WO2008037576A1

WO2008037576A1 - Process for production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer

Info

Publication number: WO2008037576A1
Application number: PCT/EP2007/059324
Authority: WO
Inventors: Michael Francis Butler; Emmanuel Heinrich; Phillippa Rayment
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 process for production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer, characterized in that the process is carried out in a reaction solution with a Ca2+ concentration of 0.1 - 10 % by weight (wt-%), based on the total weight of the reaction solution.

Description

^PROCESS FOR PRODUCTION OF COMPOUNDS, WHICH ARE STARCH CONTAINING _PARTICLES COATED, EMBEDDED OR ENCAPSULATED BY AT LEAST ONE BIOP_OLYMER

The present invention relates to process for production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer, characterized in that the process is carried out in a reaction solution with a Ca²⁺ concentration of 0.1 - 10 % by weight (wt- %), based on the total weight of the reaction solution.

Starches as carbohydrates are the preferred energy source for the body. Starches, which 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. Starches 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 starches 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 starches 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 when the process of production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer is carried out in out in a reaction solution with a Ca²⁺ concentration of 0.1 - 10 wt-%, based on the total weight of the reaction solution, the properties of the compounds in view of the energy release as well as the mouthfeeling are good.

Therefore, the present invention relates to a process of production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer, characterized in that the process carried out in a reaction solution with a Ca²⁺ concentration of 0.1 - 10 wt-%, based on the total weight of the reaction solution.

Obtained by the reaction according to the invention are compounds which are starch containing particles 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.

Preferred is a Ca²⁺ concentration in the reaction solution of 0.1 to 9 wt-%, more preferred from 0.1 to 8 wt-%, especially preferred are concentrations of 0.1 to 2.5 wt-% and 3.5 to 9 wt-%. The Ca²⁺ can be added in form of any soluble salt. The counterion of the Ca²⁺SaIt does not affect the reaction, 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 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 - A -

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.

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 100nm thickness were prepared and stained in lead citrate. The sections were then examined in Jeol 1200 TEM at 100KV.

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 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 there 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-("M)-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.

The process of production of the compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer, is carried out in an solution with a Ca²⁺ concentration of 0.1 - 10 % wt-%, based on the total weight of the reaction solution. The process of production can be any suitable process. Preferred processes are extrusion processes or emulsion processes. In case that the process is carried out in an emulsion, suitable oils are any kind of liquid vegetable and animal oils.

Preferred oils are vegetable oils, like Coconut oil, Com oil, Cottonseed oil, Canola oil, Olive oil, Palm oil, Peanut oil (Ground nut oil), Safflower oil, Sesame oil, Soybean oil, Sunflower oil, Almond oil, Cashew oil, Hazelnut oil, Macadamia oil, Pecan oil, Pistachio oil, Walnut oil, Acai oil, Blackcurrant seed oil, Borage seed oil, Evening primrose oil, Carob seed pods, Amaranth oil, Apricot oil, Argan oil, Avocado oil, Babassu oil, Ben oil, Carob pod oil (Algaroba oil), Coriander seed oil, False flax oil (made of the seeds of Camelina sativa), Hemp oil, Kapok seed oil, Meadowfoam seed oil, Mustard oil (pressed), Okra seed oil (Hibiscus seed oil), Perilla seed oil, Pine seed oil, Poppyseed oil, Prune kernel oil, Pumpkin seed oil, Quinoa oil, Ramtil oil, Rice bran oil, Tea oil (Camellia oil), Thistle oil, Wheat germ oil, Castor oil, radish oil, Ramtil oil and Tung oil, Animal oils, like tallow oil and fish oil (for example cod liver oil).

Therefore a further embodiment relates to a process as described above wherein the oil are vegetable oils, like Coconut oil, Corn oil, Cottonseed oil, Canola oil, Olive oil, Palm oil, Peanut oil (Ground nut oil), Safflower oil, Sesame oil, Soybean oil, Sunflower oil, Almond oil, Cashew oil, Hazelnut oil, Macadamia oil, Pecan oil, Pistachio oil, Walnut oil, Acai oil, Blackcurrant seed oil, Borage seed oil, Evening primrose oil, Carob seed pods, Amaranth oil, Apricot oil, Argan oil, Avocado oil, Babassu oil, Ben oil, Carob pod oil (Algaroba oil), Coriander seed oil, False flax oil (made of the seeds of Camelina sativa), Hemp oil, Kapok seed oil, Meadowfoam seed oil, Mustard oil (pressed), Okra seed oil (Hibiscus seed oil), Perilla seed oil, Pine seed oil, Poppyseed oil, Prune kernel oil, Pumpkin seed oil, Quinoa oil, Ramtil oil, Rice bran oil, Tea oil (Camellia oil), Thistle oil, Wheat germ oil, Castor oil, radish oil, Ramtil oil and Tung oil, or Animal oils, like tallow oil and fish oil (for example cod liver oil). Mixtures of these oils can be used as well.

For preparing the emulsion a surfactant compound is also necessary. For that purpose any commonly known and commonly used surfactant can be used.

The concentration of the physically and/or covalently crosslinkable and β-linked polysaccharide can range from 0.1 to 5 wt-% based on the total weight of the reaction solution.

Very preferred is an extrusion process for producing compounds, which are starch containing particles coated, embedded or encapsulated by at least one physically and/or covalently crosslinkable and β-linked polysaccharide, characterized in that the process is carried out in a reaction solution with a Ca²⁺ concentration of 0.02 - 5 wt-%, based on the total weight of the reaction solution, together with

0.02- 2 wt-% based on the total weight of the reaction solution, of at least one physically and/or covalently crosslinkable and β-linked polysaccharide,

91 - 99.94 wt-%based on the total weight of the reaction solution, of water and 0.02 - 2 wt-% based on the total weight of the reaction solution, of at least one starch comprising particle.

Also very preferred is a emulsion process for producing compounds, which are starch containing particles coated, embedded or encapsulated by at least one physically and/or covalently crosslinkable and β-linked polysaccharide, characterized in that the process is carried out in an solution with a Ca²⁺ concentration of 0.1 - 10 wt-%, based on the total weight of the water phase, together with 0.25 - 20 wt-% based on the total weight of the water phase of the reaction solution, of at least one biopolymer, and 50 - 99.5 wt-% based on the total weight of the water phase of the reaction solution, of water, and 0.05 - 20 wt-% based on the total weight of the water phase of the reaction solution, of at least one starch, characterized in that the process is carried out in an emulsion, whereby the ratio of water phase to oil phase goes from 5: 95 to 60:40, preferably from 50:50 to 20:80..

As already stated the starch can be added in pure form or in the form of a mixture with other (non carbohydrate) ingredients.

The compounds are isolated and if necessary washed and/or purified.

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. 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 compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer 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 particle 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: % Hydrolysis of free and encapsulated starch (circles = encapsulated starch; squares = unencapsulated starch). Error bars represent standard deviation from the mean of duplicate samples. Fig. 2.: % Hydrolysis of encapsulated starch as a function of CaCb concentration (circles = 6.82mM; squares = 13.64mM). Error bars represent standard deviation from the mean of duplicate samples.

Fig. 3.: Glucose release for alginate microbeads with encapsulated (circles) and unencapsulated (squares) starch. Error bars represent standard deviation from the mean of triplicate samples Fig. 4.: Confocal scanning light microscopy images of Ca-alginate gel beads containing entrapped rice starch granules (staining with acridine orange for 3 days); initial water phase contained 2% alginate and 2.5% starch.

Fig. 5.: Starch hydrolysis kinetic curves for rice starch granules entrapped in Ca-alginate beads (average size of 480 micron) versus 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. 6.: Starch hydrolysis kinetic curves for rice starch granules entrapped in Ca-alginate beads (average size of 480 micron) versus non encapsulated rice starch (in-vito 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.

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 slurry was first prepared by mixing 5% whole rice starch (ex. Remy Industries, Belgium) into a 2% alginate solution (Manugel DMB, ex ISP alginates, G content = 72%) at room temperature. Beads were prepared by extruding the 2% alginate/5% rice starch solution from a syringe into a gelling bath containing 6.82mM calcium chloride. The beads were left in the gelling bath for 4 hours to harden and then removed and stored in fresh calcium chloride solution at 5⁰C for 18 hours before use.

Example 1a (COMPARISON TEST)

Beads were also prepared from 2% alginate solution (without starch) and 5% rice starch was added to the surrounding solution to compare the digestibility of unencapsulated starch. The beads prepared were approximately 3.5mm in size.

The digestibility of the starch was determined as maltose release over time using a digestion assay. 2Og of starch-containing beads were placed in a 250ml glass bottle with 8Og deionised water and stirred gently at 37⁰C. 1 ml of the digest was removed for time = 0. 2ml of diluted amylase (Sigma No A6255; 1370U/mg protein; diluted ¹/ioo_,o∞ to a physiological concentration) was added and the stop-clock started. Stirring was continued and 2ml of digest were removed at the following time- points: 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300 minutes and overnight. This experiment was repeated using 19g beads without starch and 1g rice starch was added to the 8Og water and stirred as described. This provided an unencapsulated starch control for our experiments.

The digests at each time-point were diluted with water, as necessary, and analysed for reducing sugar concentration using the Dinitrosalicyclic acid (DNS) assay (Wood & Bhat (1988)). Fig. 1 shows the maltose release over time represented as % hydrolysis for these systems.

Example 2:

A slurry was first prepared by mixing 5% whole rice starch (ex. Remy Industries, Belgium) into a 2% alginate solution (Manugel DMB, ex ISP alginates, G content = 72%) at room temperature. Beads were prepared by extruding a 2% alginate solution from a syringe into a gelling bath containing two calcium chloride concentrations, 6.82 mM and 13.64 mM, respectively. The beads were left in the gelling bath for 4 hours to harden and then removed and stored in fresh calcium chloride solution at 5⁰C for 18 hours before use. The beads prepared were approximately 3.5mm in size.

The digestibility of the encapsulated starch was determined as maltose release over time using a digestion assay. 2Og of starch-containing beads were placed in a 250ml glass bottle with 8Og deionised water and stirred gently at 37⁰C. 1 ml of the digest was removed for time = 0. 2ml of diluted amylase (Sigma No A6255; 1370U/mg protein; diluted ¹/ioo_,o∞ to a physiological concentration) was added and the stop-clock started. Stirring was continued and 2ml of digest were removed at the following time-points: 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300 minutes and overnight.

The digests at each time-point were diluted with water, as necessary, and analysed for reducing sugar concentration using the Dinitrosalicyclic acid (DNS) assay (Wood & Bhat (1988)). Fig. 2 shows the maltose release over time represented as % hydrolysis for these systems.

It can be seen that the starch encapsulated within an alginate matrix gelled at higher CaCb concentration is liberated more quickly overtime. This has been related to a difference in porosity of the gel matrix from microstructural analysis of transmission electron micrographs (Soille, 1999). The system gelled with higher concentrations of CaCI₂ was found to contain larger pores than the system gelled with lower concentrations. Example 3:

Alginate microbeads containing starch were prepared using an emulsion route. Table 1 gives the ingredients used.

Sunflower oil and surfactant, Admul WoI, were mixed well at 500rpm using an overhead stirrer with a propeller blade. An alginate/starch slurry was prepared by mixing 1% rice starch (ex. Remy Industries, Belgium) into a 2% alginate (Manugel DMB, ex. ISP Alginates) solution. This slurry was then poured into the sunflower oil phase with continual mixing at 500rpm using the overhead stirrer. This formed a water in oil (W/O) emulsion. A 2.7M calcium chloride solution was added to the emulsion whilst stirring (concentration based on dilution to 0.68M with the dispersed aqueous alginate/starch phase). The mixture was then stirred for 2 hours at 500rpm to allow the dispersed aqueous phase to gel. The microbeads were separated from the emulsion by centrifugation for 30 minutes at 3000 rpm. The microbeads were washed to remove any remaining oil with a 1% Tween 60 solution containing 0.68M CaCb. The mixture was centrifuged to separate the microbeads. This washing step was repeated and then the microbeads were further washed with 0.68M CaCI₂ solution. The beads were stored in 0.68M CaCI₂ solution at 5⁰C overnight before use. Alginate microbeads without starch were prepared in the same way by adding alginate to the oil phase (without starch).

3.5g of alginate/1 % rice starch microbeads were placed in a container with 23ml deionised water and stirred gently at 37⁰C. Glucose concentration at time zero was determined using the glucose sensor. 10μl α-amylase (Sigma-Aldrich A6255; 1370U/mg protein; 32mg protein/ml) was diluted in 10ml deionised water and 3ml starch assay maltase (Sigma-Aldrich A9144-1VL Starch assay reagent 1 ; Reconstituted with 20ml de-ionised water) was added. 1 ml of this enzyme solution was added to the container with the microparticles. The amylase was diluted such that the concentration was at a physiologically relevant concentration (5-15nM) (Ferraris et al, 1990). Stirring was continued and glucose was determined over 40 minutes. The effect of encapsulation was determined by comparing alginate-starch microparticles and alginate microparticles with unencapsulated starch present. Fig. 3 shows the glucose release over time.

Despite the reduction in diffusion path length provided by the microbeads compared to the larger beads prepared in previous examples, glucose release was reduced by 50% due to encapsulation of starch within a biopolymer gelled matrix.

Example 4:

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 CaCI₂ solution or pure water. The microbeads were finally stored overnight in a 0.1 M CaCI₂ solution at refrigerated temperature before use. Fig. 4 shows the spherical shape of the alginate beads as well as the homogeneous distribution of the starch granules (green-yellow spots) inside the beads.

The hydrolysis rate of encapsulated starch was evaluated by means of an in-vitro test using alpha-amylase enzyme. First a suspension of gel 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.

The starch hydrolysis data of the Fig. 5 show that encapsulating starch in calcium- alginate beads allow to control the digestion rate of starch by amylase in-vitro. They also show that the alginate gel encapsulates can sustain a heating / cooking step and still lead to a retarded hydrolysis with fully gelatinised starch.

Example 5:

Emulsion made alginate beads containing rice starch granules were produced via the same method described in example 4 except that there was no overnight hardening step in 0.1 M CaCI₂ and except that the emulsifier dispersed in the oil phase; Hymono 8903 was replaced by Span 80 (Sorbitan monooleate, no. 85548, ex. Fluka Chemica) at the same concentration of 0.2 weight % with respect to the water phase. Two different high M alginate products varying in molecular weight were utilised: a high Mw, alginate HM (Sigma-Aldrich no. A-7128: alginic acid sodium salt, with a viscosity of 1400 cP at 2% in water) and a low Mw, alginate LM (Sigma-Aldrich no. A-5158: alginic acid sodium salt, with a viscosity of 25 cP at 2% in water). The initial concentration of alginate HM in the water phase of the emulsion was 2 % by weight and for alginate LM it was increased to 8% by weight (so that the rheological properties of alginate LM solution such as viscosity and elastic modulus were as close as possible to that of alginate HM solution). The microbeads HM (made with alginate HM) and the microbeads LM (with alginate LM) were produced with the same applied palette rotational speed of 800 rpm. The average particle size of the gel beads were measured by small angle light scattering of suspended encapsulates in water: mean diameter of ca. 220 micron for beads HM and ca. 400 micron for beads LM.

The effect of alginate concentration in the gel beads (network density) on starch hydrolysis rate was assessed in-vitro by applying the same procedure described in example 4. Fig. 6 displays the hydrolysis profiles of heat-treated microbeads HM and LM produced at 800 rpm, having an average size of 200 micron and 400 micron respectively. In addition it also shows the data obtained for alginate HM beads made at 300 rpm, leading to an average diameter of 740 micron. The hydrolysis of starch entrapped in microbeads LM appeared much more delayed compared to microbeads HM prepared at the same rotational speed. However the mean bead diameter was also 2 times higher for the LM system compared to HM. Therefore the contribution of the gel network density and the contribution of the bead size to the retarded hydrolysis could not be separated. The third microbeads sample produced, with alginate HM, at a lower stirring speed, had a bigger mean diameter of 740 micron and led to a close hydrolysis profile compared to that of the LM system. The only way to explain this result was seen as being due to the higher density of the gel network in the LM system (or lower porosity), hence a retarded diffusion of amylase through the gel network. If this would not be the case, the 400 micron beads would not lead to the same delayed starch hydrolysis than for the 740 micron beads. This example also shows the effect of the average bead size on the starch digestion rate.

Claims

1. A process for production of compounds, which are starch containing particles coated, embedded or encapsulated by at least one biopolymer, characterized in that the process is carried out in a reaction solution with a Ca²⁺ concentration of 0.1 - 10 wt-%, based on the total weight of the reaction solution.

2. A process according to claim 1 , wherein the compounds can have any shape.

3. A process according to any of the preceeding claims, wherein the starch can be chosen from the group consisting of raw starches, modified starches, and pregelatinized starches.

4. A process according to any of the preceeding claims, wherein the biopolymer is present in the form of a gel.

5. A process according to claim 4, wherein the gel comprises

0.5 - 20 wt-%, based on the total weight of the biopolymer gel of at least one biopolymer, and

80 - 99.5 wt-%, based on the total weight of the biopolymer gel, of water.

6. A process according to any of the preceeding claims, wherein the content of starch is 0.1 - 20 wt-%, based on the total weight of the compounds.

7. A process according to any of the preceeding claims, wherein the size of the compounds is less than 1000 microns.

8. A process according to any of the preceeding claims, wherein the biopolymers are physically and/or covalently crosslinkable polysaccharides.

9. A process according to any of the preceeding claims wherein the Ca²⁺ concentration is 0.1 to 2.5 wt-%, based on the total weight of the reaction solution.

10. A process according to claims 1-8, wherein the Ca²⁺ concentration is 3.5 to 9 wt-%, based on the total weight of the reaction solution.

11. A process according to any of the preceeding claims, wherein the process is an extrusion process or an emulsion process.

12. A process according to any of the preceeding claims, wherein the process is an extrusion process, characterized in that the process is carried out in a reaction solution with a Ca²⁺ concentration of 0.02 - 5 wt-%, based on the total weight of the reaction solution, together with

91 - 99.94 wt-% based on the total weight of the reaction solution, of water and

0.02 - 2 wt-% based on the total weight of the reaction solution of at least one starch comprising particle.

13. A process according to claims 1 - 11 , wherein the process is an emulsion process, characterized in that the process is carried out in an solution with a Ca²⁺ concentration of 0.1 - 10 wt-%, based on the total weight of the water phase, together with 0.25 - 20 wt-% based on the total weight of the water phase of the reaction solution, of at least one biopolymer, and 50 - 99.5 wt-% based on the total weight of the water phase of the reaction solution, of water, and 0.05 - 20 wt-% based on the total weight of the water phase of the reaction solution, of at least one starch, characterized in that the process is carried out in an emulsion, whereby the ratio of water phase to oil phase goes from 5: 95 to 60:40, preferably from 50:50 to 20:80.

14. Food products comprising compounds obtained by the process according to any of claims 1 - 13.

15. Food products according to claim 14, which are chosen from the group consisting of 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.