WO2011004375A1

WO2011004375A1 - Heat resistant probiotic compositions and healthy food comprising them

Info

Publication number: WO2011004375A1
Application number: PCT/IL2010/000550
Authority: WO
Inventors: Adel Penhasi; Yohai Zorea
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-07-09
Filing date: 2010-07-08
Publication date: 2011-01-13
Anticipated expiration: 2012-01-09
Also published as: IL217449A0; CA2767602A1; AU2010269814A8; EP2451300A1; AU2010269814B8; US9961910B2; AU2010202518B2; IL199781A0; US20110008493A1; CN102595938A; CN105105104A; ES2700879T3; RU2012104796A; NZ597992A; AU2010202518A1; EP2451300A4; AU2010269814B2; US20100303962A1; EP2451300B2; AU2010269814A1

Abstract

The invention provides granules of probiotic microorganisms for admixing into healthy food, wherein the microorganisms are stabilized to survive heat processing of the food. Healthy food provided by the invention includes pastry, bread, dairy products, and others.

Description

HEAT RESISTANT PROBIOTIC COMPOSITIONS

AND HEALTHY FOOD COMPRISING THEM

Field of the Invention

The present invention relates to the health food products, particularly to bakery probiotic products. Provided is a method of preparing a product which undergoes heat treatment in at least one stage of its preparation, while keeping alive a sufficient amount of probiotic microorganisms.

Background of the Invention

Probiotics are live microbial food supplements which beneficially affect the host by supporting naturally occurring gut flora, by competing harmful microorganisms in the gastrointestinal tract, by assisting useful metabolic processes, and by strengthening the resistance of the host organism against toxic substances. A number of organisms is used in probiotic foods, an example being microbial genera Lactobacillus or Bifidobacterium. Probiotic organisms should survive for the lifetime of the product, in order to be effective, and further they should survive the whole way through the gastrointestinal tract to the colon. Probiotic organisms are usually incorporated into milk products, such as yogurts. The need is felt to deliver the beneficial microorganisms in other foodstuff types, for example in baked products. However, the main problem in preparing baked health food is the processing temperature, which is usually so high (exceeding 180⁰C) that it nearly sterilizes the products. WO 94/00019 describes a method of preparing a baked product containing living microorganisms, comprising cooling a baked product and injecting into it a living suspension. WO 2009/069122 of the same inventors as the present invention describes a process for preparing baked food, comprising encapsulating probiotic granules, thereby enhancing their stability. It is an object of the present invention to provide a process for preparing a nutritionally acceptable composition comprising probiotic microorganisms, the composition being resistant to high temperatures. Another object of the invention is to provide a bakery product comprising viable microorganisms in a sufficient amount.

It is a still other object of the invention to provide a process for preparing a probiotic bakery product, without need of injecting viable microorganisms into the bakery product after the baking process.

It is a further object of the invention to provide bakery products containing live probiotic microorganisms during the whole process of baking.

It is a still further object of the invention to provide bakery products comprising heat-stabilized probiotic composition.

It is also an object of the invention to provide probiotic bakery products exhibiting a long shelf life.

Other objects and advantages of present invention will appear as description proceeds.

Summary of the Invention

The present invention provides a probiotic granule comprising i) a core comprising probiotic microorganisms and a substrate in which said microorganisms are absorbed; ii) an inner oily layer coating said core; and iii) a first outer layer and a second outer layer, which outer layers coat said core and said inner layer, comprising at least two different polymers. Said substrate and said two different polymers may comprise three nutritionally acceptable polysaccharides. Said core may further comprise one or more supplemental agents for said microorganisms. Preferably, the agents support the growth of said microorganisms, and may comprise prebiotic materials, such as oligosaccharides. Said inner oily layer may comprise a material selected from fatty acids, waxes, fats, oils, and lipids. Said first outer layer confers stability to said microorganisms under the conditions of upper gastrointestinal tract, and said second outer layer increases the stability of said microorganisms in said core at an increased temperature. Said substrate comprises one or more components selected from saccharides and additional agents. Said agents are selected from stabilizer, chelator, synergistic agent, antioxidant, and pH regulator, and said saccharides preferably comprise prebiotic oligosaccharides.

Said two outer layers in the granules of the invention comprise two different polymers; the polymers may be of fibrous or of gelatinous character. In one embodiment, at least one of the outer layers comprises a fibrous polysaccharide, and at least one of the outer layers comprises a gelatinous polysaccharide. A probiotic granule according to the invention may comprise additional layers, for example at least one intermediate layer positioned between said oily layer and said second outer layer. The probiotic granule of the present invention comprises a probiotic microorganism; the organism is preferably bacterial. Said microorganism advantageously comprises a genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces, or a mixture thereof. The microorganisms are in a preferred embodiment probiotic bacteria.

The invention provides a method for manufacturing a probiotic granule comprising a core, containing probiotic microorganisms and a substrate in which said microorganisms are absorbed, surrounded by an inner oily layer and two outer polymer layers, the method comprising i) mixing a suspension of probiotic microorganisms with a cellulose-based substrate and with supplemental agents for the microorganisms, thereby obtaining a core mixture; ii) coating particles of said core mixture with an oily layer, thereby obtaining oil-coated particles; iii) coating said oil-coated particles with a first polymer layer, which first polymer layer confers stability to said microorganisms under the conditions of upper gastrointestinal tract, thereby obtaining particles coated with two layers; and iv) coating said two-layer particles with a second polymer layer, which second polymer layer increases the stability of the microorganisms in said core under the conditions of baking. Said microorganisms may comprise one or more microbial strains, and are admixed to and absorbed on a microbiologically compatible polymer, which as also nutritionally acceptable and approvable, an example being a polysaccharide, such as cellulose based material. Materials supporting the stability or growth of said microorganisms may be added into the mixture. Preferably included are probiotic supporters known as prebiotics, for example maltodextrin, trehalose, etc. The coating steps may utilize techniques known in the field, including fluidized bed coating, spraying, etc. When creating the coated layers, solutions or suspensions may be employed, as well as powders, etc. Said coating steps ii) to iv) result in a mass increase of from 10 to 100% relatively to the mass of the core, for example between 15 and 50%. In a preferred embodiment, a method for manufacturing a probiotic granule comprises i) mixing an aqueous suspension of probiotic microorganisms comprising at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces, or a mixture thereof with at least one polysaccharide and at least one oligosaccharide (an example being microcrystalline cellulose with maltodextrin and trehalose), thereby obtaining a core mixture; ii) coating particles of said core mixture with an oily layer, thereby obtaining oil-coated particles; iii) coating said oil-coated particles with a first polymer layer and with a second polymer layer; iv) wherein said two polymers layers are different and comprise at least two materials selected from cellulose, modified cellulose, polysaccharide and or synthetic polymers and a mixture thereof.

Importantly, the invention relates to a probiotic composition comprising granules having a core, containing probiotic microorganisms and a substrate in which said microorganisms are absorbed, surrounded by an inner oily layer and two outer polymer layers. The prebiotic composition according to the invention exhibits a high resistance to the increased temperature. When relating here to a high resistance to the increased temperature, or when relating to a high heat-stability, intended is survival of the probiotic microorganisms within the granules compared to free microorganisms, and particularly survival of the probiotic microorganisms within the granules admixed in a food product compared to free microorganisms. In one aspect of the invention, the probiotic microorganisms in the core of the three-layer granule survives exposures of the granules to temperatures higher than ambient temperature. The heat stability of the probiotic composition according to the invention is sufficiently high to ensure that a part of the initial bacterial load admixed in a probiotic food product of the invention remains viable even after all necessary manufacturing steps. Such steps may include baking.

The invention provides a healthy food product or a food additive comprising a probiotic composition, as above described, comprising the stable probiotic granules. Said product may preferably comprise a bakery product, for example pastry or bread. Said product may also comprise tuna, chocolate, fruit juices, and dairy products.

The invention provides a healthy food product comprising pastry, bread, flour, flour products, baked goods, frozen baking products, yogurt, dairy products, chocolate, nectars, fruit juices, and tuna. The food product according to the invention, comprising probiotic granules, may be exposed to higher than ambient temperature during the production process.

Brief Description of the Drawing

The above and other characteristics and advantages of the invention will be more readily apparent through the following examples, and with reference to the appended drawing, wherein: Fig. 1. shows a schema of a multiple-layered capsule according to one embodiment of the invention, to be comprised in healthy food; the encapsulation is designed to provide probiotic microorganisms with maximum heat resistance during the heating step of the manufacturing process, when providing said food, and also with high biological efficacy in the lower GI tract after leaving the stomach intact; the white core comprises probiotic microorganisms and absorbing substrate! the first dark layer adjacent to the core is an oily layer! a light layer adjacent to the oily layer is an acid-resistant layer," a dark layer adjacent to the acid-resistant layer is an optional intermediate layer,' and the outer light layer is a heat-resistant layer.

Detailed Description of the Invention

It has now been surprisingly found that probiotic microorganisms may be formulated in the cores of granules having at least three layers, thus providing probiotic compositions of viable probiotic organisms even after baking, the compositions being further stable on storage and capable of administering viable microorganisms to colon after the oral administration.

The invention provides granular probiotics to be used as healthy food additives. The present invention is particularly directed to a process for the preparation of baked food, such as probiotic pastry. A suspension of probiotic microorganisms is mixed and granulated with a suitable carrier material to form a core part of particles to be coated. The probiotic microorganism may be selected from Bacillus coagulans GBI-30, 6086; Bacillus subtilis var natt; Bifidobacterium sp LAFTI B94; Bifidobacterium bifidum; Bifidobacterium bifidum rosell-71; Bifidobacterium breve; Bifidobacterium breve Yakult; Bifidobacterium breve Rosell-70; Bifidobacterium infantis; Bifidobacterium infantis 35624; Bifidobacterium lactis; Bifidobacterium longum; Bifidobacterium longum Rosell-175; Bifidobacterium longum BB536; Bifidobacterium animalis; Bifidobacterium animalis subsp. lactis BB- 12; Bifidobacterium animalis subsp. lactis HN019; Escherichia coli M-17; Escherichia coli Nissle 1917; Lactobacillus acidophilus; Lactobacillus acidophilus DDS-I; Lactobacillus acidophilus LAFTI® LlO; Lactobacillus acidophilus LA-5; Lactobacillus acidophilus NCFM; Lactobacillus casei; Lactobacillus casei LAFTI® L26; Lactobacillus casei CRL431; Lactobacillus casei DNl 14-001 (Lactobacillus casei Immunitas(s) / Defensis); Lactobacillus breυis; Lactobacillus bulgaricus; Lactobacillus gasseri; Lactobacillus paracasei; Lactobacillus casei F19; Lactobacillus casei Shirota; Lactobacillus paracasei StIl (or NCC2461); Lactobacillus plantarum; Lactobacillus plantarum 299V; Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri SD2112J; Lactobacillus rhamnosus; Lactobacillus saliυarius; Lactobacillus delbrueckii; Lactobacillus fermentum; Lactococcus lactis; Lactococcus lactis LlA; Lactococcus lactis subsp; Lactococcus lactis Rosell-1058; Lactobacillus paracasei StIl (or NCC24610; Lactobacillus johnsonii LaI (= Lactobacillus LCIj; Lactobacillus johnsonii LaI (= Lactobacillus LCl, Lactobacillus johnsonii NCC 533); Lactobacillus rhamnosus Rosell-11; Lactobacillus acidophilus Rosell-52; Streptococcus thermophilus; Diacety lactis; Lactobacillus rhamnosus ATCC 53013 (discovered by Gorbach & Goldin(=LGG)); Lactobacillus rhamnosus LB21; Lactobacillus rhamnosus GR-I & Lactobacillus reuteri RC- 14; Lactobacillus acidophilus NCFM & Bifidobacterium bifidum BB- 12; Saccharomyces cerevisiae; Saccharomyces cerevisiae (boulardii) lyo; and a mixture thereof.

The granulation process may employ a suitable granulator, or alternatively a fluidized bed. The drying process may comprise lyophilization. The probiotic granules according to the invention may have a wide range of dimensions. A non-limiting example of a probiotic granule according to the invention is an essentially spherical particle having a mean diameter of about from 0.1 to about 1000 microns. Without wishing to be limited by any theory, it is believed that mixing the probiotic microorganisms with a microbiologically acceptable polymer, such as a cellulose derivative, in a particle core to be further coated with a triple layer of microbiologically acceptable materials results in an increased heat resistance of the microorganism, wherein the increased resistance may result both from lowered heat conductivity and from cell stabilization. The probiotic microorganisms processed according to the invention resist baking heat for a predetermined baking temperature and baking time. It is believed that the inner oily layer, and the first outer layer (enteric coating layer) further protect the probiotic microorganisms during their passage through the upper gastrointestinal tract, enabling the release of the probiotics in either small intestine, or colon or both. The structure of the granular probiotic composition of the invention ensures a relatively high stability (microbial viability) on storage before its use in preparing food products, as well as inside food products on their storage. Furthermore, said structure ensures desirable release of the viable microorganisms in the lower gastrointestinal tract of a person eating the healthy food, for example healthy bakery product. Furthermore, the whole beneficial effect may be further enhanced when including in the probiotic composition also oligosaccharides (called prebiotics) supporting the growth of the beneficial microorganism. Optionally, said first outer layer (the gastrointestinal resistant coating layer, called the enteric coating layer) may be separated from said second outer layer (the outer heat resistant coating layer) by an intermediate inert coating layer in order to prevent any possible reaction between them.

Said oily inner layer may be selected from the group consisting of bee wax, carnauba wax, japan wax, bone wax, paraffin wax, Chinese wax, lanolin (wool wax), shellac wax, spermaceti, bayberry wax, candelilla wax, castor wax, esparto wax, jojoba oil, ouricury wax, rice bran wax, soy wax, ceresin waxes, montan wax, ozocerite, peat waxes, microcrystalline wax, petroleum jelly, polyethylene waxes, fischer-tropsch waxes, chemically modified waxes, substituted amide waxes, polymerized α-olefins, vegetable oil, hydrogenated vegetable oil, hydrogenated castor oil, fatty acids, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, stearic acid, sodium stearate, calcium stearate, magnesium stearate, palmitate, palmitoleate, hydroxypalmitate, oleate esters of long-chain, aliphatic alcohols, hydroxyl- octacosanylhydroxy stearate, phospholipids, lecithin, phosphatidyl- choline.

Said first outer layer may comprise one or more pH-sensitive coatings, commonly referred to in the art as "enteric coatings," according to conventional procedures in order to delay the release of pro-biotic microorganisms. Suitable pH-sensitive polymers include those which are relatively insoluble and impermeable at the pH of the stomach, but which are more soluble or disintegrate or permeable at the pH of the small intestine and colon. Such pH-sensitive polymers include polyacrylamides, phthalate derivatives such as acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), other cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (HPCP), hydroxypropylethyl cellulose phthalate (HPECP), hydroxylpro- plymethylcellulose phthalate (HPMCP), HPMCAS, methylcellulose phthalate (MCP), polyvinyl acetate phthalate (PVAcP), polyvinyl acetate hydrogen phthalate, sodium CAP, starch acid phthalate, cellulose acetate trimellitate (CAT), styrene-maleic acid dibutyl phthalate copolymer, styrene-maleic acid/polyvinylacetate phthalate copolymer, styrene and maleic acid copolymers, polyacrylic acid derivatives such as acrylic acid and acrylic ester copolymers, polymethacrylic acid and esters thereof, polyacrylic and methacrylic acid copolymers, shellac, and vinyl acetate and crotonic acid copolymers. Preferred pH-sensitive polymers include shellac, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, particularly copolymers comprising acrylic acid and at least one acrylic acid ester, polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, and vinyl acetate, crotonic acid copolymers, alginic acid and alginates such as ammonia alginate, sodium, potassium, magnesium or calcium alginate. A particularly preferred group of pH-sensitive polymers includes CAP, PVAcP, HPMCP, HPMCAS, anionic acrylic copolymers of methacrylic acid and methylmethacrylate, and osmopolymers comprising acrylic acid and at least one acrylic acid ester. Cellulose acetate phthalate may be applied as an enteric coating to the encapsulated pro-biotic compositions of the invention to provide delayed release of pro-biotic microorganisms until the dosage form has exited the stomach. The CAP coating solution may also contain one or more plasticizers, such as diethyl phthalate, polyethyleneglycol-400, triacetin, triacetin citrate, propylene glycol, and others as known in the art. Preferred plasticizers are diethyl phthalate and triacetin. The CAP coating formulation may also contain one or more emulsifiers, such as polysorbate-80.

Anionic acrylic copolymers of methacrylic acid and methylmethacrylate are also particularly useful enteric coating materials for delaying the release of pro-biotic microorganisms until they have moved to a position in the GI tract which is distal to the stomach. Copolymers of this type are available from Rohm America, Inc., under the trade names EUDRAGIT-L and EUDRAGIT- S. EUDRAGIT-L and EUDRAGIT-S are anionic copolymers of methacrylic acid and methylmethacrylate. The ratio of free carboxyl groups to the esters is approximately 1:1 in EUDRAGIT-L and approximately 1:2 in EUDRAGIT- S. Mixtures of EUDRAGIT-L and EUDRAGIT-S may also be used. For coating these acrylic coating polymers can be dissolved in an organic solvent or mixture of organic solvents or suspended in aqueous media. Useful solvents for this purpose are acetone, isopropyl alcohol, and methylene chloride. It is generally advisable to include 5-20 wt% plasticizer in coating formulations of acrylic copolymers. Useful plasticizers include polyethylene glycols, propylene glycols, diethyl phthalate, dibutyl phthalate, castor oil, and triacetin. EUDRAGIT-L is preferred because it dissolves relatively quickly at intestinal pH. In addition to the pH-sensitive polymers listed above, delayed release coatings may consist of a mixture or blend of two or more pH-sensitive polymers or may consist of a mixture of one or more pH- sensitive polymers and one or more non-pH-sensitive polymers. Addition of a non-pH-sensitive polymer to the pH-sensitive polymer is useful in modulating the duration of the delay or rate of release of pro-biotic microorganisms from the granule, bead or pellets. For example, the delay can be lengthened by blending an aqueous-insoluble polymer with the pH- sensitive polymers, while the delay can be shortened by blending a water- soluble polymer with the pH-sensitive polymers. Preferred non-pH-sensitive aqueous insoluble polymers include cellulose esters, cellulose ethers, polyacrylates, polyamides, polyesters, and vinyl polymers. Preferred non-pH- sensitive aqueous-soluble polymers include hydroxyalkyl-substituted cellulosics such as HPC, HEC and HPMC, PVA, PEG, PEO, PEG/PPG copolymers, and aqueous-soluble polyamides, polysaccharides, and polyacrylates.

Various additives may be included in such coatings, including emulsifiers, plasticizers, surfactants, fillers and buffers. Finally, the polymeric coating may be described as being "quasi-enteric" in the sense that it remains substantially intact for a significant period of time (e.g., greater than an hour) after the dosage form exits the stomach, thereafter becoming sufficiently pro-biotic microorganisms-permeable to permit gradual release of pro-biotic microorganisms by diffusion through the coating.

Optionally a formulation according to the present invention features an intermediate layer between the enteric layer and the outer heat resistant layer. The intermediate coating layer of the composition according to the present invention substantially entirely covers the enteric coating of each individual unit. The intermediate layer is provided in order to prevent direct contact between the enteric layer and the outer heat resistant layer thus preventing any interaction between them. The intermediate coating layer according to any of the embodiments of the present invention optionally and preferably comprises one of aqueous soluble polymers which includes but is not limited to polyvinyls such as povidone (PVP: polyvinyl pyrrolidone), polyvinyl alcohol, copolymer of PVP and polyvinyl acetate, cross-linked polyvinyls, HPC (hydroxypropyl cellulose) (more preferably a low molecular weight), HPMC (hydroxypropyl methylcellulose) (more preferably a low molecular weight), CMC (carboxy methyl cellulose) (more preferably a low molecular weight), ethylcellulose, MEC (methylethyl cellulose), CMEC (carboxy methyl ethyl cellulose), HEC (hydroxyethyl cellulose) HEMC (hydroxy methyl ethyl cellulose), polyethylene oxide, acacia, dextrin, magnesium aluminum silicate, starch, polyacrylic acid, polyhydroxy- ethylmethacrylate (PHEMA), polymethacrylates and their copolymers, gum, water soluble gum, polysaccharides, cross-linked polysaccharides, peptides or cross-linked peptides, protein or cross-linked proteins, gelatin or cross- linked gelatin, hydrolyzed gelatin or cross-linked hydrolyzed gelatin, collagen or cross-linked collagen, modified cellulose, polyacrylic acid or cross-linked polyacrylic acid and/or mixtures thereof.

Said second outer layer, outer heat resistant coating, may comprise linear, branched, or crosslinked polymers. They may be homopolymers or copolymers or graft copolymers or block copolymers, single or a blend. Although they may be synthetic polymers, preferably, such polymers may be naturally occurring polymers such as polysaccharides, cross-linked polysaccharides, gums, modified polysaccharides modified starch and modified cellulose, polysaccharide can be selected from the group consisting of chitin, chitosan, dextran, pullulan, gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum and scleroglucan, starches, dextrin and maltodextrin, hydrophilic colloids such as pectin, high methoxy pectin, and low methoxy pectin. Phosphatides such as lecithin may be employed in the composition. The cross-linked polysaccharide can be selected from the group consisting of insoluble metal salts or cross-linked derivatives of alginate, pectin, xantham gum, guar gum, tragacanth gum, and locust bean gum, carrageenan, metal salts thereof, and covalently cross-linked derivatives thereof. The modified cellulose may be selected from the group consisting of cross-linked derivatives of hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, methylcellulose, carboxymethyl cellulose, and metal salts of carboxymethylcellulose. More preferably such polymers may be cationic polymers. Samples of cationic polymers include but are not limited to cationic polyamines, cationic polyacrylamide, cationic polyethyleneimine, cationic polyvinyl alcohol which is a methyl chloride quaternary salt of poly(dimethylamino ethyl acrylate/polyvinyl alcohol graft copolymer or a methyl sulfate quaternary salt of poly(dimethylamino ethyl acrylate)/polyvinyl alcohol graft copolymer, a series of dry blends of PVA with N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride, available from Dow Chemical Company under the name QUAT.RTM.-188, containing varying amounts of water and of NaOH, cationic polyvinylpyrrolidone, gelatin, polyvinylpyrrolidone, copolymer of polyvinylacetate and polyvinylpyrrolidone, copolymer of polyvinylalcohol and polyvinylpyrrolidone, polyethyleneimine, polyallylamine and its salts, polyvinylamine and its salts, dicyandiamide-polyalkylenepolyamine condensate, polyalkylenepolyamine- dicyandiamideammonium condensate, dicyandiamide-formalin condensate, an addition polymer of epichlorohydrin-dialkylamine, a polymer of diallyldimethylammonium chloride ("DADMAC"), a copolymer of dimethylaminoethyl methacrylate and neutral methacrylic esters available from Rohm Pharma (Degusa) under the name Eudragit E, a copolymer of diallyldimethylammonium chloride-SO2, polyvinylimidazole, polyvinyl pyrrolidone, a copolymer of vinylimidazole, polyamidine, chitosan, cationized starch, cationic polysaccharides such as cationic guar and cationic hydroxypropyl guar, polymers of vinylbenzyltrimethylqammoniumchloride, (2-methacryloyloxy ethyl) trimethyl-ammoniumchloride, polymers of dimethylaminoethyl methacrylate, a polyvinylalcohol with a pendant quaternary ammonium salt, cationic polyvinylformamide cationic poly- vinylacetamide, cationic polyvinylmethylformamide, cationic polyvinylme- thylacetamide, poly (dimethylaminopropylmethacrylamide) (DMAPMAM), poly(dimethyl aminoethylacrylate), poly(acryloylethyl trimethylammonium chloride), poly(acrylamidopropyltrimethylammonium chloride) (poly APTAC), poly (methacrylamidopropyltrimethylammonium chloride) (poly MAPT AC), and its salts, poly(vinylpyridine) and its salts, poly(dimethylamine-co- epichlorohydrin), poly(dimethylamine-co-epichlorohydrin-co-ethylene diami- ne), polyζamidoamine-epichlorohydrin), cationic starch, copolymers which contain N-vinylformamide, allylamine, diallyldimethylammonium chloride, N-vinylacetamide, N-vinylpyrrolidone, N-methyl-N-vinylformamide, N- methyl-N-vinylacetamide, dimethylamino propyl methacrylamide, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, acryloyl- ethyltrimethylammonium chloride or methacryl amidopropyltri- methylammonium chloride in'the form of polymerized units and, if required, in cleaved form, and salts thereof and combinations thereof. The chitosan may have a deacetylation degree ranging from 80% to more than 95%. The chitosan may also optionally have a viscosity ranging from 50 mpa to 800 mpa. The chitosan may optionally be trimethylchitosan or quaternised chitosan. The polymer may also optionally be polyglucosamine, one of the components of chitosan. For example, the polymer may optionally be the β- 1,4 polymer of D-glucosamine or the β-1,4 polymer of D-glucosamine and N- acetyl-D-glucosamine.

According to a preferred embodiment of the invention, the probiotic microorganisms in said granule core are mixed with a substrate. Said substrate may comprise monosaccharides such as trioses including ketotriose (dihydroxyacetone) and aldotriose (glyceraldehyde), tetroses such as ketotetrose (erythrulose), aldotetroses (erythrose, threose) and ketopentose (ribulose, xylulose), pentoses such as aldopentose (ribose, arabinose, xylose, lyxose), deoxy sugar (deoxyribose) and ketohexose (psicose, fructose, sorbose, tagatose), hexoses such as aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose), deoxy sugar (fucose, fuculose, rhamnose) and heptose such as (sedoheptulose), and octose and nonose (neuraminic acid). The substarte may comprise multiple saccharides such as 1) disaccharides, such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose,

2) trisaccharides such as raffinose, melezitose and maltotriose,

3) tetrasaccharides such as acarbose and stachyose, 4) other oligosaccharides such as fructooligosaccharide (FOS), galactooligosaccharides (GOS) and mannan-oligosaccharides (MOS), 5) polysaccharides such as glucose-based polysaccharides/glucan including glycogen ^• starch (amylose, amylopectin), cellulose, dextrin, dextran, beta-glucan (zymosan, lentinan, sizofiran), and maltodextrin, fructose-based polysaccharides/fructan including inulin, levan beta 2-6, mannose-based polysaccharides (mannan), galactose-based polysaccharides (galactan), and N-acetylglucosamine-based polysaccharides including chitin. Other polysaccharides may be comprised, including gums such as arabic gum (gum acacia).

According to a preferred embodiment of the invention, the probiotic microorganisms in said inner core are mixed with a substrate which may further comprise additional components. The components may be selected from chelating agents. Preferably, the chelating agent is selected from the group consisting of antioxidants, dipotassium edetate, disodium edetate, edetate calcium disodium, edetic acid, fumaric acid, malic acid, maltol, sodium edetate, trisodium edetate.

According to some embodiments of the present invention, the core further comprises both a chelator and a synergistic agent (sequestrate). Without wishing to be limited by a single hypothesis or theory, chelating agents and sequestrates may optionally be differentiated as follows. A chelating agent, such as citric acid is intended to help in chelation of trace quantities of metals thereby assisting to prevent the loss of the active ingredient(s), such as simvastatin, by oxidation. A sequestrate such as ascorbic acid, optionally and preferably has several hydroxyl and/or carboxylic acid groups, which can provide a supply of hydrogen for regeneration of the inactivated antioxidant free radical. A sequestrate therefore preferably acts as a supplier of hydrogen for rejuvenation of the primary antioxidant. According to preferred embodiments of the present invention, the core further comprises an antioxidant. Preferably, the antioxidant is selected from the group consisting of cysteine hydrochloride, 4,4 (2,3 dimethyl tetramethylene dipyrochatechol), tocopherol-rich extract (natural vitamin E), α-tocopherol (synthetic Vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, butylhydroxinon, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), propyl gallate, octyl gallate, dodecyl gallate, tertiary butylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid (Vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate, ascorbyl palmitate, and ascorbyl stearate. Comprised in the core may be citric acid, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, anoxomer, erythorbic acid, sodium erythorbate, erythorbin acid, sodium erythorbin, ethoxyquin, glycine, gum guaiac, sodium citrates (monosodium citrate, disodium citrate, trisodium citrate), potassium citrates (monopotassium citrate, tripotassium citrate), lecithin, polyphosphate, tartaric acid, sodium tartrates (monosodium tartrate, disodium tartrate), potassium tartrates (monopotassium tartrate, dipotassium tartrate), sodium potassium tartrate, phosphoric acid, sodium phosphates (monosodium phosphate, disodium phosphate, trisodium phosphate), potassium phosphates (monopotassium phosphate, dipotassium phosphate, tripotassium phosphate), calcium disodium ethylene diamine tetra-acetate (calcium disodium EDTA), lactic acid, trihydroxy butyrophenone and thiodipropionic acid. According to one preferred embodiment, the antioxidant is BHA.

According to preferred embodiments of the present invention, the core further comprises a stabilizer. Preferably, the stabilizer can be a basic substance which can elevate the pH of an aqueous solution or dispersion of the formulation to at least about 6.8. Examples of such basic substances include but are not limited to antiacids such as magnesium aluminometasilicate, magnesium aluminosilicate, magnesium aluminate, dried aluminum hydroxide, synthetic hydrotalcite, synthetic aluminum silicate, magnesium carbonate, precipitated calcium carbonate, magnesium oxide, aluminum hydroxide, and sodium hydrogencarbonate, and mixtures thereof; and pH-regulator agents such as L-arginine, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogenphosphate, potassium phosphate, dipotassium hydrogenphosphate, potassium dihydrogenphosphate, disodium citrate, sodium succinate, ammonium chloride, and sodium benzoate and mixtures thereof. The basic substance can be selected from the group consisting of an inorganic water-soluble or inorganic water- insoluble compound. Examples of inorganic water-soluble basic substance includes but are not limited to carbonate salt such as sodium or potassium carbonate, sodium bicarbonate, potassium hydrogen carbonate, phosphate salts selected from, e.g., anhydrous sodium, potassium or calcium dibasic phosphate, trisodium phosphate, alkali metal hydroxides, selected from sodium, potassium, or lithium hydroxide, and mixtures thereof. Sodium bicarbonate advantageously serves to neutralize acid groups in the composition in the presence of moisture that may adsorb onto particles of the composition during storage. The calcium carbonate exerts a buffering action in the stored composition, without apparent effect on material release upon ingestion. It has further been discovered that the carbonate salts sufficiently stabilize the composition. Examples of inorganic water-insoluble basic substance include but not limited to suitable alkaline compounds capable of imparting the requisite basicity, include certain pharmaceutically acceptable inorganic compounds commonly employed in antiacid compositions e.g., magnesium oxide, magnesium hydroxide, or magnesium carbonate, magnesium hydrogen carbonate, aluminum or calcium hydroxide or carbonate, composite aluminum-magnesium compounds, such as magnesium aluminum hydroxide, silicate compound such as magnesium aluminum silicate (Veegum F), magnesium aluminometasilicate (Nesulin FH2), magnesium aluminosilicate (Nisulin A); as well as pharmaceutically acceptable salts of phosphoric acid such as tribasic calcium phosphate; and mixtures thereof.

The invention enables to manufacture various healthy food products without separating the admixing heating steps. Enables is, for example, the preparation of bread dough containing the probiotic granules, avoiding any awkward injecting steps of prior art methods. The mass ratio between the probiotic composition and the rest of the dough may be, for example, 1:100. The encapsulated pro-biotic microorganisms according to the present invention may be incorporated into flour, flour products, bake goods, yogurt, tuna, frozen baking products, chocolate, hot drinks, nectars and fruit juices, and other products that during the handling and/or production process may be exposed to higher temperature than an ambient (room temperature).

The invention will be further described and illustrated in the following examples.

Examples

Example 1

Materials

Method

1. Absorption of microorganisms on microcrystalline core substrate

Lactobacillus acidophilus and Bifidobacterium were absorbed on MCC substrate based on a ratio of 38:62 respectively. For this purpose an aqueous- based suspension of 30% of the microorganisms and maltodextrin and trehalose was prepared. The concentration of microorganisms was about 15% (w/w) in that suspension. The absorption process was carried out at an outlet temperature <35°C. 2. The first coating layer using a hvdrogenated vegetable oil

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose hydrogenated vegetable oil was sprayed on the Bacteria-absorbed MCC substrate at 40⁰C to obtain a 40% weight gain. The inlet air flow was adjusted to be low.

3. The second coating layer - an enteric coating

The coating was carried out using a solution of ethylcellulose ElOO and sodium alginate with a ratio of 85:15 respectively in ethanol with a concentration of total solid of 6% (w/w). The end point of the coating process was targeted to obtain a 20% weight gain by the coating. The coating process was performed using a fluidized bed coater at 40⁰C.

4. The third coating layer - heat resistant coating

Calcium alginate was used as heat-resisting polymer for the third coating layer. First an aqueous solution of sodium alginate (3% w/w) and calcium chloride (5% w/w) were separately prepared. Then both sodium alginate and calcium chloride solutions were alternatively sprayed on the resulting coated microorganisms until a weight gain of 20% (w/w) was obtained.

Example 2

Materials

Method

1. Absorption of microorganisms on microcrvstalline core substrate

Lactobacillus acidophilus and Bifidobacterium were absorbed on MCC substrate based on a ratio of 38:62 respectively. For this purpose an aqueous- based suspension of 30% of the microorganisms and maltodextrin and trehalose was prepared. The concentration of microorganisms was about 15% (w/w) in that suspension. The absorption process was carried out at an outlet temperature <35°C in order to avoid the exposure of microorganisms to high temperatures and thus high-temperature damage.

2. The first coating layer using a hydrogenated vegetable oil

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose hydrogenated vegetable oil was sprayed on the Microorganisms-absorbed MCC substrate at 4O⁰C to obtain a 40% weight gain. The inlet air flow was adjusted to be low.

3. The second coating layer - an enteric coating

Sodium alginate was used as an enteric polymer. An aqueous solution of sodium alginate (2% w/w) was prepared. The sodium alginate solution was sprayed on resulting coated microorganisms until a weight gain of 15% was obtained.

4. The third coating layer- heat resistant coating

Chitosan was used as the heat-resisting polymer for the third coating layer. First an aqueous solution of chitosan (4% w/w) in pH 5 using HCl was prepared. The resulting solution was sprayed on the resulting coated microorganisms until a weight gain of 20% (w/w) was obtained.

Example 3

Materials

Lactobacillus acidophilus and Bifidobacterium were absorbed on MCC substrate based on a ratio of 38:62 respectively. For this purpose an aqueous- based suspension of 30% of the bacteria and maltodextrin and trehalose was prepared. The concentration of bacteria was about 15% (w/w) in that suspension. The absorption process was carried out at an outlet temperature <35°C in order to avoid the exposure of bacteria to high temperatures and thus high-temperature damage.

2. The first coating layer using a hydrogenated vegetable oil

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose hydrogenated vegetable oil was sprayed on the Microorganisms-absorbed MCC substrate at 40⁰C to obtain a 40% weight gain. The inlet air flow was adjusted to be low.

3. The second coating layer - an enteric coating

Sodium alginate was used as an enteric polymer. An aqueous solution of sodium alginate (2% w/w) was prepared. The sodium alginate solution was sprayed on resulting coated bacteria until a weight gain of 15% was obtained.

4. The third coating layer- heat resistant coating

Example 4

Materials

Method

1. Absorption of microorganisms on microcrvstalline core substrate

2. The first coating layer using a saturated vegetable oil

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose saturated vegetable oil was sprayed on the Microorganisms-absorbed MCC substrate at 40°C to obtain a 40% weight gain. The inlet air flow was adjusted to be low. 3. The second coating layer - an enteric coating

4. The third coating layer- heat resistant coating

Chitosan was used as the heat-resisting polymer for the third coating layer. First an aqueous solution of chitosan (4% w/w) in pH 5 using HCl was prepared. Then after complete dissolution of chitosan silicon dioxide (1% w/w) was added. The resulting solution was sprayed on the resulting coated microorganisms until a weight gain of 25% (w/w) was obtained.

Example 5

Materials:

Method:

1. Absorption of microorganisms on microcrystalline core substrate

2. The first coating layer using a hydrogenated vegetable oil

3. The second coating layer - an enteric coating

4. The third coating layer- heat resistant coating

Chitosan was used as the heat-resisting polymer for the third coating layer. First an aqueous solution of chitosan (4% w/w) in pH 5 using HCl was prepared. The resulting solution was sprayed on the resulting coated microorganisms until a weight gain of 30% (w/w) was obtained.

Example 6

Materials

Method

1. Absorption of microorganisms on microcrvstalline core substrate

2. The first coating layer using a hydrogenated vegetable oil

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose hydrogenated vegetable oil was sprayed on the Microorganisms-absorbed MCC substrate at 40°C to obtain a 40% weight gain. The inlet air flow was adjusted to be low.

3. The second coating layer - an enteric coating

Sodium alginate was used as an enteric polymer. An aqueous solution of sodium alginate (2% w/w) was prepared. The sodium alginate solution was sprayed on resulting coated microorganisms until a weight gain of 25% was obtained.

4. The third coating layer- heat resistant coating

Example 7

Encapsulated probiotic microorganisms granules were tested for heat resistance. Accordingly, the resulting encapsulated microorganisms granules from Example 6 were exposed to 85°C for 45 minutes. Then CFU/g was determined using a counting procedure described as follows.

Lactobacillus acidophilus and Lactobacillus bifidus counting procedure:

10 g of sample was suspended in 90 ml phosphate buffer and placed in a Stomacher for 10 min. Then the resulting suspension was shacked for 90 min. The mixture was then serially (decimally) diluted and finally poured into an appropriate plate culture media. MRS growth media containing either cystein or maltose were respectively used for acidophilus and bifidus. The resulting plates were then incubated for 3 days under anaerobic conditions. Finally the microorganisms were counted and CFU/g was calculated accordingly.

Results:

* The weight ratio between two microorganisms types in the final product is i:i.

** The microorganisms blend constitutes 10% (w/w) of the final product.

*** The heating process was carried out at 80⁰C for 45 minutes.

Example 8

A probiotic biscuit

This probiotic biscuit is made up of 0.3 g of filling and 30 g of Biscuit. Filling: The following ingredients are mixed at room temperature (percentages are weight percentages, based on the total filling weight): Biscuit recipe: 1 part sugar, 2 parts margarine, 3 parts flour mixed with 1 percent of the Probiotic powder. Microorganisms survival

The maximal temperature was about 200°C, applied for up to 4.5 minutes; that suits most industrial biscuit production. About 50% of live

microorganisms was maintained after baking.

Example 9

Probiotic bread

This probiotic bread is made up of 0.3 g of filling and 30 g of bread.

Microorganisms survival

The level of microorganisms survival obtained in simulation was between

50% to 80%. Up to 83% live microorganisms have been obtained after 10 minutes baking at 200 °C with a starting point of 10⁹ microorganisms per gram.

Example 10

Heat resistance assessment of encapsulated pro-biotic microorganisms according to the present invention in dry condition

Objective

To assess the heat resistance and survival of encapsulated pro-biotic microorganisms using the technology based on the present invention in comparison to uncoated pro-biotic microorganisms in a dry condition.

Summation

Both encapsulated and unencapsulated pro-biotic microorganisms (L. Acidophillus and Bifidobacteria) were placed in an oven which was preheated to 80 degree C for either 30 minutes or 45 minutes. The probiotics were then drawn out and CFU test was performed to determine the survival of microencapsulated microorganisms versus unencapsulated. The results showed that exposure of unencapsulated probiotics to such heat conditions can be catastrophic, wherein no CFU/g could be calculated which indicates that total destruction of unencapsulated microorganisms occurred. On the contrary, the encapsulated probiotics based on microencapsulation process according to the present invention did not show significant reduction in vitality test upon such heat treatment conditions. Based on these results one can conclude that microencapsulation process using multilayered coating based on the present invention provides heat resistance to probiotics under conditions described above.

Materials

2 Grams of Coated- Probiotic mix microorganisms (L. Acidophillus and

Bifidobacteria) according to the present invention. The composition of coating layers has been presented in Table 1.

2 Grams uncoated mix bacteria (L. Acidophillus and Bifidobacteria)

Method

The microencapsulation process was carried out according to manufacturing master processing record batch numbers RDEN 90405 land RDEN 904051.

Table 1: The components of different steps of microencapsulation process

Heating test

Both microencapsulated and unencapsulated (control) probiotics were introduced in an oven which was preheated to 80 degree C for either 30 or 45 minutes. CFU test

CFU tests were performed for the bacteria before and after heating process using the method described as follows;

1) 10 g of sample with 90 ml phosphate buffer.

2) Stomacher 10 min.

3) Shake the samples for 90 min.

4) Decimal dilutions.

5) Pour plate methods.

6) For acidophilus use MRS with cystein.

7) For bifidus use MRS with maltose instead of lactose.

8) Incubation 3 days in anaerobic conditions.

9) Count the bacteria and calculate the CFU/g. The method has been described in details elsewhere (K. G. de C. Lima et al. / LWT - Food Science and Technology 42 (2009), 491-494).

For encapsulated probiotic bacteria first the multi-layer shell surrounding the microorganisms was broken using a mortar and pestle before applying the above CFU method.

Results

Table 2. The effect of encapsulation process on CFU

* The weight ratio between two microorganisms in the final product is 1:1. ** The microorganisms blend compound constitutes 10% (w/w) of the final product. Table 3. The effect of heat treatment in dry condition in survival of pro-biotic microorganisms

Conclusion

Based on the results of Table 3 one can conclude that microencapsulation process using multilayered coating process according to the present invention provides heat resistance to probiotics under dry condition.

Example 11

Heat resistance assessment of encapsulated pro-biotic microorganisms according to the present invention in semi-baking condition

Objective

To assess the heat resistance and survival of encapsulated pro-biotic microorganisms using the technology according to the present invention in comparison to uncoated pro-biotic microorganisms in a semi-baking condition.

Summation

Both encapsulated and unencapsulated pro-biotic microorganisms (L. Acidophillus and Bifidobacteria) mixed with white bread ingredients and underwent baking at 180⁰C, 70% Humidity for 40 minutes. In order to enable collecting the microorganisms from the baked dough, both encapsulated and unencapsulated microorganisms were inserted into dough using two different methods being named as "Cheese cloth" and "Ravioli". Accordingly, the microorganisms were added into dough either indirectly by using cheese cloth to isolate the microorganisms from dough (cheese cloth method Experiment I) or directly by creating a separated pocket (Ravioli method Experiment II), made of the same dough, containing previously the microorganisms. According to the cheese cloth method the microorganisms either were previously encased in a cheese cloth which was then inserted into the dough before baking process (Experiment Ia) or the microorganisms were placed on a thin piece of cheese cloth which was previously inserted into dough by creating a small bowl in the center of the dough loaf and padding it by the thin piece of cheese cloth (Experiment Ib). According to "Ravioli" method a small pocket like Ravioli was first formed from the dough in which 2 Grams of coated mix microorganisms were placed and closed. The pocket was then placed in the center of the dough loaf. By these means one could also prevent the adherence of the dough to the microorganisms after baking process. It is important to prevent the adherence of the dough to the microorganisms since in such an experiment the dough may constitute a mechanical barrier against crushing force, during the crushing process, acting as a "Shock absorber ". By this way one may make sure that the coating is wholly broken during the crushing process before testing CFU. After baking, the microorganisms were pulled out and CFU/g was determined for each microorganisms strain and both encapsulated and unencapsulated microorganisms.

CFU results clearly show that uncoated microorganisms could not survive the baking condition whereas the encapsulated microorganisms demonstrated heat resistance during the baking process and high survival value after the baking process.

Materials

3 cups of flour

10 Grams Yeast 2 Tbs. Olive oil

1/8 Tsp. Salt

Water

2 Grams of Coated- Probiotic mix microorganisms (L. Acidophillus and

Bifidobacteria)

2 Grams uncoated mix bacteria (L. Acidophillus and Bifidobacteria)

Methods

Baking process

The bread ingredients were mixed all together and after a few minutes of kneading the dough was left to rise. The dough was then divided into separate loafs. The microorganisms were inserted into the dough loafs by using two different "cheese cloth" and "Ravioli" methods as described below

(remarked by Experiment I and Experiment II respectively).

Experiment I- "Cheese cloth" method

Experiment Ia - Both encapsulated and unencapsulated microorganisms were inserted into the dough when they were previously encased in a "Cheese

Cloth". 2 g of either encapsulated or unencapsulated microorganisms were placed in the middle of each dough loaf.

Experiment Ib- 2 g of encapsulated microorganisms were placed on the surface of a thin piece of cheese cloth which previously inserted in the middle of dough loaf by creating a bowl and padding with the thin piece of cheese cloth. The bowl was then covered with the remaining dough.

Experiment II- "Ravioli" method

A small pocket like Ravioli was first formed from the dough in which 2 g of encapsulated mix microorganisms were placed and closed. The pocket was then placed in the center of the dough loaf.

The dough was left to rise for additional 15 minutes.

The baking was carried out at 180°C for 40 minutes.

On the bottom shelf of the oven a metal tray with 1/2 a liter water was placed to create humidity inside the oven prior to inserting the bread loafs into the oven. The humidity created inside the oven was measured before inserting the bread loafs into the oven. In order to get the optimal baking humidity standard the bread loafs were inserted into the oven when the humidity reached between 60-70%. Once the dough loafs were baked the microorganisms were easily pulled out and sent to CFU test.

CFU test

CFU tests were performed for the microorganisms before and after baking process using the method described as follows;

1. 10 g of sample with 90 ml phosphate buffer.

2. Stomacher 10 min.

3. Shake the samples for 90 min.

4. Decimal dilutions.

5. Pour plate methods.

6. For acidophilus use MRS with cystein.

7. For bifidus use MRS with maltose instead of lactose.

8. Incubation 3 days in anaerobic conditions.

9. Count the microorganisms and calculate the CFU/g

The method has been described in detail elsewhere ((K. G. de C. Lima et al. / LWT - Food Science and Technology 42 (2009), 491-494)

For encapsulated probiotic microorganisms first the multi-layer shell surrounding the microorganisms was broken using a mortar and pestle before applying the above CFU method.

Results

Table 4: CFU/g of encapsulated and unencapsulated before and after baking condition

Conclusion

The results obtained above show that the encapsulated probiotic microorganisms using the technology according to the present invention are resistant to heat of baking in exposure to humidity existing in dough during baking process.

Example 12

Heat resistance assessment of encapsulated pro-biotic microorganisms according to the present invention in a full baking condition

Objective

To assess the heat resistance and survival of encapsulated pro-biotic microorganisms using the technology according to the present invention in a full baking condition using a commercial procedure. This study was designed to show feasibility of the concept of encapsulated probiotics according to the present invention which are resistant to a baking process in which they are subjected to shear forces, humidity, and heat.

Abstract

The purpose of this study was to assess the resistance of the encapsulated probiotics according to the present invention in a commercially used baking process. Accordingly, the encapsulated probiotics was directly added to dough being exposed first to shear forces of kneading and subsequently heat and humidity of baking process. This process was planned in order to mimic the baking process which is done in a commercial procedure. For this purpose the encapsulated probiotics according to the present invention were directly added to flour and other ingredients onto which water was then added (direct addition method) and subsequently kneaded and baked. Accordingly, the encapsulated probiotics were added directly to dough making ingredients and then distributed homogenously in the dough through kneading where they are exposed to moist environment during dough making step followed by heating of baking process. After the baking process CFU test was performed to determine the survival of encapsulated microorganisms. CFU results obviously showed that encapsulated microorganisms demonstrated high survival value after the baking process. Therefore, one can conclude that encapsulated probiotics according to the present invention are definitely resistant to moist environment under high shear existing during dough kneading, as well as to the heat of baking process.

Materials

Bread ingredients:

White flour: 231 Grams

Olive Oil: 18.7 Grams

Salt: 2 Grams

Yeast: 5 Grams

Encapsulated probiotics: 2 Grams

Dough before baking: 398.7 Grams

Bread after baking: 364.5 Grams

General method of baking process

Encapsulated pro-biotic microorganisms L. Addophillus and Bifidobacteria were homogenously mixed with all the rest of bread ingredients (white bread). Water was added and dough was then kneaded. The resulting dough was then baked at 180⁰C, 70% humidity for 40 minutes. This process was as follows: Equipments

Kenwood Mixer: 5 Liter bowl.

Dough preparation

Place flour, yeast and the encapsulated microorganisms in a mixing bowl

Mix all ingredients together.

Add oil and salt.

Add water gradually until the flour mixture forms firm dough.

Allow the mixer to knead the dough for 10 minutes.

Turn off the mixer and allow the dough to rest in the bowl cover and rise for

30 minutes.

Switch the mixer on for several seconds to "Punch Down" the dough.

Baking procedure

First the oven was preheated to 180⁰C prior to inserting the dough. The baking was carried out at 180 degrees C for 40 minutes. A metal tray containing 1/2 liter water was placed on the bottom shelf of the oven to create appropriate humidity (-70% RH) inside the oven prior to inserting the dough. The humidity created inside the oven was measured before baking. The dough was shaped in a baking pan and baked for 40 minutes (180 degree C and 70% RH). At the end of baking the humidity was checked again.

Baking conditions

Humidity before baking: 70% (RH).

Humidity after baking: 70% (RH).

Baking temperature: 180 degree C.

Baking duration: 40 minutes.

After baking a sample of the baked bread was taken to determine CFU/g for the encapsulated microorganisms.

CFU test

CFU tests were performed for the encapsulated probiotics after baking process using the CFU method described as follows: 1) 20 g of sample (baked bread) was taken to which 90 ml sterile phosphate buffer was added.

2) The mixture was then crushed using a mortar and pestle for a few minutes.

3) Additional 160 ml sterile phosphate buffer was added to Stomacher disposable sterile bag.

The mixture was then homogenized for 2 min using the Stomacher.

The CFU/g test was performed using the following regular procedure:

1- Decimal dilutions.

2- Pour plate methods.

3- For acidophilus use MRS with cy stein.

4- For bifidus use MRS with maltose instead of lactose.

5- Incubation 3 days in anaerobic conditions.

6- Count the microorganisms and calculate the CFU/g.

The method has been described in details elsewhere (K. G. de C. Lima et al. / LWT - Food Science and Technology 42 (2009), 491-494).

Results

CFU results before and after baking are summarized in Table 5. The CFU results clearly show that encapsulated microorganisms demonstrated heat resistance during the full baking process and high survival value after the baking process.

Table 5: CFU/g of encapsulated probiotics under full baking conditions

Conclusion

Encapsulated probiotics according to the present invention are resistant to heat of baking during a commercial preparation where encapsulated probiotics are added directly to all ingredients and then subjected to kneading process under humidity existing in dough and subsequently to the heat of baking process. These findings visibly indicate that the coating-layers formulations of the invention provide the probiotics with the needed protection to withstand all stages of baked product preparation, including shear forces of kneading, relatively high humidity, and the heat of baking.

Example 13

Materials:

Method:

1. Absorption of microorganisms on microcrystalline core substrate

Lactobacillus acidophilus and Bifidobacterium were absorbed on MCC substrate based on a ratio of 38:62 respectively. For this purpose an aqueous- based suspension of 30% of the microorganisms and maltodextrin and trehalose was prepared. The concentration of microorganisms was about 15% (w/w) in that suspension. The absorption process was carried out at an outlet temperature <35°C in order to avoid the exposure of microorganisms to high temperatures and thus high-temperature damage. 2. The first coating layer using stearic acid

The coating was carried out using a fluidized bed coater based on a Hot-Melt method. For this purpose stearic acid was sprayed on the microorganisms- absorbed MCC substrate at 4O⁰C to obtain a 40% weight gain. The inlet air flow was adjusted to be low.

3. The second coating layer - an enteric coating

4. The third coating layer- heat resistant coating

While this invention has been described in terms of some specific examples, many modifications and variations are possible. It is therefore understood that within the scope of the appended claims, the invention may be realized otherwise than as specifically described.

Claims

1. A probiotic granule comprising

i) a core comprising probiotic microorganisms and a substrate in which said microorganisms are absorbed;

ii) an inner oily layer coating said core; and

iii) a first outer layer and a second outer layer, which outer layers coat said core and said inner layer, comprising at least two different polymers.

2. A probiotic granule according to claim 1, wherein said first outer layer confers stability to said microorganisms under the conditions of upper gastrointestinal tract, and said second outer layer increases the stability of said microorganisms in said core at an increased temperature.

3. A probiotic granule according to claim 1, wherein said substrate comprises one or more components selected from saccharides and additional agents.

4. A probiotic granule according to claim 3, wherein said agents are selected from stabilizer, chelator, synergistic agent, antioxidant, and pH regulator, and wherein said saccharides comprise prebiotic oligos accharide s .

5. A probiotic granule according to claim 1, wherein one of said outer layers comprises a fibrous polysaccharide.

6. A probiotic granule according to claim 1, wherein one of said outer layers comprises a gelatinous polysaccharide.

7. A probiotic granule according to claim 1, comprising at least one intermediate layer positioned between said oily layer and said second outer layer.

8. A probiotic granule according to claim 1, wherein said microorganisms comprise a genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces, or a mixture thereof.

9. A method for manufacturing the granule of claim 1, comprising

i) mixing a suspension of probiotic microorganisms with a cellulose- based substrate and with supplemental agents for the microorganisms, thereby obtaining a core mixture;

ii) coating particles of said core mixture with an oily layer, thereby obtaining oil-coated particles;

iii) coating said oil-coated particles with a first polymer layer, which first polymer layer confers stability to said microorganisms under the conditions of upper gastrointestinal tract, thereby obtaining particles coated with two layers; and

iv) coating said two-layer particles with a second polymer layer, which second polymer layer increases the stability of the microorganisms in said core under the conditions of baking.

10. A method according to claim 9, wherein each of said coating steps ii) to iv) results in a mass increase of from 10 to 100 % relatively to the mass of said core.

11. A method according to claim 9, comprising

i) mixing an aqueous suspension of probiotic microorganisms comprising at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces, or a mixture thereof with at least one polysaccharide and at least one oligosaccharide, thereby obtaining a core mixture;

iii) coating said oil-coated particles with a first polysaccharide layer and with a second polysaccharide layer;

wherein said two polysaccharides layers are different and comprise at least two of cellulose, alginate, chitosan, or a mixture thereof.

12. A probiotic composition comprising granules according to claim 1, said composition exhibiting high heat resistance and long storage stability.

13. A food product or a food additive comprising a probiotic granule according to claim 1.

14. A food product according to claim 13, selected from the group consisting of pastry, bread, flour, flour products, baked goods, frozen baking products, yogurt, dairy products, chocolate, nectars, fruit juices, and tuna.

15. A food product according to claim 14, exposed to higher than ambient temperature during the production process.