WO2008037578A1

WO2008037578A1 - Compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer in a multilayer arrangement

Info

Publication number: WO2008037578A1
Application number: PCT/EP2007/059329
Authority: WO
Inventors: Michael Francis Butler; Emmanuel Heinrich; Stive Pregent; Phillippa Rayment; Peter Conrad Schuetz
Original assignee: Unilever Plc; Unilever N.V.; Hindustan Unilever Limited
Priority date: 2006-09-29
Filing date: 2007-09-06
Publication date: 2008-04-03

Abstract

The present invention relates to compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a multilayer arrangement, as well as to its production and, as well as to the use of these particles in food products.

Description

COMPOUNDS, WHICH ARE STARCH CONTAINING PARTICLES COATED, EMBEDDED OR ENCAPSULATED BY AT LEAST ONE BIOPOLYMER IN A MULTILAYER ARRANGEMENT

The present invention relates to compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a multilayer arrangement, as well as to its production and, as well as to the use of these compounds in food products.

Starches as carbohydrates are the preferred energy source for the body. Starches occur naturally in vegetables and grains During digestion starches are broken down into glucose, which provides essential energy for brain, central nervous system and for muscles during activity. Starches are one prime source of energy. The other source are sugars. Carbohydrates are far easier to break down in the digestive tract (producing less metabolic waste products) than either fat or protein and as a result the body's reserves of carbohydrate energy (stored in the blood, liver and muscles) are utilised first and are rapidly depleted during exercise. If these carbohydrates are not converted to energy, they are mostly converted and stored as fat, with a small amount stored as glycogen in the liver and muscles. When the body calls for more fuel (such as during exercise), the fat or glycogen is converted back to glucose and used accordingly.

It is desirable that the digestibility of starch be slowed to provide a controlled and/or steady release of glucose to the body over a period of several hours. Contrarily to the transit time of a meal in the stomach that can vary mainly in function of the type and quantity of food ingested, the transit time in the small intestine where most of carbohydrate digestion occurs is in average of 2 hours roughly before large intestine is reached. Therefore it is desirable that starch hydrolysis happens in a controlled and gradual way in the small intestine so that sustained energy can be delivered to the body. Additionally, the starch digestion should be complete or close to complete after 2 hours transit in the small intestine in order to minimise the quantity of undigested starch entering into the colon.

The slow release should work in any kind of food product in which these compounds according to the present invention are incorporated in. The food products can be food for humans (human food product) as well as for animals. The food product, which contains the compounds according to the present invention, can have any physical form, which is common for food products. The food product can be solid or liquid, soft or hard, gel-like, frozen, cooked, boiled, pasteurised, unpasteurised, etc. The compounds according to the present invention must be incorporated into the food products without being destroyed. When the compounds according to the present invention, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer are incorporated into food products, especially in food for humans, then it is desirable that these compounds can not be detected in mouth during the consumption. The mouthfeel of the food product is a very important criteria.

For a slow release of starch larger compounds are preferred, because the digestion takes place slower. But for a good mouthfeel smaller particles are usually preferred. Therefore a compromise regarding the dimension of the compounds has to be found.

It has to be said that the mouthfeel also depends on the softness of the compounds as well as on the format of food product wherein the compounds are to be incorporated in.

Surprisingly, it has been found out that compounds, which are starch containing particles coated, embedded or encapsulated in a multilayer arrangement allow the release of the starch in an excellent manner.

In the context of the present invention the term compound means the coated, embedded or encapsulated product. In the context of the present invention the term starch containing particle means the particle, which is not (yet) coated, embedded or encapsulated.

Compounds according to the present invention are starch containing particles, which are coated, embedded, encapsulated by at least one biopolymer. Starches are not part of the coating, embedding or encapsulating layer. That means that the biopolymer are no starches or do not comprise starches.

The compounds as well as the starch containing particle can have any physical form. Usually the starch containing particles are solid or liquid. The starch could also be crystallised.

The preferred form of the starch containing particle is the solid and crystallised form.

The starch containing particles can have any shape, such as spheres, tubes, fibres, as well as ill- defined forms. The same applies for the compounds. The compounds according to the present invention are usually solid, gel or in a liquid form. Preferably they are solid or gel-like.

The compounds can have any shape, such as spheres, tubes, fibres, as well as ill-defined forms.

The term "starch containing particles" means particles which are made out of starch material, but which can comprise further non-starch material. These materials are not carbohydrates. This means that the term "starch containing particles" covers particles, which are pure starch or mixtures of different starches as well as starch (or mixture of starches) mixed with other non- carbohydrate compounds. The particles do not comprise any sugar compounds.

Furthermore it has to be stated that the coated, embedded or encapsulated particles are either compounds wherein the starches are concentrated in the core of the particle (coated, encapsulated) or they are starch particles dispersed in a matrix of biopolymeric material (embedded). Under term "coated and encapsulated starch containing particles" we defined compounds wherein the starch(es) (or starch mixed with other ingredients) is located in the middle (core) of the compound and it is coated or encapsulated by at least one biopolymer. The starch is not part of the coating or the encapsulating material and vice versa the biopolymer is not part of the core. Under the term "embedded starch containing particles, we defined compounds wherein the starch(es) (or starch mixed with other ingredients) are dispersed, so that the starches are always concentrated at certain spots in the matrix. In the sense of the present invention the starches are not part of the matrix and vice versa the biopolymer is not part of the starch.

The compounds according to the present invention are starch containing particles coated, embedded or encapsulated by at least one biopolymer, wherein the starch containing particles can optionally comprise at least one non carbohydrate compounds and wherein the biopolymer is no starch or do not contain starch.

The compounds according to the present invention can contain starch, which is from natural or synthetic origin. Of course in case that the starch comes from a natural source there are always other compounds present. But it is also possible to mix the starch with other useful compounds, which are not harmful to the animal or human body. Such compounds could be for example proteins, peptides, vitamins, probiotics, etc.

Therefore under the term "starch containing particles" falls pure starch as such well as starch mixed with other compounds. - A -

The starch can be raw starches, modified starches, and pregelatinized starches. Preferred are raw and modified starches are preferred.

The compounds do not coat, embed or capsulate the particles in a permanent way. That means the resulting compounds release the starch during time as already stated above. This release happens inside the human or animal body after the intake of the compounds according to the present invention.

If compounds according to the present invention are eaten the starch is released by the help of enzymes.

The compounds according to the present invention can be described as sponge-like compounds. Therefore the compounds according to the present invention have pores, which are about between 50nm and 100nm. The pores sizes can be measured by Transmission Electron Microscopy (TEM). The following procedure has been used to determine the pore sizes. To enhance fixation, the beads were cut in half and then placed in 0.1 % ruthenium tetroxide for 90 minutes. The beads were then rinsed using distilled water for 20 minutes and this was repeated. The beads were then stained in 1% aq. uranyl acetate overnight. The beads were dehydrated in ethanol and infiltrated with epoxy resin, which was polymerised at 6O⁰C for 48 hours. Sections of approximately 10Onm thickness were prepared and stained in lead citrate. The sections were then examined in Jeol 1200 TEM at 100KV.

The size of the starch containing particles can be a few microns as well as a few millimetres. For the purpose of the present invention the size of the starch containing particles is less than 1000 microns. Usually their size is between 5 and 1000 microns, preferably between 10 and 800 microns, more preferably between 20 and 500 microns.

The size of the compounds according to the present invention can be a few microns as well as a few millimetres. For the purpose of the present invention the size of the compounds according to the present invention is less than 1000 microns. Usually it is between 5 and 1000 microns, preferably between 10 and 800 microns, more preferably between 20 and 500 microns. Of course a compound is always larger than the corresponding starch containing particle. When the starch containing particles are embedded by at least one biopolymer the compounds is usually much larger than the starch containing particles, which are embedded therein. The compounds as well as the starch containing particles can have any form. They can be a bead, a sphere, a fibre, or any other form. When these coated embedded particles are used it is obvious that mixture of several forms can be used.

The biopolymer can be any biopolymer which is able to coat, embed or encapsulate starch containing particles. Additionally, because the compounds according to the present invention are incorporated into food products, the biopolymer should be not harmful to humans and animals. The biopolymer is no starch or does not comprise starch.

Preferred biopolymers are physically (such as ionically) and/or covalently crosslinkable polysaccharides.

More preferred biopolymers are physically and/or covalently crosslinkable polysaccharides which are β-linked polysaccharides.

Such crosslinkable polysaccharide includes food hydrocolloids such as agarose, chitin, carrageenan, pectins, amidated pectines, xanthan, alginates, gum arabic, galactomannans like locust bean gum, guar and tara gum, and cellulosics like carboxymethylcellulose, methylcellulose, hydroxypropylcellulose and methylhydroxypropylcellulose as well as gellans, ispaghula, β-glucans, konjacglucomannan, gum tragacanth, detarium and tamarind.

Another preferred biopolymer is chitosan, which is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is produced commercially by deacetylation of chitin (can be produced from chitin also), which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.). The degree of deacetylation (%DA) can be determined by NMR spectroscopy, and the %DA in commercial chitosans is in the range 60-100 %

The most preferred physically and/or covalently crosslinkable and β-linked polysaccharide are alginates. Chemically, alginate is a linear copolymer with homopolymeric blocks of (1-4)-linked β-D- mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. The ratio of the different blocks can differ widely. The relative amount of each block type varies both with the origin of the alginate. Preferred are alginates with a M:G ratio of 80:20 to 20:80. Alginates are commercially available. Preferably, the starch containing particle is coated, embedded or encapsulated by a multilayer arrangement, wherein the biopolymers have opposite charges in each layer. That means that for example the first layer is built up by a biopolymer (or a mixture of biopolymers) which has mainly negative charges and the following layer is built up by a biopolymer (or a mixture of biopolymers) which has mainly positive charges and so on. The first layer has preferably the opposite charge than the starch containing particle. Preferably the first layer is a negative charged biopolymer.

Negative charged biopolymers are generally understood to mean biopolymers having ionically dissociable groups which may be a component or substituent of the polymer chain. The number of these ionically dissociable groups in the biopolymers is usually large enough to ensure the water- solubility of the polymers in a dissociated form (also referred to as polyions). The term biopolymer as used herein also refers to ionomers in which the concentration of the ionic groups is not sufficient to make them water soluble but they have sufficient charges for a self-assembly.

Negative charged biopolymers which can be inorganic as well as organic polymers can be formed from polyacids when they dissociate with cleavage of protons.

Suitable biopolymers include naturally occurring polyanions and as well as synthetic modified polyanions. Examples of naturally occurring polyanions are alginate, carboxymethylamylose, carboxymethylcellulose, carboxymethyldextran, carageenan, cellulose sulfate, chrondroitin sulfate, chitosan sulfate, dextran sulfate, gum arabic, guar gum, gellan gum, heparin, hyaluronic acid, pectin, amidated pectines, xanthan and proteins at an appropriate pH.

Suitable positive charged biopolymers include naturally occurring polycations and synthetic modified polycations. Examples of suitable naturally occurring polycations are chitosan, modified dextrans, e.g. diethylaminoethyl-modified dextrans, hydroxymethylcellulose trimethylamine, lysozyme, polylysine, protamine sulfate, hydroxyethylcellulose trimethylamine and proteins at appropriate pH values.

Linear or branched biopolymers can be used. The use of branched biopolymers leads to less compact polyelectrolyte multifilms having a higher degree of wall porosity. The capsule stability can be increased by cross-linking the biopolymers within or/and between the individual layers.

In the present invention the biopolymer is usually present in the form of a gel, where gel comprises 0.5 - 20 wt-% of at least one biopolymer and 80 - 99.5 wt-% of water.

The weight percentages are based on the total weight of the biopolymer gel.

Preferably, the gel comprises 0.5 - 15 wt-%, more preferred 0.5 - 10 wt-%, especially preferred 1 - 10 wt-%, very especially preferred 1 - 5 wt-% of at least one biopolymer. The weight percentages are based on the total weight of the biopolymer gel.

As a consequence thereof, the water content is preferably 85 - 99.5 wt-%, more preferred 90 - 99.5 wt-%, especially preferred 90 - 99 wt-%, very especially preferred 95 - 99 wt-% of water. The weight percentages are based on the total weight of the biopolymer gel.

The content of starch in the compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer is 0.1 - 20 wt-%, based on the total weight of the compounds. Preferably the content of starch is 0.5 - 15 wt-%, more preferably, 0.5 - 10 wt-% equally preferred 1 - 15 wt-%, especially preferred 1 - 10 wt-%, based on the total weight of the compounds.

The permeability or other properties of the coating, embedment (=matrix) or encapsulation can be adjusted in a defined manner by using copolymers which change their structure as a function of the external conditions.

After application of each layer the excessive molecules which have not contributed to forming the layer are preferably separated off before the next layer is applied. Such separation can be done according to any known method, particularly centrifugation, filtration or/and dialysis. A preferred embodiment relates to compounds, which are starch containing particles firstly coated, embedded or encapsulated with alginate and secondly coated, embedded or encapsulated with a positive charged biopolymer. Optionally more layers may be added, wherein the third layer is built up by a negative charged biopolymer, the fourth one by a positive charged biopolymer and so on.

A more preferred embodiment relates to compounds, which are starch containing particles firstly coated, embedded or encapsulated with alginate and secondly coated, embedded or encapsulated with chitosan, modified dextrans, e.g. diethylaminoethyl-modified dextrans, hydroxymethylcellulose trimethylamine, lysozyme, polylysine, protamine sulfate, hydroxyethylcellulose trimethylamine or proteins at appropriate pH values, as well as a mixture of these biopolymers. Optionally more layers may be added, wherein the third layer is built up by a negative charged biopolymer, the fourth one by a positive charged biopolymer and so on.

An especially preferred embodiment relates to compounds, which are starch containing particles firstly coated, embedded or encapsulated with alginate and secondly coated, embedded or encapsulated with chitosan.

Optionally more layers may be added, wherein the third layer is built up by a negative charged biopolymer, the fourth one by a positive charged biopolymer and so on.

As already mentioned the compounds, which are starch containing particle can be coated, embedded or encapsulated. The first layer can therefore be the coating (or encapsulation) of discrete starch containing particles or starch containing particles which are embedded in the biopolymer of the first layer. In such a matrix the starch particles are randomly distributed. Such embodiments (discrete coated or embedded particles) are then coated, embedded or encapsulated by the second layer.

The process of production of the compounds, which are starch containing particles which are coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers, can be done according to well known processes, such as extrusion processes or emulsion processes. It is possible that the different layers are built up by using different processes.

A suitable generic process for multilayer compounds according to the present invention is the following:

1. The first layer, which may or may not be part of the described process, is the dispersion of starch containing particles (CCP) in a semi-concentrated biopolymer matrix / solution followed by break up into individual entities (extrusion / emulsification). This forms the first biopolymer barrier layer that can have variable thickness and may contain and agglomerate multiple CCPs. If desired an immersion in a diluted electrolyte solution may follow this step to consolidate or change the obtained structure.

2. The template material (i.e. the untreated CCPs or the entities formed as described in 1.) are then immersed in a dilute solution of a charged biopolymer that may or may not contain other electrolytes. This biopolymer solution then interacts with the added template and forms an additional barrier layer. 3. The process described in 2. can be repeated to add consecutive barrier layers that can further change the overall barrier functionality.

In short the process can be summarized as the build-up of multiple barrier layers around CCPs by repeated interactions of a template material with different charged biopolymers from dilute solutions possibly combined with embedding the template or CCPs in a biopolymer matrix that may be modified using further reactants.

Therefore the present invention relates to a process of production of compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers wherein

(i) the starch containing particles are coated, embedded or encapsulated with a first layer of at least one biopolymer and then (ii) coated, embedded or encapsulated with a second layer of at least one biopolymer of the opposite charge than the first layer, (iii) and optionally repeat the steps (i) and (ii).

When a mixture of biopolymer is used then preferably the biopolymers should have the same charge.

A specific method how to produce a multilayer arrangement in one step is the following: Starch is dispersed in a solution of polyanion biopolymer (such as alginate). This solution is extruded drop wise through a syringe into a solution of polycation biopolymer (such as chitosan). Beads with a liquid core of polyanion biopolymer (such as alginate) -starch solution, and coated with a polyanion-polycation (such as chitosan-alginate) complex shell, are formed. The addition of calcium chloride in the polycation biopolymer (such as chitosan) solution prior to the beads formation allows a gelled core of polyanion biopolymer (such as alginate). The compounds obtained by that process can be further coated, embedded or encapsulated by biopolymers.

Therefore a further embodiment of the present invention relates to a process for the production of compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a two-layer arrangement, wherein (i) the starch containing particle is dispersed in a solution comprising at least one polyanion biopolymer, and (ii) this solution is extruded drop wise into a solution comprising at least one polycation biopolymer.

The compounds obtained by that process can be further coated, embedded or encapsulated by biopolymers.

Therefore a further embodiment of the present invention relates to a process for the production of compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a two-layer arrangement, wherein (i) the starch containing particle is dispersed in a solution comprising at least one polycation biopolymer, and (ii) this solution is extruded drop wise into a solution comprising at least one polyanion biopolymer.

A further embodiment of the present invention relates to a process for the production compounds , which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a two-layer arrangement, wherein

(i) the starch containing particle is dispersed in a solution comprising at least one polyanion biopolymer, and

(ii) this solution is extruded drop wise into a solution comprising at least one polycation biopolymer and Ca²⁺ cations.

A further embodiment of the present invention relates to a process for the production of compounds , which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers in a two-layer arrangement, wherein

(i) the starch containing particle is dispersed in a solution comprising at least one polycation biopolymer, and

(ii) this solution is extruded drop wise into a solution comprising at least one polyanion biopolymer and Ca²⁺ cations. The compounds obtained by that process can be further coated, embedded or encapsulated by biopolymers.

The reaction can also comprise a posthardening step. Such a posthardening step is carried out in a Ca²⁺ solution after the coating, embedding or encapsulating process. The duration of the posthardening step can vary a lot. Such a process can be carried within minutes or it can be carried out during several hours. In some cases the posthardening process can be continued when the end product is stored (or sold) in a Ca²⁺ solution. The posthardening reaction solution can also contain in addition to (or as a replacement of) Ca²⁺ other alkaline-earth metals.

The Ca²⁺ can be added in form of any soluble salt. The counterion of the Ca²⁺ salt does not affect the posthardening step, therefore the choice of a Ca²⁺ salt is not dependent on the counterion. Suitable Ca²⁺ salts are for example CaCI₂, CaSO₄. It is also possible to use salt which are usually hardly soluble or even insoluble in water, but when using a low pH they become soluble. An example for such a salt is CaCO₃.

The compounds according to the present invention are incorporated into food products. Food products for animals as well as humans can be provided. Preferably food products for humans are provided. The compounds obtained by the inventive process are very stable so they can be used in any food product which needs to have starch in it. The term food products covers any kind of drinks or other liquid food product, snacks, candies and confections, dessert mixes, granola bars, energy bars, various beverages, shelf stable powders, ready to eat foods such as puddings, frozen yogurts, ice creams, frozen novelties; cereals, snacks, meal replacements, baked goods, pasta products, confections, military rations, specially formulated foods for children, and specialized gastric enteral feeding formulations.

The food product can be treated with any usually used food technology process like cooking, baking, freezing, pasterizing, etc. without destroying the compounds obtained by the inventive process.

Because the active component is encapsulated by the method of the subject invention, the resulting coated compounds are relatively inert and bland in both aroma and taste. This allows the compounds of the subject invention to be incorporated in the disclosed foods without affecting the characteristic properties and flavours of the food. In WO 2005/020717 (examples 1 , 2 and 3), WO 2005/020718 (examples 1 and 2) and WO 2005/020719 (examples 1 , 2 and 3) suitable food forms can be found, wherein the particles obtained by the process according to the present invention can be put in.

Description of the figures:

Fig. 1 : Confocal scanning light microscopy images of shrunken chitosan-alginate encapsulates containing entrapped rice starch granules (staining with acridine orange for 3 days); initial water phase for making the plain calcium-alginate beads contained 2% alginate and 5% starch. Fig. 2: Starch hydrolysis kinetic curves for rice starch granules entrapped in chitosan- alginate encapsulates, plain Ca-alginate beads and non encapsulated rice starch (in-vitro test data); initial water phase in the emulsion method contained 2% alginate and 5% starch; pretreatment in temperature before hydrolysis assay: 15 min at 75°C. Fig. 3: TEM image of the perimeter of a sliced gel bead. The dense biopolymer multilayer film at the perimeter is clearly visible between the arrows. Fig. 4: Glucose release curve comparing the digestion of multilayer coated and uncoated calcium alginate beads containing rice starch granules.

Fig. 5: CLSM image of multilayer coated agar beads. The alginate used for the last layer is labelled with fluorescein.

Fig. 6: TEM and CLSM microscopy images of starch granules coated with chitosan / alginate multilayer films (left TEM of rice starch; right CLSM of potato starch, fluorescently labelled alginate was used for the film build-up).

Fig. 7: Glucose release as a function of time for digestion of starch, starch encapsulated in beads with a liquid core, starch encapsulated in beads with a gelled core.

The following examples serve to illustrate the invention without limiting the invention to them.

If not otherwise stated the percentages are weight percentages and the temperatures are given in Celsius.

Example 1 :

A 1-2% alginate solution (Sigma-Aldrich no. A-7128: alginic acid sodium salt, high mannuronic acid content) containing 1 to 10% rice starch granules (Remy DR, ex. Remy,

Orafti Group, Belgium) and 0.2% Tween 20 (Polysorbate 20, no. 233360010, ex. Acros Organics) was first emulsified at ambient temperature in sun flower oil. The water phase volume fraction was 30% and the oil phase contained monoglycerides, Hymono 8903 (ex. Quest International, The Netherlands), predispersed at 60⁰C at a concentration of 0.2 weight % with respect to the water phase. The water phase emulsification in oil was performed during 25 min with a rotating palette in combination with four wall baffles at a constant speed of rotation between 300 and 1000 rpm. A 1 M solution of calcium chloride was then added quickly along the side of the beaker to reach a final calcium ions concentration of 0.1 M in the aqueous phase. The break-up of the water-in-oil emulsion was obtained as the alginate fine droplets start to gel. After full phase separation the oil was removed by repetitive washing of the gel beads on a filter with a 0.1 M CaC^ solution or pure water. The 'plain' calcium-alginate microspheres were finally stored overnight in a 0.1 M CaCb solution at refrigerated temperature (hardening phase).

Then the plain alginate beads were coated with chitosan. The coating was achieved by incubating the alginate beads into a 1.5% solution of chitosan having a degree of deacetylation of 91% (ChitoClear, ex. Primex, Norway). The chitosan solution was prepared by first hydrating the chitosan powder in acidified deionised water using 0.5% glacial acetic acid (Sigma-Aldrich no. 537020). After adjusting the pH to 5.5, the solution was stirred during 5 hours at 60⁰C to achieve maximal swelling of the chitosan. Then the chitosan solution was filtered (0.45 micron pore size filter). The coating of the alginate beads with chitosan was performed by immersing 15g of plain Ca-alginate beads into 100 ml of the chitosan solution. The suspension of encapsulates was gently agitated during

45 min on an orbital shaker. After this incubation period the chitosan-alginate encapsulates were washed on a filter with a 0.1 M CaC^ solution and then kept overnight in the same solution at refrigerated temperature (rehardening).

The hydrolysis rate of encapsulated starch was evaluated by means of an in-vitro test using alpha-amylase enzyme. First a suspension of chitosan-coated or non-coated alginate beads containing 1% starch (or a 1 % starch granules suspension as a control) was prepared in Pipes buffer (piperazine-1 ,4-bis(2-ethanesulfonic acid), ex. Acros Organics) at pH 6.9. The starch-containing samples were then preheated at 75°C or 95°C during 15 min before cooling down to 37°C. 5 ml of the starch sample was added to 5 ml of a freshly made alpha-amylase solution prepared by adding 0.1026 g of porcine pancrease alpha-amylase (Sigma-Aldrich no. A-6255; 700-1400 units/mg protein) to 40 ml of a 0.9% NaCI solution. The starch/amylase mixture was then incubated straightaway at 37°C under gentle stirring. 500 μl_ samples were collected at different times in order to follow the kinetic of hydrolysis and build full hydrolysis profiles. 500 μl_ of DNSA solution (Aldrich no. 128848: 3,5-dinitrosalicylic acid) was added to each 500μl_ collected sample and the resulting mixture was heated up to 99°C during 10 min so that the DNSA reagent could react with the reducing end groups. After a ten or twenty-fold dilution step, the absorbance at 540 nm of the reacted samples was measured and compared to calibration curve of maltose conversion.

As a result of the coating with chitosan the alginate beads were shrunken from 480 micron down to 200 micron as measured by small angle light scattering. As can be observed at the Fig. 1 the resulting shape of the chitosan-alginate encapsulates is rather ill-defined and certainly no longer spherical. Furthermore the starch granules became much more closely packed after the shrinking of the encapsulates. The better compaction of the starch granules and/or the presence of the semi-permeable membrane of chitosan-alginate complex at the outer of the encapsulates did lead to a further delay / control in the hydrolysis of the entrapped starch as can be seen at the Fig. 2 (by comparison to the case of the plain alginate beads).

In addition it was found that the stability of the chitosan-alginate encapsulates was excellent in presence of various chelating agents / anions forming unsoluble salt complexes with calcium ions, which was not the case with the plain calcium-alginate system. This is a serious limitation for using alginate beads in products but also in-vivo since sodium bicarbonate in particular is secreted by the pancreas and delivered to the duodenum (bicarbonate ions do complex with calcium ions). The Table 1 displays the improved stability performance after chitosan coating in different aqueous media (salts) and under gentle stirring.

Tablei : Encapsulate stability performance in presence of salts and under gentle stirring; performance of chitosan-alginate particles versus plain calcium- alginate beads.

Example 2:

Calcium/alginate gel beads are prepared using an emulsion method. 396 g sunflower oil and 4g Admul WoI (non-ionic surfactant, Quest) was mixed well with an overhead stirrer. Then a solution of 4g alginate and 2g rice starch in 150ml of water was added under mixing. A CaCI₂ solution 2Og in 50 ml water was added to the mixture and stirred for another 2h. To separate and clean the beads, the mixture was centrifuged and the supernatant oil was removed. The beads were then twice re- suspended in 11 of an aqueous solution containing 1% Tween 60 and 20 g CaCI₂ using high speed Silverson mixer with a high shear cage. This process was repeated another two times using a CaCI₂ solution without added surfactant. Spherical microparticles of approximately 50-100 microns diameter are obtained. A dispersion of these calcium/alginate gel beads was prepared with ca. 20 volume % bead content. A 0.1% solution of chitosan (ex Primex, Chitoclear, fg 95 gras) at pH 5.5 containing 0.2M sodium chloride was added in a 10 fold excess (10 ml to 1 ml bead dispersion). The resulting dispersion was agitated for 5 to 15 min to prevent sedimentation and aggregation. The excess chitosan was removed by three washing steps that consisted of centrifugation of the sample followed by re-dispersion in water (or an appropriate salt solution). To the obtained beads a 0.1% solution of alginate (Manugel DMB, ex ISP alginates, G content = 72%) at pH 5.5 containing 0.2M sodium chloride was added in a 10 fold excess (10 ml to 1 ml bead dispersion). The resulting dispersion was agitated for 5 to 15 min to prevent sedimentation and aggregation. The excess alginate was removed by tree washing steps that consisted of centrifugation of the sample followed by re-dispersion in water (or a appropriate salt solution). To maintain the stability of the gel beads a solution of 10 mM calcium chloride was added at this point and the dispersion was agitated for 15 min to let the calcium diffuse into the gel beads and "re-harden" them. For the build-up of multilayers the described procedure was repeated until the desired layer number was achieved. After 10 consecutive adsorption steps as described, TEM images (Fig. 3) revealed a ca. 50 nm thick dense film at the perimeter of the sliced gel beads.

The digestibility of the encapsulated starch was determined as maltose release over time using a digestion assay. A defined volume of coated gel beads was put into a suitable mixture of amylase (Sigma No A6255), PBS buffer and Amyloglucosidase (sigma, starch assay reagent SA-20). An electrochemical microdialysis glucose sensor (Sycopel, Jarrow, UK) sensor was used to monitor the release of glucose from the treated beads and from a control of untreated beads. Fig. 4 shows that the release from the coated beads is much lower and doesn't reach a saturation level after 1500 min.

As a prove that the same amount of starch was encapsulated the same samples were boiled at 80 ⁰C for 1 h to gelatinise the starch and speed up the digestion rate. Here the same final values of released glucose were reached, showing that samples contained similar levels of starch.

Example 3:

Spherical gelled agar beads were made via a water-in-oil emulsion route. Biopolymer solutions were prepared by dissolving agar (Luxara 1253, ArthurBranwell) in deionised water at 98°C at concentrations ranging from 0.5 to 5 wt-%. The solution was then cooled to 85°C. A water-in-oil emulsion was formed by adding the biopolymer solution to oil containing 1 wt % surfactant (Admul WOL, Quest) at 85 ⁰C. The mixture was then stirred rapidly using a Silverson SLR4 at 85°C for 30 minutes to allow an equilibrium particle size to develop. The stirring speed was varied to control the average particle size obtained. The emulsion was then quenched by placing in an iced water bath to allow the aqueous phase to gel, stirring at a slower speed was continued for a further 2 hours to prevent aggregation and coalescence. The spherical particles produced were then separated from the oil phase by centrifugation, followed by several washes with deionised water to remove residual oil and surfactant. Microgel suspensions were then made by re-dispersing the particles in deionised water.

Gelled agar beads with diameters around 50 μm are obtained and dispersed in water at a concentration of up to 10 weight %. To this dispersion a 10 fold excess of chitosan solution (0.05% wt , 0.2 M sodium chloride, pH 6.0) was added and the resulting dispersion was stirred for 10 minutes. Then the supernatant was removed by filtration through a 5 μm mesh. Fresh water was added 3 times just before the filter residue was about to get dry to wash off any not interacting material. Alginate solution (0.05% wt , 0.2 M sodium chloride, pH 6.0) was then added in a similar fashion followed by washing as described above. The whole process described above was then repeated until the desired thickness of the multilayer film was obtained. To visualize the successful coating, during one reaction step, a solution of fluorescently labelled alginate was used. As seen in Fig. 5, confocal scanning laser microscopy revealed, that the surface of the beads was successfully coated.

To test the stability of the coating of the beads, they were heated to 80 ⁰C in a water bath alongside a control sample of uncoated beads. As this temperature is above the gellation temperature of agar, the uncoated beads dissolved completely. In contrast to this after re-cooling and thus re-gelling of the core matrix, the coated beads could be recovered unchanged.

Example 4:

Starch granules (rice, potato and wheat starch) were coated by alternative adsorption of the biopoymers chitosan (Primex, Chitoclear) (polycationic) and alginate (DMB manugel) (polyanionic). The pH values of the biopolymer solution were both adjusted to pH 6. The protocol for the multilayer coating sequence was the following:

1) 0.5 g of starch granules were dispersed well in 20 ml of water.

2) The sample was concentrated by centrifugation and the supernatant discarded.

3) The granules were dispersed in 20 ml of a solution of chitosan (1 g/l chitosan; 0.2 M sodium chloride) and stirred for 5 to 15 min. 4) The sample was concentrated by centrifugation and the supernatant discarded.

5) The granules were dispersed in water (washing step).

6) Steps 4 and 5 were repeated another 2 times to remove all not adsorbed biopolymer from the system.

7) The granules were dispersed in 20 ml of a solution of alginate (1 g/l alginate; 0.2 M sodium chloride) and stirred for 5 to 15 min.

8) The sample was concentrated by centrifugation and the supernatant discarded.

9) The granules were dispersed in water (washing step).

10) Steps 4 and 5 were repeated another 2 times to remove all not adsorbed biopolymer from the system. 11) Steps 3 to 10 were repeated until the desired number of layers (typically 8 layers) was reached.

The resulting structure was imaged using confocal microscopy (CLSM) and electron microscopy (TEM). (Fig.6). Both methods clearly showed the successful formation of a ca. 50 nm thick biopolymer film on the granule surface.

Instead of a pH value of 6 any pH value between 4.5 and 6.5 could be used.

Example 5: A slurry was first prepared by mixing 5% whole rice starch (Remy industries, Belgium) into a 2% alginate solution (Manugel DMB, ex ISP Alginates, G content =72%), at room temperature. Beads were prepared by extruding this solution from a syringe into a solution containing 0.5% chitosan (Chitoclear, Primex Ingredients) and 1% acetic acid (acetic acid glacial, Fisher Scientific UK Limited). Beads with a liquid core of alginate/starch and a solid shell are formed and stored in the chitosan solution.

Alternatively, calcium chloride (6.82mM) is added to the chitosan solution in order to get a gelled core. The beads prepared were approximately 3.5mm in size for the liquid core ones and 2mm for the gelled one.

The digestability of the starch was determining using an enzymatic digestion assay. Stock solutions were used to prepare the reagent mix:

Amylase from aspergillus oryzae (150-250 units/mg, Sigma-Aldrich) diluted to 0.04 mg/mL (8 units/mL, ca. 710 nM assuming a Mw of 56000) Amyloglucosidase from aspergillus niger in solution (50 units/mL, Sigma-Aldrich: SA-20)

Glucose oxidase (GOD, 12.5 units/mL, aspergillus niger) and peroxidase (POD, 2.5 units/mL, horseradish) as glucose oxidase / peroxidase reagent from Sigma-Aldrich (G3660) 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt 50mg/ml_ solution (ABTS, 9OmM, Fluka).

The reagent is prepared by mixing 4 ml. of the GOD/POD solution, 480 μl_ of ABTS solution, 200 μl_ of amyloglucosidase solution and 200 μL of amylase with 4 ml. of deionised water.

0.8 ml. of this reagent is the diluted with 3.2 ml. of water in a vial. One bead is placed in the vials and UV scans in the range of 400 to 600 nm are taken at regular intervals. Vials containing 12.5 μL of a 5% starch solution are used as control, as 12.5 μL is the average volume of alginate/starch solution used to make one bead. For each system, the experiment is repeated 3 times. The release of glucose is shown in Figure 7.

The chitosan/alginate coating of the beads slows down the digestability of the starch and gelation of the core slows it down further.

Claims

1. Compounds, which are starch containing particles coated, embedded or encapsulated by a biopolymer or a mixture of biopolymers, characterized in that more than one layer of at least one biopolymer are used

2. Compounds according to claim 1 , wherein the starch containing particles are solid or liquid.

3. Compounds according to any of the preceding claims, wherein the starch include raw starches, modified starches, and pregelatinized starches.

4. Compounds containing particles according to any of the preceding claims, wherein the starch are raw and/or modified starches.

5. Compounds according to any of the preceding claims, wherein the size of the compounds is less than 1000 microns.

6. Compounds according to any of the preceding claims, wherein the biopolymers are physically and/or covalently crosslinkable polysaccharides.

7. Compounds according to any of the preceding claims, wherein the biopolymers are is chosen from the group consisting of includes food hydrocolloids such as agarose, chitin, carrageenan, pectins, xanthan, alginates, gum arabic, galactomannans like locust bean gum, guar and tara gum, and cellulosics like carboxymethylcellulose, methylcellulose, hydroxypropylcellulose and methylhydroxypropylcellulose.

8. Compounds according to any of the preceding claims, wherein the biopolymers have opposite charges in each layer.

9. Compounds according to any of the preceding claims, wherein the first layer is a negative charged biopolymer.

10. Compounds according to any of the preceding claims, wherein negative charge biopolymers are chosen from the group consisting of alginate, carboxymethyl-amylose, carboxymethylcellulose, carboxymethyldextran, carageenan, cellulose sulfate, chrondroitin sulfate, chitosan sulfate, dextran sulfate, gum arabic, guar gum, gellan gum, heparin, hyaluronic acid, pectin, xanthan and proteins at an appropriate pH.

11. Compounds according to any of the preceding claims, wherein positive charged biopolymers are chosen from the group consisting of chitosan, modified dextrans, e.g. diethylaminoethyl-modified dextrans, hydroxymethylcellulose trimethylamine, lysozyme, polylysine, protamine sulfate, hydroxyethylcellulose trimethylamine and proteins at appropriate pH values.

12. Process of production of compounds according to any of the preceeding claims, wherein

(i) the starch containing particles are coated, embedded or encapsulated with a first layer of at least one biopolymer of the same charge and then

(ii) coated, embedded or encapsulated with a second layer of at least one biopolymer of the opposite charge than the first layer,

(iii) and optionally repeat the steps (i) and (ii).

13. Process of production of compounds according to claim 1 - 11 , wherein

(ii) this solution is extruded drop wise into a solution comprising at least one polycation biopolymer.

14. Process of production of compounds according to claim 1 - 11 , wherein (i) the starch containing particle is dispersed in a solution comprising at least one polycation biopolymer, and

(ii) this solution is extruded drop wise into a solution comprising at least one polyanion biopolymer.

15. Process of production of compounds according to claim 1 - 11 , wherein wherein

16. Process of production of compunds according to claim 1 - 11 , wherein wherein

(i) the starch containing particle is dispersed in a solution comprising at least one polycation biopolymer, and (ii) this solution is extruded drop wise into a solution comprising at least one polyanion biopolymer and Ca²⁺ cations.

17. Use of the compounds obtained by the process according to claims 1 - 11 in food products.

18. Use according to claim 17, wherein the food product is a human food product.

19. Use according to claim 17 or 18, wherein the food products are any kind of drinks, snacks, candies and confections, dessert mixes, granola bars, energy bars, various beverages, shelf stable powders, ready to eat foods such as puddings, frozen yogurts, ice creams, frozen novelties; cereals, snacks, meal replacements, baked goods, pasta products, confections, military rations, specially formulated foods for children, and specialized gastric enteral feeding formulations.