the sugar dosages, according to its known sweetness equivalent relative to sucrose. Five different tastants were used, namely sucrose (6g, Sigma-Aldrich, St. Louis, MO, USA), aspartame (0.03g), acesulfame K (0.04g), saccharine (0.012g) (all from Supelco, Bellefonte, PA, USA), and sucralose (0.01 g, Tate&Lyle, London, UK). From previous studies it was shown that pea protein is a very potent protein to stimulate the release of CCK and GLP-1 , and it also reduces food intake in male subjects, when infused intraduodenally (unpublished data). Pea protein (0.1 mg/ml, Dutch Protein Services, Tiel, The Netherlands) was added to the sweeteners. Cell culture conditions

The STC-1 cell line is derived from an intestinal endocrine tumor that developed in a double-transgenic mouse expressing the rat insulin promotor linked to the simian virus 40 large T antigen and the polyoma small T antigen [20]. STC-1 cells (kindly provided by Dr. D. Hanahan, University of California, San Francisco) were maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units ml-1 penicillin, and 100 pg ml-1 streptomycin as additional supplements, at 37°C in 5% C02/air humidity. All products were obtained from Invitrogen, Carlsbad, California, USA, unless stated otherwise. Secretion of CCK and GLP-1 from STC-1 cells

To determine the secretion of CCK and GLP-1 from STC-1 cells, suspensions of these cells were plated at 1.0 x 105 cells/well in 24-well plates (Costar) and assays were performed on cultures that reached at least 80% confluency. Before treatment with the test products, culture medium was removed and dishes were rinsed with HBSS. Cells were then incubated with HBSS (negative control), the sweeteners, and the combination of sweeteners with pea protein, and were incubated at 37°C for 2 hours. The supernatant was collected for the measurement of CCK and GLP-1. All analysis was performed in triplicate, using three biological replicate samples.. Statistical Analyses

The descriptive and statistical analyses were performed with SPSS, version 11.0. With regard to the cell culture results, the means of the secreted hormones were compared using the one-sample t-test. Means of the secreted hormones between groups were compared using an unpaired Student t-test. Means of the secreted hormones within a group was were compared using a paired Student t-test. All Student t-tests were corrected for multiple testing using the Bonferroni correction. The means of the variables are presented with their standard error (mean ± SEM). A P-value of less than 0.05 was considered statistically significant. RESULTS

Hormone release from STC-1 cells

The release of CCK and GLP-1 from the enteroendocrine STC-1 cells are presented in Figure 1. As can be seen in Figure 1A, CCK release was significantly increased after addition of aspartame, sucralose, sucrose, pea, and pea with sucralose, when compared to the negative control. The highest levels of CCK release were found after addition of the combination of pea with sucralose (82.5 pM ± 0.7). As can be seen in Figure 1 B, addition of all test products significantly increased the release of GLP-1 from the STC-1 cells when compared with the negative control (only HBSS buffer). Addition of acesulfame K, pea with acesulfame K, pea with sucrose, or pea with sucralose (2442.2 pM ± 60, 3039.5 pM ± 22, 4336.6 pM ± 93, and 4690.9 pM ± 38, respectively) induced the highest levels of GLP-1.

Example 2

The sweeteners that gave the best results in Example 1 in combination with pea protein were then tested in Ussing Chambers.

MATERIAL AND METHODS

Human Ex vivo assay

Subjects

In this example, ten (five lean and five obese) healthy male subjects were recruited. Selection took place according to health criteria (no diabetes, no gastrointestinal diseases, and no medical treatment) and body weight (BW) criteria (for lean subjects: body mass index (BMI) 18-25 kg/m2, and for obese subjects: BMI<30 kg/m2). Baseline characteristics of the subjects are presented in Table 2. The nature and risks of the experimental procedure were explained to the subjects, and all subjects gave their written informed consent. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the Medical Ethical Committee of the University Hospital Maastricht approved all procedures involving human subjects. Lean (n=5) Obese (n=5)

Age (years) 27 ± 5 37 ± 10

B I (kg/m²) 24.2 ± 1 32.8 ± 2 ^*

HbAl c (%) 4.6 ± 0.2 4.4 ± 0.2

Basal glucose (mmol/L) 4.9 ± 0.2 4.8 ± 0.2

Table 2. Subject characteristics. All data are mean ± SEM. * Difference between lean and obese subjects (p<0.05) Duodenal tissue sampling for ex vivo experiments

All subjects received a standardized meal (9 g protein, 39.5 g carbohydrates, 16 g fat) for the evening prior to the test day to standardize macronutrient intake. After an overnight fast, eight mucosal tissue samples from the horizontal part of the duodenum were obtained by flexible gastroduodenoscopy using standard biopsy forceps. During this procedure, no sedatives were given to the subjects. The diameter of the biopsies varied from 2.0 mm to 2.2 mm. After sampling, the biopsies were placed in ice-cold Krebs-Ringer bicarbonate buffer (KRB) and arrived at the laboratory within 15 min.

Ussing Chamber experiments

Duodenal biopsies were mounted in modified Ussing Chambers (Harvard Apparatus Inc., Holliston, Mass., USA) with a ø 9-mm opening and reduced to an exposed tissue area of 1.76 mm2, using a technique previously described by Wallon et al [21 , 22]. Mucosal compartments were filled with 1.5ml 10mM mannitol in KRB and the serosal compartments were filled with 10mM glucose in KRB. The chambers were kept at 37°C and continuously oxygenated with 95% 0₂ / 5% C0₂ and circulated by gas flow. Before the experiments were started, tissues equilibrated for 40 min in the chambers to achieve steady-state conditions in transepithelial potential difference (PD), with replacement of mannitol of glucose KRB at 20 min. A four-electrode system was used, as described previously [23]. One pair of Ag/CI electrodes with 3M NaCI / 2% agar bridges was used for measurement of transepithelial PD and another pair of Ag/CI electrodes was used to monitor current. The electrodes were coupled to an external 6- channel electronic unit with a voltage controlled current source. Data sampling was computer controlled via an A/D D/A board (Lab NB, National Instruments, USA) by a program developed in LabVIEW (National Instruments, USA) by Wikman-Larhed et al

[24]. Every other minute, direct pulses of -3, 3, and 0 μΑ, with a duration of 2 seconds each, were sent across the tissue segments and the voltage response was measured. In each measurement, the mean voltage response of 2 seconds was calculated. A linear-squares fit was performed on the current (I) - voltage (U) pair relationship: U = PD + TER x I. The TER was obtained from the slope of the l-U line and the PD from the intersection of the voltage.

After the equilibration period, the mucosal side of the biopsies were exposed to sucrose, sucralose, pea, pea with sucrose, and pea with sucralose. Serosal samples (1.25 ml) were collected the end of the experiment (after 2h) for CCK- and GLP-1 analysis.

Biopsies with PD less negative than -0.5mV were excluded from all tested (n=3) because of malfunction in the ability to uphold normal electrophysiology. Electrical parameters were measured. These parameters are widely accepted for monitoring the viability and integrity of tissue in the Ussing Chambers. In general, PD reflects the voltage gradient generated by the tissue, TER reflects the tissue integrity, and Isc reflects the ionic fluxes across the epithelium [35-37]. Basal electrical parameters varied over a wide range. This variability has also been noticed in previous studies on human tissue samples from jejunum [38] and colon [39]. Moreover, the reported electrical parameters from investigators using different Ussing chambers on biopsy specimens from the same gastrointestinal region have been associated with a large variability. To correct for this variability, the areas under the curves have been calculated, after correcting for baseline values for each biopsy. The electrophysiology results from the study were comparable with those found in the literature, and showed that all biopsies used in this study were viable throughout the experiments. Addition of sweeteners, pea protein, or a combination of both did not affect the resistance of the tissue, but the Isc was significantly increased compared to the negative control. The Isc is a marker for transepithelial ion transport. Once food compounds stimulate enteroendocrine cells, Ca2+ will be transported into the cell, resulting in release of satiety hormones [40]. This influx may have been the cause of the increase in Isc.

Hormone assays

CCK levels were determined using the RIA from Euria-CCK, Euro-Diagnostica AB, Malmo, Sweden. According to the manufacturer's instructions, the detection limit of this kit was 0.3 pmol/L. The intra-assay variation ranges from 2.0 to 5.5% and the inter- assay variation from 4,1 to 13,7%. Cross-reaction with gastrin is≤ 0.5%. Total GLP-1 levels were determined using the RIA from Linco Research, Missouri, USA. The detection limit of this kit was 3 to 333 pM. The intra-assay variation ranges from 10 to 23% and the inter-assay variation from 22 to 38%. There is no cross-reaction with GLP-2 and glucagon (0.01 % and 0.2%, respectively). GLP-1 samples were spiked with 100 pM of GLP-1 to be within range of the detection limit. Both RIAs can be used for the analysis of both rat and human samples. Statistical Analyses

The same analyses and tests were used as in Example 1. With regard to the Ussing chambers, the electrophysiological parameters were compared using the Wilcoxon signed rank test.

RESULTS

Hormone release from Ussing chamber experiments

In duodenal tissue of lean subjects basal CCK secretion levels of 6.4 pM ± 2 in lean subjects were observed, whereas basal CCK levels in obese subjects were 9.7 pM ± 4 (Figure 2A). After addition of pea, pea with sucrose, or pea with sucrose, the levels of CCK were significantly increased compared to the negative control, for both lean (27.1 pM ± 1 , 22.6 pM ± 3, and 56.7 pM ± 1 , respectively) and obese subjects (37.3 pM ± 2, 38.2 pM ± 3, and 74.7 pM ± 2, respectively). Also, addition of these compounds to the duodenal biopsies resulted in significantly increased CCK levels in obese subjects when compared with lean subjects. Addition of sucrose and sucralose alone did not affect CCK release when compared with the negative control.

GLP-1 secretion from duodenal biopsies is presented in Figure 2B. Basal GLP-1 secretions levels of 3.7 pM ± 0.4 from lean subjects and 4.1 pM ± 0.5 from obese subjects were observed. Addition of intact pea protein to the luminal side significantly increased GLP-1 levels (8.4 pM ± 0.2 in lean subjects, and 9.3 pM ± 0.2 in obese subjects) when compared with negative control and with addition of sucrose. Also, addition of intact pea protein induced significantly higher levels of GLP-1 when compared with lean subjects. Addition of the combination of pea with sucrose or pea with sucraiose also induces significant elevated levels of GLP-1 (14.4 pM ± 0.1 and 23.8 pM ± 1 , respectively, for lean subjects, 16.6 pM ± 0.7 and 24.7 pM ± 1 , respectively, for obese subjects), when compared with the negative control and addition of only sucrose.

Electrical Measurements

The electrical parameters PD, Isc, and TER were followed over time. Basal electrical properties of all biopsies were measured. After an equilibration period of 40 min, the mean PD of -1.4 mV ± 0.2 was observed. Overall, no changes in PD were observed in the following 120 minutes. A decrease in TER and an increase in Isc were observed. The TER was significantly less decreased after addition of pea with sucrose (-29.2 Ω.ατι2 ± 1), when compared with the negative control (-51.4 Ω.ατι2 ± 3) in obese subjects. All other products did not affect TER when compared with the negative control for both lean and obese subjects.

Addition of sucrose, sucraiose, and pea with sucrose resulted in an increased Isc (242.7 Q.cm2 ± 32, 246.9 Q.cm2 ± 25, and 235.4 Q.cm2 ± 26, respectively) when compared with the negative control (49.7 Q.cm2 ± 19) in lean subjects. Also, addition of sucraiose or pea with sucrose to biopsies from lean subjects resulted in increased Isc when compared with obese subjects. Addition of sucrose or pea with sucraiose to the luminal side of biopsies from obese subjects resulted in an increased Isc (195.9 μΑ/αη2 ± 34 and 387.1 A/cm2 ± 32, respectively) when compared with the negative control (31.8 pA/cm2 ± 1 1).

DISCUSSION OF RESULTS FROM EXAMPLES 1 AND 2

In Examples 1 and 2, the effects of five sweeteners in the presence or absence of pea protein on satiety hormone release were investigated. It was demonstrated that the combination of pea protein with the artificial sweeteners sucraiose and sucrose induce stronger effects on satiety hormone release compared with the compounds separately, both in vitro and ex vivo. It was also shown that addition of sucrose or sucraiose to STC-1 cells stimulated the release of CCK and GLP-1 , whereas addition of the sweeteners alone to human duodenal biopsies did not result in hormone release. Carbohydrate is an adequate stimulus for secretion of GLP-1. Failure of non-nutritive sweeteners to elicit the release of such peptides could theoretically result in lower satiety and augment energy intake. It has previously been shown that sucralose induces GLP-1 secretion [19]. In contrast, aspartame does not stimulate GLP-1 secretion [25]. Examples 1 and 2 demonstrate that most artificial sweeteners are able to induce CCK release, and that all sweeteners stimulate GLP-1 release from enteroendocrine STC-1 cells, but when human duodenal tissue is exposed to sucrose or sucralose, both CCK and GLP-1 release is not affected. Overall, sucrose, sucralose, and aspartame were the most potent sweeteners to stimulate satiety hormone release from STC-1 cells. Since sucrose was the only caloric sweetener and the most commonly used, this sweetener was used in the Ussing chambers as a control in Example 2.

Pea protein contains large amounts of arginine, asparagine, and glutamine, and is digested in the stomach for approximately 93% [28]. Unlike other common protein sources such as milk, soy, or wheat proteins, pea protein has a very low allergenic potential, which makes this protein more suitable for dietary interventions compared with wheat protein.

Examples 1 and 2 show that most sweeteners are able to induce secretion of CCK and GLP-1 from STC-1 cells. However, combined with pea protein, the positive effects on the hormone release were diminished. Tested on human duodenal tissue, sucrose and sucralose did not affect hormone secretion when compared with the negative control. Combining the sweeteners, and in particular sucralose with pea protein, strongly activated hormone release.

In Example 2, both lean and obese subjects were tested. There are indications that obese subjects are less sensitive to satiety signals compared with lean subjects. The results from Example 2 indicate that obese subjects have higher release of CCK after exposure to the pea protein compared with lean subjects. Also, the combination of pea protein with either sucrose or sucralose resulted in higher levels of CCK in obese subjects. Indeed, combining pea protein with sucralose induced the strongest effects on CCK and GLP-1 release by both STC-1 cells and human duodenal tissue samples. References

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Claims

CLAIMS:

1. An edible product comprising:

i) intact plant protein;

ii) a sugar in an amount greater than 2 times the amount of intact plant protein by weight or a sugar substitute; and

iii) a delivery vehicle for at least partially protecting the plant protein from hydrolysis.

2. An edible product according to claim 1 wherein the delivery vehicle comprises an enteric coating of at least the plant protein.

3. An edible product according to claim 1 or 2 wherein the product comprises at least 5g of the plant protein, preferably at least 10g of the plant protein.

4. An edible product according to any one of the preceding claims wherein the sugar is in an amount of greater than 2.5 or 3 times the amount of intact plant protein by weight.

5. An edible product according to any one of the preceding claims wherein the product further comprises a delivery vehicle for at least partially protecting the sugar or sugar substitute from hydrolysis

6. A method of inducing or increasing satiety in an individual comprising the steps of:

i) delivering an intact plant protein to the duodenum of the individual; and ii) delivering a sugar in an amount greater than 2 times the amount of plant protein by weight or a sugar substitute to the duodenum of the individual.

7. A method according to claim 6 wherein the method is non-therapeutic.

8. A method of treating obesity in an individual comprising the steps of:

i) delivering an intact pea protein to the duodenum of the individual; and ii) delivering a sugar in an amount greater than 2 times the amount of plant protein by weight or a sugar substitute to the duodenum of the individual.

9. A method according to claim 8 wherein the method is a cosmetic method.

10. A method according to any one of claims 6 to 9 wherein the individual has a body mass index of greater than 25, preferably greater than 30.

11. An edible product according to any one of claims 1 to 5 or a method according to any one of claims 6 to 10 wherein the intact plant protein comprises or consists of wheat protein, soy protein and/or pea protein.

12. An edible product or method according to claim 11 wherein the intact pea plant protein comprises or consists of a proteinaceous extract of a plant of the species of Pisum sativum.

13. An edible product according to any one of claims 1 to 5, 1 1 or 12 or a method according to any one of claims 6 to 12 wherein the sugar substitute is a high intensity sweetener or a sugar alcohol.

14. An edible product or method according to claim 13 wherein the high intensity sweetener is a naturally occurring or non-naturally occurring high intensity sweetener.

15. An edible product or a method according to claim 14, wherein the naturally occurring high intensity sweetener comprises a protein, preferably Thaumatin, Brazzein or Monellin.

16. An edible product or a method according to claim 14, wherein the naturally occurring high intensity sweetener comprises Stevia , a Steviol glycoside, Mogroside V (Lo Han Guo ), Monatin or Glycyrrhizin.

17. An edible product or a method according to claim 16, wherein the Steviol glycoside is stevioside, steviol, Rebaudioside A , Rebaudioside B, Rebaudioside C, Rebaudioside D or Rebaudioside E.

18. An edible product or a method according to claim 14, wherein the non-naturally occurring high intensity sweetener comprises a peptide, preferably aspartame, neotame, advantame or alitame.

19. An edible product or a method according to claim 14, wherein the non-naturally occurring high intensity sweetener comprises a halosugar, preferably sucralose.

20. An edible product or a method according to claim 14, wherein the non-naturally occurring high intensity sweetener comprises Acesulfame-K, Saccharin, Cyclamate or neohesperidin DC.

21. An edible product according to any one of claims 1 to 5, 1 1 or 12 or a method according to any one of claims 6 to 12 wherein the sugar comprises sucrose, glucose or fructose.

22. An edible product or a method according to claim 13 wherein the sugar alcohol comprises xylitol, sorbitol, maltitol, erythritol, isomalt or lactitol.

23. An edible product according to any one of claims 1 to 5, 1 1 or 12 or a method according to any one of claims 6 to 12 wherein the sugar substitute comprises a precursor of a sugar or a sugar substitute.

24. An edible product or a method according to claim 23 wherein the precursor comprises maltodextrin.

25. A method according to any one of claims 6 to 10 comprising administering to the individual an edible product according to any one of claims 1 to 5 and 1 1 to 24.